bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2021–08–01
forty-six papers selected by
Sean Rudd, Karolinska Institutet
  1. An extending ATR-CHK1 circuitry: the replication stress response and beyond.
  2. Understanding the DNA double-strand break repair and its therapeutic implications.
  3. Replication protein A: a multifunctional protein with roles in DNA replication, repair and beyond.
  4. Homologous recombination, cancer and the 'RAD51 paradox'.
  5. DNA polymerase η is a substrate for calpain: a possible mechanism for pol η retention in UV-induced replication foci.
  6. RAD51 paralog function in replicative DNA damage and tolerance.
  7. Genetic vulnerabilities upon inhibition of DNA damage response.
  8. Physical interactions between MCM and Rad51 facilitate replication fork lesion bypass and ssDNA gap filling by non-recombinogenic functions.
  9. To Join or Not to Join: Decision Points Along the Pathway to Double-Strand Break Repair vs. Chromosome End Protection.
  10. Biomolecular condensates at sites of DNA damage: More than just a phase.
  11. DNA end resection during homologous recombination.
  12. Recombination-mediated genome rearrangements.
  13. The dark side of homology-directed repair.
  14. BARD1 reads H2A lysine 15 ubiquitination to direct homologous recombination.
  15. FANCD2 limits acetaldehyde-induced genomic instability during DNA replication in esophageal keratinocytes.
  16. SMARCA4 deficiency-associated heterochromatin induces intrinsic DNA replication stress and susceptibility to ATR inhibition in lung adenocarcinoma.
  17. Mechanisms of BRCA1-BARD1 nucleosome recognition and ubiquitylation.
  18. Genome instability and pressure on non-homologous end joining drives chemotherapy resistance via a DNA repair crisis switch in triple negative breast cancer.
  19. Loss of Cyclin C or CDK8 provides ATR inhibitor resistance by suppressing transcription-associated replication stress.
  20. The HelQ human DNA repair helicase utilizes a PWI-like domain for DNA loading through interaction with RPA, triggering DNA unwinding by the HelQ helicase core.
  21. PCNA inhibition enhances the cytotoxicity of β-lapachone in NQO1-Positive cancer cells by augmentation of oxidative stress-induced DNA damage.
  22. Interplay between Sae2 and Rif2 in the regulation of Mre11-Rad50 activities at DNA ends.
  23. The role of Sp1 in the detection and elimination of cells with persistent DNA strand breaks.
  24. Epigenetic Mechanisms in DNA Double Strand Break Repair: A Clinical Review.
  25. The DNA damage inducible lncRNA SCAT7 regulates genomic integrity and topoisomerase 1 turnover in lung adenocarcinoma.
  26. FAN1-MLH1 interaction affects repair of DNA interstrand cross-links and slipped-CAG/CTG repeats.
  27. Apurinic/Apyrimidinic Endonuclease 2 (APE2): An ancillary enzyme for contextual base excision repair mechanisms to preserve genome stability.
  28. The PI3K/mTOR Inhibitor Ompalisib Suppresses Nonhomologous End Joining and Sensitizes Cancer Cells to Radio- and Chemotherapy.
  29. The repair gene BACH1 - a potential oncogene.
  30. A Minimalistic Coumarin Turn-On Probe for Selective Recognition of Parallel G-Quadruplex DNA Structures.
  31. Replication-dependent histone biosynthesis is coupled to cell-cycle commitment.
  32. Defining R-loop classes and their contributions to genome instability.
  33. IMPDH2: a new gene associated with dominant juvenile-onset dystonia-tremor disorder.
  34. The major mechanism of melanoma mutations is based on deamination of cytosine in pyrimidine dimers as determined by circle damage sequencing.
  35. SUMOylation of SAMHD1 at Lysine 595 is required for HIV-1 restriction in non-cycling cells.
  36. TDRD3 promotes DHX9 chromatin recruitment and R-loop resolution.
  37. Mechanistic insights into the three steps of poly(ADP-ribosylation) reversal.
  38. Tumor-selective, antigen-independent delivery of a pH sensitive peptide-topoisomerase inhibitor conjugate suppresses tumor growth without systemic toxicity.
  39. PRMT1-dependent regulation of RNA metabolism and DNA damage response sustains pancreatic ductal adenocarcinoma.
  40. An enzymatic activation of formaldehyde for nucleotide methylation.
  41. Structural determinants and distribution of phosphate specificity in ribonucleotide reductases.
  42. Long noncoding RNA NIHCOLE promotes ligation efficiency of DNA double-strand breaks in hepatocellular carcinoma.
  43. ADP-ribosyltransferases, an update on function and nomenclature.
  44. BRCA2 binding through a cryptic repeated motif to HSF2BP oligomers does not impact meiotic recombination.
  45. Hepatic mTORC1 signaling activates ATF4 as part of its metabolic response to feeding and insulin.
  46. Sirt3 increases CNPase enzymatic activity through deacetylation and facilitating substrate accessibility.
  1. Curr Opin Genet Dev. 2021 Jul 27. pii: S0959-437X(21)00089-7. [Epub ahead of print]71 92-98
    An extending ATR-CHK1 circuitry: the replication stress response and beyond.
    Antoine Simoneau, Lee Zou.
      The maintenance of genomic integrity relies on the coordination of a wide range of cellular processes and efficient repair of DNA damage. Since its discovery over two decades ago, the ATR kinase has been recognized as the master regulator of the circuitry orchestrating the cellular responses to DNA damage and replication stress. Recent studies reveal that ATR additionally functions in the unperturbed cell cycle through its control of replication fork speed and stability, replication origin firing, completion of genome duplication, and chromosome segregation. Here, we discuss several recently discovered mechanisms through which ATR safeguards genomic integrity during the cell cycle, from S phase to mitosis.
    DOI:  https://doi.org/10.1016/j.gde.2021.07.003
  2. DNA Repair (Amst). 2021 Jul 09. pii: S1568-7864(21)00133-6. [Epub ahead of print]106 103177
    Understanding the DNA double-strand break repair and its therapeutic implications.
    Ujjayinee Ray, Sathees C Raghavan.
      Repair of DNA double-strand breaks (DSBs) and its regulation are tightly integrated inside cells. Homologous recombination, nonhomologous end joining and microhomology mediated end joining are three major DSB repair pathways in mammalian cells. Targeting proteins associated with these repair pathways using small molecule inhibitors can prove effective in tumors, especially those with deregulated repair. Sensitization of cancer to current age therapy including radio and chemotherapy, using small molecule inhibitors is promising and warrant further development. Although several are under clinical trial, till date no repair inhibitor is approved for commercial use in cancer patients, with the exception of PARP inhibitors targeting single-strand break repair. Based on molecular profiling of repair proteins, better prognostic and therapeutic output can be achieved in patients. In the present review, we highlight the different mechanisms of DSB repair, chromatin dynamics to provide repair accessibility and modulation of inhibitors in association with molecular profiling and current gold standard treatment modalities for cancer.
    Keywords:  Cancer therapy; Chromatin accessibility; DSB repair; Double-strand break; Genomic instability; Homologous recombination; MMEJ; Microhomology mediated end joining; NHEJ; Nonhomologous DNA end joining
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103177
  3. NAR Cancer. 2020 Sep;2(3): zcaa022
    Replication protein A: a multifunctional protein with roles in DNA replication, repair and beyond.
    Rositsa Dueva, George Iliakis.
      Single-stranded DNA (ssDNA) forms continuously during DNA replication and is an important intermediate during recombination-mediated repair of damaged DNA. Replication protein A (RPA) is the major eukaryotic ssDNA-binding protein. As such, RPA protects the transiently formed ssDNA from nucleolytic degradation and serves as a physical platform for the recruitment of DNA damage response factors. Prominent and well-studied RPA-interacting partners are the tumor suppressor protein p53, the RAD51 recombinase and the ATR-interacting proteins ATRIP and ETAA1. RPA interactions are also documented with the helicases BLM, WRN and SMARCAL1/HARP, as well as the nucleotide excision repair proteins XPA, XPG and XPF-ERCC1. Besides its well-studied roles in DNA replication (restart) and repair, accumulating evidence shows that RPA is engaged in DNA activities in a broader biological context, including nucleosome assembly on nascent chromatin, regulation of gene expression, telomere maintenance and numerous other aspects of nucleic acid metabolism. In addition, novel RPA inhibitors show promising effects in cancer treatment, as single agents or in combination with chemotherapeutics. Since the biochemical properties of RPA and its roles in DNA repair have been extensively reviewed, here we focus on recent discoveries describing several non-canonical functions.
    DOI:  https://doi.org/10.1093/narcan/zcaa022
  4. NAR Cancer. 2021 Jun;3(2): zcab016
    Homologous recombination, cancer and the 'RAD51 paradox'.
    Gabriel Matos-Rodrigues, Josée Guirouilh-Barbat, Emmanuelle Martini, Bernard S Lopez.
      Genetic instability is a hallmark of cancer cells. Homologous recombination (HR) plays key roles in genome stability and variability due to its roles in DNA double-strand break and interstrand crosslink repair, and in the protection and resumption of arrested replication forks. HR deficiency leads to genetic instability, and, as expected, many HR genes are downregulated in cancer cells. The link between HR deficiency and cancer predisposition is exemplified by familial breast and ovarian cancers and by some subgroups of Fanconi anaemia syndromes. Surprisingly, although RAD51 plays a pivotal role in HR, i.e., homology search and in strand exchange with a homologous DNA partner, almost no inactivating mutations of RAD51 have been associated with cancer predisposition; on the contrary, overexpression of RAD51 is associated with a poor prognosis in different types of tumours. Taken together, these data highlight the fact that RAD51 differs from its HR partners with regard to cancer susceptibility and expose what we call the 'RAD51 paradox'. Here, we catalogue the dysregulations of HR genes in human pathologies, including cancer and Fanconi anaemia or congenital mirror movement syndromes, and we discuss the RAD51 paradox.
    DOI:  https://doi.org/10.1093/narcan/zcab016
  5. J Cell Sci. 2021 Jul 01. pii: jcs258637. [Epub ahead of print]134(13):
    DNA polymerase η is a substrate for calpain: a possible mechanism for pol η retention in UV-induced replication foci.
    Jo-Ann Nettersheim, Régine Janel-Bintz, Lauriane Kuhn, Agnès M Cordonnier.
      DNA polymerase η (pol η) is specifically required for translesion DNA synthesis across UV-induced DNA lesions. Recruitment of this error-prone DNA polymerase is tightly regulated during replication to avoid mutagenesis and perturbation of fork progression. Here, we report that pol η interacts with the calpain small subunit-1 (CAPNS1) in a yeast two-hybrid screening. This interaction is functional, as demonstrated by the ability of endogenous calpain to mediate calcium-dependent cleavage of pol η in cell-free extracts and in living cells treated with a calcium ionophore. The proteolysis of pol η was found to occur at position 465, leading to a catalytically active truncated protein containing the PCNA-interacting motif PIP1. Unexpectedly, cell treatment with the specific calpain inhibitor calpeptin resulted in a decreased extent of pol η foci after UV irradiation, indicating that calpain positively regulates pol η accumulation in replication foci.
    Keywords:  CAPNS1; Calpain protease; DNA damage response; DNA polymerase η; Replication foci; Translesion DNA synthesis
    DOI:  https://doi.org/10.1242/jcs.258637
  6. Curr Opin Genet Dev. 2021 Jul 23. pii: S0959-437X(21)00080-0. [Epub ahead of print]71 86-91
    RAD51 paralog function in replicative DNA damage and tolerance.
    Hayley L Rein, Kara A Bernstein, Robert A Baldock.
      RAD51 paralog gene mutations are observed in both hereditary breast and ovarian cancers. Classically, defects in RAD51 paralog function are associated with homologous recombination (HR) deficiency and increased genomic instability. Several recent investigative advances have enabled characterization of non-canonical RAD51 paralog function during DNA replication. Here we discuss the role of the RAD51 paralogs and their associated complexes in integrating a robust response to DNA replication stress. We highlight recent discoveries suggesting that the RAD51 paralogs complexes mediate lesion-specific tolerance of replicative stress following exposure to alkylating agents and the requirement for the Shu complex in fork restart upon fork stalling by dNTP depletion. In addition, we describe the role of the BCDX2 complex in restraining and promoting fork remodeling in response to fluctuating dNTP pools. Finally, we highlight recent work demonstrating a requirement for RAD51C in recognizing and tolerating methyl-adducts. In each scenario, RAD51 paralog complexes play a central role in lesion recognition and bypass in a replicative context. Future studies will determine how these critical functions for RAD51 paralog complexes contribute to tumorigenesis.
    DOI:  https://doi.org/10.1016/j.gde.2021.06.010
  7. Nucleic Acids Res. 2021 Jul 28. pii: gkab643. [Epub ahead of print]
    Genetic vulnerabilities upon inhibition of DNA damage response.
    Chao Wang, Mengfan Tang, Zhen Chen, Litong Nie, Siting Li, Yun Xiong, Klaudia Anna Szymonowicz, Jeong-Min Park, Huimin Zhang, Xu Feng, Min Huang, Dan Su, Traver Hart, Junjie Chen.
      Because of essential roles of DNA damage response (DDR) in the maintenance of genomic integrity, cellular homeostasis, and tumor suppression, targeting DDR has become a promising therapeutic strategy for cancer treatment. However, the benefits of cancer therapy targeting DDR are limited mainly due to the lack of predictive biomarkers. To address this challenge, we performed CRISPR screens to search for genetic vulnerabilities that affect cells' response to DDR inhibition. By undertaking CRISPR screens with inhibitors targeting key DDR mediators, i.e. ATR, ATM, DNAPK and CHK1, we obtained a global and unbiased view of genetic interactions with DDR inhibition. Specifically, we identified YWHAE loss as a key determinant of sensitivity to CHK1 inhibition. We showed that KLHL15 loss protects cells from DNA damage induced by ATM inhibition. Moreover, we validated that APEX1 loss sensitizes cells to DNAPK inhibition. Additionally, we compared the synergistic effects of combining different DDR inhibitors and found that an ATM inhibitor plus a PARP inhibitor induced dramatic levels of cell death, probably through promoting apoptosis. Our results enhance the understanding of DDR pathways and will facilitate the use of DDR-targeting agents in cancer therapy.
    DOI:  https://doi.org/10.1093/nar/gkab643
  8. Cell Rep. 2021 Jul 27. pii: S2211-1247(21)00857-3. [Epub ahead of print]36(4): 109440
    Physical interactions between MCM and Rad51 facilitate replication fork lesion bypass and ssDNA gap filling by non-recombinogenic functions.
    María J Cabello-Lobato, Cristina González-Garrido, María I Cano-Linares, Ronald P Wong, Aurora Yáñez-Vílchez, Macarena Morillo-Huesca, Juan M Roldán-Romero, Marta Vicioso, Román González-Prieto, Helle D Ulrich, Félix Prado.
      The minichromosome maintenance (MCM) helicase physically interacts with the recombination proteins Rad51 and Rad52 from yeast to human cells. We show, in Saccharomyces cerevisiae, that these interactions occur within a nuclease-insoluble scaffold enriched in replication/repair factors. Rad51 accumulates in a MCM- and DNA-binding-independent manner and interacts with MCM helicases located outside of the replication origins and forks. MCM, Rad51, and Rad52 accumulate in this scaffold in G1 and are released during the S phase. In the presence of replication-blocking lesions, Cdc7 prevents their release from the scaffold, thus maintaining the interactions. We identify a rad51 mutant that is impaired in its ability to bind to MCM but not to the scaffold. This mutant is proficient in recombination but partially defective in single-stranded DNA (ssDNA) gap filling and replication fork progression through damaged DNA. Therefore, cells accumulate MCM/Rad51/Rad52 complexes at specific nuclear scaffolds in G1 to assist stressed forks through non-recombinogenic functions.
    Keywords:  Cdc7; DNA damage; MCM; Rad51; Rad52; homologous recombination; replication
    DOI:  https://doi.org/10.1016/j.celrep.2021.109440
  9. Front Cell Dev Biol. 2021 ;9 708763
    To Join or Not to Join: Decision Points Along the Pathway to Double-Strand Break Repair vs. Chromosome End Protection.
    Stephanie M Ackerson, Carlan Romney, P Logan Schuck, Jason A Stewart.
      The regulation of DNA double-strand breaks (DSBs) and telomeres are diametrically opposed in the cell. DSBs are considered one of the most deleterious forms of DNA damage and must be quickly recognized and repaired. Telomeres, on the other hand, are specialized, stable DNA ends that must be protected from recognition as DSBs to inhibit unwanted chromosome fusions. Decisions to join DNA ends, or not, are therefore critical to genome stability. Yet, the processing of telomeres and DSBs share many commonalities. Accordingly, key decision points are used to shift DNA ends toward DSB repair vs. end protection. Additionally, DSBs can be repaired by two major pathways, namely homologous recombination (HR) and non-homologous end joining (NHEJ). The choice of which repair pathway is employed is also dictated by a series of decision points that shift the break toward HR or NHEJ. In this review, we will focus on these decision points and the mechanisms that dictate end protection vs. DSB repair and DSB repair choice.
    Keywords:  DNA repair; double-strand break; homologous recombination; non-homologous end joining; telomeres
    DOI:  https://doi.org/10.3389/fcell.2021.708763
  10. DNA Repair (Amst). 2021 Jul 14. pii: S1568-7864(21)00135-X. [Epub ahead of print]106 103179
    Biomolecular condensates at sites of DNA damage: More than just a phase.
    Vincent Spegg, Matthias Altmeyer.
      Protein recruitment to DNA break sites is an integral part of the DNA damage response (DDR). Elucidation of the hierarchy and temporal order with which DNA damage sensors as well as repair and signaling factors assemble around chromosome breaks has painted a complex picture of tightly regulated macromolecular interactions that build specialized compartments to facilitate repair and maintenance of genome integrity. While many of the underlying interactions, e.g. between repair factors and damage-induced histone marks, can be explained by lock-and-key or induced fit binding models assuming fixed stoichiometries, structurally less well defined interactions, such as the highly dynamic multivalent interactions implicated in phase separation, also participate in the formation of multi-protein assemblies in response to genotoxic stress. Although much remains to be learned about these types of cooperative and highly dynamic interactions and their functional roles, the rapidly growing interest in material properties of biomolecular condensates and in concepts from polymer chemistry and soft matter physics to understand biological processes at different scales holds great promises. Here, we discuss nuclear condensates in the context of genome integrity maintenance, highlighting the cooperative potential between clustered stoichiometric binding and phase separation. Rather than viewing them as opposing scenarios, their combined effects can balance structural specificity with favorable physicochemical properties relevant for the regulation and function of multilayered nuclear condensates.
    Keywords:  Biomolecular condensates; DNA damage response (DDR); DNA repair; Genome stability; Higher-order assemblies; Intrinsically disordered regions (IDR); Liquid-liquid phase separation (LLPS); Low complexity domains (LCD); Multivalent interactions
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103179
  11. Curr Opin Genet Dev. 2021 Jul 27. pii: S0959-437X(21)00090-3. [Epub ahead of print]71 99-105
    DNA end resection during homologous recombination.
    Robert Gnügge, Lorraine S Symington.
      Exposure to environmental mutagens but also cell-endogenous processes can create DNA double-strand breaks (DSBs) in a cell's genome. DSBs need to be repaired accurately and timely to ensure genomic integrity and cell survival. One major DSB repair mechanism, called homologous recombination, relies on the nucleolytic degradation of the 5'-terminated strands in a process termed end resection. Here, we review new insights into end resection with a focus on the mechanistic interplay of the nucleases, helicases, and accessory factors involved.
    DOI:  https://doi.org/10.1016/j.gde.2021.07.004
  12. Curr Opin Genet Dev. 2021 Jul 26. pii: S0959-437X(21)00078-2. [Epub ahead of print]71 63-71
    Recombination-mediated genome rearrangements.
    Jérôme Savocco, Aurèle Piazza.
      Homologous recombination (HR) is a universal DNA double-strand break (DSB) repair pathway that uses an intact DNA molecule as a template. Signature HR reactions are homology search and DNA strand invasion catalyzed by the prototypical RecA-ssDNA filament (Rad51 and Dmc1 in eukaryotes), which produces heteroduplex DNA-containing joint molecules (JMs). These reactions uniquely infringe on the DNA strands association established at replication, on the basis of substantial sequence similarity. For that reason, and despite the high fidelity of its templated nature, DSB repair by HR authorizes the alteration of genome structure, guided by repetitive DNA elements. The resulting structural variations (SVs) can involve vast genomic regions, potentially affecting multiple coding sequences and regulatory elements at once, with possible pathological consequences. Here, we discuss recent advances in our understanding of genetic and molecular vulnerabilities of HR leading to SVs, and of the various fidelity-enforcing factors acting across scales on the balancing act of this complex pathway. An emphasis is put on extra-chomosomal DNAs, both product of, and substrate for HR-mediated chromosomal rearrangements.
    DOI:  https://doi.org/10.1016/j.gde.2021.06.008
  13. DNA Repair (Amst). 2021 Jul 17. pii: S1568-7864(21)00137-3. [Epub ahead of print]106 103181
    The dark side of homology-directed repair.
    Amr M Al-Zain, Lorraine S Symington.
      DNA double strand breaks (DSB) are cytotoxic lesions that can lead to genome rearrangements and genomic instability, which are hallmarks of cancer. The two main DSB repair pathways are non-homologous end joining and homologous recombination (HR). While HR is generally highly accurate, it has the potential for rearrangements that occur directly or through intermediates generated during the repair process. Whole genome sequencing of cancers has revealed numerous types of structural rearrangement signatures that are often indicative of repair mediated by sequence homology. However, it can be challenging to delineate repair mechanisms from sequence analysis of rearrangement end products from cancer genomes, or even model systems, because the same rearrangements can be generated by different pathways. Here, we review homology-directed repair pathways and their consequences. Exploring those pathways can lead to a greater understanding of rearrangements that occur in cancer cells.
    Keywords:  Break-induced replication; Chromosome rearrangement; Homologous recombination; Microhomology-mediated end joining; Single-strand annealing
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103181
  14. Nature. 2021 Jul 28.
    BARD1 reads H2A lysine 15 ubiquitination to direct homologous recombination.
    Jordan R Becker, Gillian Clifford, Clara Bonnet, Anja Groth, Marcus D Wilson, J Ross Chapman.
      Protein ubiquitination at sites of DNA double-strand breaks (DSBs) by RNF168 recruits BRCA1 and 53BP11,2, which are mediators of the homologous recombination and non-homologous end joining DSB repair pathways, respectively3. Non-homologous end joining relies on 53BP1 binding directly to ubiquitinated lysine 15 on H2A-type histones (H2AK15ub)4,5 (which is an RNF168-dependent modification6), but how RNF168 promotes BRCA1 recruitment and function remains unclear. Here we identify a tandem BRCT-domain-associated ubiquitin-dependent recruitment motif (BUDR) in BRCA1-associated RING domain protein 1 (BARD1) (the obligate partner protein of BRCA1) that, by engaging H2AK15ub, recruits BRCA1 to DSBs. Disruption of the BUDR of BARD1 compromises homologous recombination and renders cells hypersensitive to PARP inhibition and cisplatin. We further show that BARD1 binds nucleosomes through multivalent interactions: coordinated binding of H2AK15ub and unmethylated H4 lysine 20 by its adjacent BUDR and ankyrin repeat domains, respectively, provides high-affinity recognition of DNA lesions in replicated chromatin and promotes the homologous recombination activities of the BRCA1-BARD1 complex. Finally, our genetic epistasis experiments confirm that the need for BARD1 chromatin-binding activities can be entirely relieved upon deletion of RNF168 or 53BP1. Thus, our results demonstrate that by sensing DNA-damage-dependent and post-replication histone post-translation modification states, BRCA1-BARD1 complexes coordinate the antagonization of the 53BP1 pathway with promotion of homologous recombination, establishing a simple paradigm for the governance of the choice of DSB repair pathway.
    DOI:  https://doi.org/10.1038/s41586-021-03776-w
  15. Mol Oncol. 2021 Jul 30.
    FANCD2 limits acetaldehyde-induced genomic instability during DNA replication in esophageal keratinocytes.
    Jasmine D Peake, Chiaki Noguchi, Baicheng Lin, Amber Theriault, Margaret O'Connor, Shivani Sheth, Koji Tanaka, Hiroshi Nakagawa, Eishi Noguchi.
      Individuals with Fanconi anemia (FA), a rare genetic bone marrow failure syndrome, have an increased risk of young-onset head and neck squamous-cell carcinomas (SCCs) and esophageal SCC. The FA DNA repair pathway is activated upon DNA damage induced by acetaldehyde, a chief alcohol metabolite and one of the major carcinogens in humans. However, the molecular basis of acetaldehyde-induced genomic instability in SCCs of the head and neck and of the esophagus in FA remains elusive. Here we report the effects of acetaldehyde on replication stress response in esophageal epithelial cells (keratinocytes). Acetaldehyde-exposed esophageal keratinocytes displayed accumulation of DNA damage foci consisting of 53BP1 and BRCA1. At physiologically relevant concentrations, acetaldehyde activated the ATR-Chk1 pathway, leading to S- and G2/M-phase delay with accumulation of the FA complementation group D2 protein (FANCD2) at the sites of DNA synthesis, suggesting that acetaldehyde impedes replication fork progression. Consistently, depletion of the replication fork protection protein Timeless led to elevated DNA damage upon acetaldehyde exposure. Furthermore, FANCD2 depletion exacerbated replication abnormalities, elevated DNA damage, and led to apoptotic cell death, indicating that FANCD2 prevents acetaldehyde-induced genomic instability in esophageal keratinocytes. These observations contribute to our understanding of the mechanisms that drive genomic instability in FA patients and alcohol-related carcinogenesis, thereby providing a translational implication in the development of more effective therapies for SCCs.
    Keywords:  Acetaldehyde; Alcohol; Esophageal squamous-cell carcinoma; FANCD2; Fanconi anemia; Genomic instability
    DOI:  https://doi.org/10.1002/1878-0261.13072
  16. NAR Cancer. 2020 Jun;2(2): zcaa005
    SMARCA4 deficiency-associated heterochromatin induces intrinsic DNA replication stress and susceptibility to ATR inhibition in lung adenocarcinoma.
    Kiminori Kurashima, Hideto Kashiwagi, Iwao Shimomura, Ayako Suzuki, Fumitaka Takeshita, Marianne Mazevet, Masahiko Harata, Takayuki Yamashita, Yusuke Yamamoto, Takashi Kohno, Bunsyo Shiotani.
      The SWI/SNF chromatin remodeling complex regulates transcription through the control of chromatin structure and is increasingly thought to play an important role in human cancer. Lung adenocarcinoma (LADC) patients frequently harbor mutations in SMARCA4, a core component of this multisubunit complex. Most of these mutations are loss-of-function mutations, which disrupt critical functions in the regulation of chromatin architecture and can cause DNA replication stress. This study reports that LADC cells deficient in SMARCA4 showed increased DNA replication stress and greater sensitivity to the ATR inhibitor (ATRi) in vitro and in vivo. Mechanistically, loss of SMARCA4 increased heterochromatin formation, resulting in stalled forks, a typical DNA replication stress. In the absence of SMARCA4, severe ATRi-induced single-stranded DNA, which caused replication catastrophe, was generated on nascent DNA near the reversed forks around heterochromatin in an Mre11-dependent manner. Thus, loss of SMARCA4 confers susceptibility to ATRi, both by increasing heterochromatin-associated replication stress and by allowing Mre11 to destabilize reversed forks. These two mechanisms synergistically increase susceptibility of SMARCA4-deficient LADC cells to ATRi. These results provide a preclinical basis for assessing SMARCA4 defects as a biomarker of ATRi efficacy.
    DOI:  https://doi.org/10.1093/narcan/zcaa005
  17. Nature. 2021 Jul 28.
    Mechanisms of BRCA1-BARD1 nucleosome recognition and ubiquitylation.
    Qi Hu, Maria Victoria Botuyan, Debiao Zhao, Gaofeng Cui, Elie Mer, Georges Mer.
      The BRCA1-BARD1 tumour suppressor is an E3 ubiquitin ligase necessary for the repair of DNA double-strand breaks by homologous recombination1-10. The BRCA1-BARD1 complex localizes to damaged chromatin after DNA replication and catalyses the ubiquitylation of histone H2A and other cellular targets11-14. The molecular bases for the recruitment to double-strand breaks and target recognition of BRCA1-BARD1 remain unknown. Here we use cryo-electron microscopy to show that the ankyrin repeat and tandem BRCT domains in BARD1 adopt a compact fold and bind to nucleosomal histones, DNA and monoubiquitin attached to H2A amino-terminal K13 or K15, two signals known to be specific for double-strand breaks15,16. We further show that RING domains17 in BRCA1-BARD1 orient an E2 ubiquitin-conjugating enzyme atop the nucleosome in a dynamic conformation, primed for ubiquitin transfer to the flexible carboxy-terminal tails of H2A and variant H2AX. Our work reveals a regulatory crosstalk in which recognition of monoubiquitin by BRCA1-BARD1 at the N terminus of H2A blocks the formation of polyubiquitin chains and cooperatively promotes ubiquitylation at the C terminus of H2A. These findings elucidate the mechanisms of BRCA1-BARD1 chromatin recruitment and ubiquitylation specificity, highlight key functions of BARD1 in both processes and explain how BRCA1-BARD1 promotes homologous recombination by opposing the DNA repair protein 53BP1 in post-replicative chromatin18-22. These data provide a structural framework to evaluate BARD1 variants and help to identify mutations that drive the development of cancer.
    DOI:  https://doi.org/10.1038/s41586-021-03716-8
  18. NAR Cancer. 2021 Jun;3(2): zcab022
    Genome instability and pressure on non-homologous end joining drives chemotherapy resistance via a DNA repair crisis switch in triple negative breast cancer.
    Adrian P Wiegmans, Ambber Ward, Ekaterina Ivanova, Pascal H G Duijf, Mark N Adams, Idris Mohd Najib, Romy Van Oosterhout, Martin C Sadowski, Greg Kelly, Scott W Morrical, Ken O'Byrne, Jason S Lee, Derek J Richard.
      Chemotherapy is used as a standard-of-care against cancers that display high levels of inherent genome instability. Chemotherapy induces DNA damage and intensifies pressure on the DNA repair pathways that can lead to deregulation. There is an urgent clinical need to be able to track the emergence of DNA repair driven chemotherapy resistance and tailor patient staging appropriately. There have been numerous studies into chemoresistance but to date no study has elucidated in detail the roles of the key DNA repair components in resistance associated with the frontline clinical combination of anthracyclines and taxanes together. In this study, we hypothesized that the emergence of chemotherapy resistance in triple negative breast cancer was driven by changes in functional signaling in the DNA repair pathways. We identified that consistent pressure on the non-homologous end joining pathway in the presence of genome instability causes failure of the key kinase DNA-PK, loss of p53 and compensation by p73. In-turn a switch to reliance on the homologous recombination pathway and RAD51 recombinase occurred to repair residual double strand DNA breaks. Further we demonstrate that RAD51 is an actionable target for resensitization to chemotherapy in resistant cells with a matched gene expression profile of resistance highlighted by homologous recombination in clinical samples.
    DOI:  https://doi.org/10.1093/narcan/zcab022
  19. Nucleic Acids Res. 2021 Jul 30. pii: gkab628. [Epub ahead of print]
    Loss of Cyclin C or CDK8 provides ATR inhibitor resistance by suppressing transcription-associated replication stress.
    Rebecca L Lloyd, Vaclav Urban, Francisco Muñoz-Martínez, Iñigo Ayestaran, John C Thomas, Christelle de Renty, Mark J O'Connor, Josep V Forment, Yaron Galanty, Stephen P Jackson.
      The protein kinase ATR plays pivotal roles in DNA repair, cell cycle checkpoint engagement and DNA replication. Consequently, ATR inhibitors (ATRi) are in clinical development for the treatment of cancers, including tumours harbouring mutations in the related kinase ATM. However, it still remains unclear which functions and pathways dominate long-term ATRi efficacy, and how these vary between clinically relevant genetic backgrounds. Elucidating common and genetic-background specific mechanisms of ATRi efficacy could therefore assist in patient stratification and pre-empting drug resistance. Here, we use CRISPR-Cas9 genome-wide screening in ATM-deficient and proficient mouse embryonic stem cells to interrogate cell fitness following treatment with the ATRi, ceralasertib. We identify factors that enhance or suppress ATRi efficacy, with a subset of these requiring intact ATM signalling. Strikingly, two of the strongest resistance-gene hits in both ATM-proficient and ATM-deficient cells encode Cyclin C and CDK8: members of the CDK8 kinase module for the RNA polymerase II mediator complex. We show that Cyclin C/CDK8 loss reduces S-phase DNA:RNA hybrid formation, transcription-replication stress, and ultimately micronuclei formation induced by ATRi. Overall, our work identifies novel biomarkers of ATRi efficacy in ATM-proficient and ATM-deficient cells, and highlights transcription-associated replication stress as a predominant driver of ATRi-induced cell death.
    DOI:  https://doi.org/10.1093/nar/gkab628
  20. NAR Cancer. 2021 Mar;3(1): zcaa043
    The HelQ human DNA repair helicase utilizes a PWI-like domain for DNA loading through interaction with RPA, triggering DNA unwinding by the HelQ helicase core.
    Tabitha Jenkins, Sarah J Northall, Denis Ptchelkine, Rebecca Lever, Andrew Cubbon, Hannah Betts, Vincenzo Taresco, Christopher D O Cooper, Peter J McHugh, Panos Soultanas, Edward L Bolt.
      Genome instability is a characteristic enabling factor for carcinogenesis. HelQ helicase is a component of human DNA maintenance systems that prevent or reverse genome instability arising during DNA replication. Here, we provide details of the molecular mechanisms that underpin HelQ function-its recruitment onto ssDNA through interaction with replication protein A (RPA), and subsequent translocation of HelQ along ssDNA. We describe for the first time a functional role for the non-catalytic N-terminal region of HelQ, by identifying and characterizing its PWI-like domain. We present evidence that this domain of HelQ mediates interaction with RPA that orchestrates loading of the helicase domains onto ssDNA. Once HelQ is loaded onto the ssDNA, ATP-Mg2+ binding in the catalytic site activates the helicase core and triggers translocation along ssDNA as a dimer. Furthermore, we identify HelQ-ssDNA interactions that are critical for the translocation mechanism. Our data are novel and detailed insights into the mechanisms of HelQ function relevant for understanding how human cells avoid genome instability provoking cancers, and also how cells can gain resistance to treatments that rely on DNA crosslinking agents.
    DOI:  https://doi.org/10.1093/narcan/zcaa043
  21. Cancer Lett. 2021 Jul 27. pii: S0304-3835(21)00373-6. [Epub ahead of print]
    PCNA inhibition enhances the cytotoxicity of β-lapachone in NQO1-Positive cancer cells by augmentation of oxidative stress-induced DNA damage.
    Xiaolin Su, Jiangwei Wang, Lingxiang Jiang, Yaomin Chen, Tao Lu, Marc S Mendonca, Xiumei Huang.
      β-Lapachone is a classic quinone-containing antitumor NQO1-bioactivatable drug that directly kills NQO1-overexpressing cancer cells. However, the clinical applications of β-lapachone are primarily limited by its high toxicity and modest lethality. To overcome this side effect and expand the therapeutic utility of β-lapachone, we demonstrate the effects of a novel combination therapy including β-lapachone and the proliferating cell nuclear antigen (PCNA) inhibitor T2 amino alcohol (T2AA) on various NQO1+ cancer cells. PCNA has DNA clamp processivity activity mediated by encircling double-stranded DNA to recruit proteins involved in DNA replication and DNA repair. In this study, we found that compared to monotherapy, a nontoxic dose of the T2AA synergized with a sublethal dose of β-lapachone in an NQO1-dependent manner and that combination therapy prevented DNA repair, increased double-strand break (DSB) formation and PARP1 hyperactivation and induced catastrophic energy loss. We further determined that T2AA promoted programmed necrosis and G1/S phase cell cycle arrest in β-lapachone-treated NQO1+ cancer cells. Our findings show novel evidence for a new therapeutic approach that combines of β-lapachone treatment with PCNA inhibition that is highly effective in treating NQO1+ solid tumor cells.
    Keywords:  Combination chemotherapy; NQO1; PCNA; T2AA; β-Lapachone
    DOI:  https://doi.org/10.1016/j.canlet.2021.07.040
  22. Curr Opin Genet Dev. 2021 Jul 23. pii: S0959-437X(21)00087-3. [Epub ahead of print]71 72-77
    Interplay between Sae2 and Rif2 in the regulation of Mre11-Rad50 activities at DNA ends.
    Diego Bonetti, Michela Clerici, Maria Pia Longhese.
      DNA double-strand breaks (DSBs) can be repaired by non-homologous end-joining (NHEJ) or homologous recombination (HR). HR is initiated by nucleolytic degradation of the DSB ends in a process termed resection. The Mre11-Rad50-Xrs2/NBS1 (MRX/N) complex is a multifunctional enzyme that, aided by the Sae2/CtIP protein, promotes DSB resection and maintains the DSB ends tethered to each other to facilitate their re-ligation. Furthermore, it activates the protein kinase Tel1/ATM, which initiates DSB signaling. In Saccharomyces cerevisiae, these MRX functions are inhibited by the Rif2 protein, which is enriched at telomeres and protects telomeric DNA from being sensed and processed as a DSB. The present review focuses on recent data showing that Sae2 and Rif2 regulate MRX functions in opposite manners by interacting with Rad50 and influencing ATP-dependent Mre11-Rad50 conformational changes. As Sae2 is enriched at DSBs whereas Rif2 is predominantly present at telomeres, the relative abundance of these two MRX regulators can provide an effective mechanism to activate or inactivate MRX depending on the nature of chromosome ends.
    DOI:  https://doi.org/10.1016/j.gde.2021.07.001
  23. NAR Cancer. 2020 Jun;2(2): zcaa004
    The role of Sp1 in the detection and elimination of cells with persistent DNA strand breaks.
    Polina S Loshchenova, Svetlana V Sergeeva, Sally C Fletcher, Grigory L Dianov.
      Maintenance of genome stability suppresses cancer and other human diseases and is critical for organism survival. Inevitably, during a life span, multiple DNA lesions can arise due to the inherent instability of DNA molecules or due to endogenous or exogenous DNA damaging factors. To avoid malignant transformation of cells with damaged DNA, multiple mechanisms have evolved to repair DNA or to detect and eradicate cells accumulating unrepaired DNA damage. In this review, we discuss recent findings on the role of Sp1 (specificity factor 1) in the detection and elimination of cells accumulating persistent DNA strand breaks. We also discuss how this mechanism may contribute to the maintenance of physiological populations of healthy cells in an organism, thus preventing cancer formation, and the possible application of these findings in cancer therapy.
    DOI:  https://doi.org/10.1093/narcan/zcaa004
  24. Front Mol Biosci. 2021 ;8 685440
    Epigenetic Mechanisms in DNA Double Strand Break Repair: A Clinical Review.
    Alejandra Fernandez, Connor O'Leary, Kenneth J O'Byrne, Joshua Burgess, Derek J Richard, Amila Suraweera.
      Upon the induction of DNA damage, the chromatin structure unwinds to allow access to enzymes to catalyse the repair. The regulation of the winding and unwinding of chromatin occurs via epigenetic modifications, which can alter gene expression without changing the DNA sequence. Epigenetic mechanisms such as histone acetylation and DNA methylation are known to be reversible and have been indicated to play different roles in the repair of DNA. More importantly, the inhibition of such mechanisms has been reported to play a role in the repair of double strand breaks, the most detrimental type of DNA damage. This occurs by manipulating the chromatin structure and the expression of essential proteins that are critical for homologous recombination and non-homologous end joining repair pathways. Inhibitors of histone deacetylases and DNA methyltransferases have demonstrated efficacy in the clinic and represent a promising approach for cancer therapy. The aims of this review are to summarise the role of histone deacetylase and DNA methyltransferase inhibitors involved in DNA double strand break repair and explore their current and future independent use in combination with other DNA repair inhibitors or pre-existing therapies in the clinic.
    Keywords:  DNA double strand breaks; DNA methyltransferase inhibitors; DNA repair; epigenetic mechanisms; histone deacetylase inhibitors
    DOI:  https://doi.org/10.3389/fmolb.2021.685440
  25. NAR Cancer. 2021 Mar;3(1): zcab002
    The DNA damage inducible lncRNA SCAT7 regulates genomic integrity and topoisomerase 1 turnover in lung adenocarcinoma.
    Luisa Statello, Mohamad M Ali, Silke Reischl, Sagar Mahale, Subazini Thankaswamy Kosalai, Maite Huarte, Chandrasekhar Kanduri.
      Despite the rapid improvements in unveiling the importance of lncRNAs in all aspects of cancer biology, there is still a void in mechanistic understanding of their role in the DNA damage response. Here we explored the potential role of the oncogenic lncRNA SCAT7 (ELF3-AS1) in the maintenance of genome integrity. We show that SCAT7 is upregulated in response to DNA-damaging drugs like cisplatin and camptothecin, where SCAT7 expression is required to promote cell survival. SCAT7 silencing leads to decreased proliferation of cisplatin-resistant cells in vitro and in vivo through interfering with cell cycle checkpoints and DNA repair molecular pathways. SCAT7 regulates ATR signaling, promoting homologous recombination. Importantly, SCAT7 also takes part in proteasome-mediated topoisomerase I (TOP1) degradation, and its depletion causes an accumulation of TOP1-cc structures responsible for the high levels of intrinsic DNA damage. Thus, our data demonstrate that SCAT7 is an important constituent of the DNA damage response pathway and serves as a potential therapeutic target for hard-to-treat drug resistant cancers.
    DOI:  https://doi.org/10.1093/narcan/zcab002
  26. Sci Adv. 2021 Jul;pii: eabf7906. [Epub ahead of print]7(31):
    FAN1-MLH1 interaction affects repair of DNA interstrand cross-links and slipped-CAG/CTG repeats.
    Antonio Porro, Mohiuddin Mohiuddin, Christina Zurfluh, Vincent Spegg, Jingqi Dai, Florence Iehl, Virginie Ropars, Giulio Collotta, Keri M Fishwick, Nour L Mozaffari, Raphaël Guérois, Josef Jiricny, Matthias Altmeyer, Jean-Baptiste Charbonnier, Christopher E Pearson, Alessandro A Sartori.
      FAN1, a DNA structure-specific nuclease, interacts with MLH1, but the repair pathways in which this complex acts are unknown. FAN1 processes DNA interstrand crosslinks (ICLs) and FAN1 variants are modifiers of the neurodegenerative Huntington's disease (HD), presumably by regulating HD-causing CAG repeat expansions. Here, we identify specific amino acid residues in two adjacent FAN1 motifs that are critical for MLH1 binding. Disruption of the FAN1-MLH1 interaction confers cellular hypersensitivity to ICL damage and defective repair of CAG/CTG slip-outs, intermediates of repeat expansion mutations. FAN1-S126 phosphorylation, which hinders FAN1-MLH1 association, is cell cycle-regulated by cyclin-dependent kinase activity and attenuated upon ICL induction. Our data highlight the FAN1-MLH1 complex as a phosphorylation-regulated determinant of ICL response and repeat stability, opening novel paths to modify cancer and neurodegeneration.
    DOI:  https://doi.org/10.1126/sciadv.abf7906
  27. Biochimie. 2021 Jul 21. pii: S0300-9084(21)00180-2. [Epub ahead of print]190 70-90
    Apurinic/Apyrimidinic Endonuclease 2 (APE2): An ancillary enzyme for contextual base excision repair mechanisms to preserve genome stability.
    Sima Chaudhari, Akshay P Ware, Pradyumna Jayaram, Sankar Prasad Gorthi, Sherif F El-Khamisy, Kapaettu Satyamoorthy.
      The genome of living organisms frequently undergoes various types of modifications which are recognized and repaired by the relevant repair mechanisms. These repair pathways are increasingly being deciphered to understand the mechanisms. Base excision repair (BER) is indispensable to maintain genome stability. One of the enigmatic repair proteins of BER, Apurinic/Apyrimidinic Endonuclease 2 (APE2), like APE1, is truly multifunctional and demonstrates the independent and non-redundant function in maintaining the genome integrity. APE2 is involved in ATR-Chk1 mediated DNA damage response. It also resolves topoisomerase1 mediated cleavage complex intermediate which is formed while repairing misincorporated ribonucleotides in the absence of functional RNase H2 mediated excision repair pathway. BER participates in the demethylation pathway and the role of Arabidopsis thaliana APE2 is demonstrated in this process. Moreover, APE2 is synthetically lethal to BRCA1, BRCA2, and RNase H2, and its homolog, APE1 fails to complement the function. Hence, the role of APE2 is not just an alternate to the repair mechanisms but has implications in diverse functional pathways related to the maintenance of genome integrity. This review analyses genomic features of APE2 and delineates its enzyme function as error-prone as well as efficient and accurate repair protein based on the studies on mammalian or its homolog proteins from model systems such as Arabidopsis thaliana, Schizosaccharomyces pombe, Trypanosoma curzi, Xenopus laevis, Danio rerio, Mus musculus, and Homo sapiens.
    Keywords:  Apurinic/Apyrimidinic endonuclease 2; Base excision repair; DNA damage response; DNA repair; Demethylation; Synthetic lethality
    DOI:  https://doi.org/10.1016/j.biochi.2021.07.006
  28. Mol Cancer Res. 2021 Jul 30. pii: molcanres.0301.2021. [Epub ahead of print]
    The PI3K/mTOR Inhibitor Ompalisib Suppresses Nonhomologous End Joining and Sensitizes Cancer Cells to Radio- and Chemotherapy.
    Jie Du, Fuqiang Chen, Jiahua Yu, Lijun Jiang, Meijuan Zhou.
      As the predominant pathway for the repair of DNA double-strand breaks (DSBs), non-homologous end joining (NHEJ) attenuates the efficacy of cancer treatment which relies on the introduction of DSBs, such as radiotherapy and genotoxic drugs. Identifying novel NHEJ inhibitors is of great importance for improving the therapeutic efficiency of radio- or chemotherapy. Here we miniaturized our recently developed NHEJ detecting system into a 96-well plate-based format and interrogated an FDA approved drug library containing 1732 compounds. A collection of novel hits were considered to be potential DSB repair inhibitors at the non-cytotoxic concentration. We identified omipalisib as an efficient sensitizer for DNA damage-induced cell death in vitro. Furthermore, in vitro analysis uncovered the repressive effect of omipalisib on the phosphorylation of DNA-dependent protein kinase catalytic subunit induced by ionizing radiation and doxorubicin, which led to the suppression of NHEJ pathway. Implications: In summary, our findings suggested the possibility for repurposing these candidates as radio- or chemosensitizers, which might extend their clinical application in cancer therapy.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-21-0301
  29. Oncol Rev. 2021 Feb 26. 15(1): 519
    The repair gene BACH1 - a potential oncogene.
    Katheeja Muhseena N, Sooraj Mathukkada, Shankar Prasad Das, Suparna Laha.
      BACH1 encodes for a protein that belongs to RecQ DEAH helicase family and interacts with the BRCT repeats of BRCA1. The N-terminus of BACH1 functions in DNA metabolism as DNA-dependent ATPase and helicase. The C-terminus consists of BRCT domain, which interacts with BRCA1 and this interaction is one of the major regulator of BACH1 function. BACH1 plays important roles both in phosphorylated as well as dephosphorylated state and functions in coordination with multiple signaling molecules. The active helicase property of BACH1 is maintained by its dephosphorylated state. Imbalance between these two states enhances the development and progression of the diseased condition. Currently BACH1 is known as a tumor suppressor gene based on the presence of its clinically relevant mutations in different cancers. Through this review we have justified it to be named as an oncogene. In this review, we have explained the mechanism of how BACH1 in collaboration with BRCA1 or independently regulates various pathways like cell cycle progression, DNA replication during both normal and stressed situation, recombination and repair of damaged DNA, chromatin remodeling and epigenetic modifications. Mutation and overexpression of BACH1 are significantly found in different cancer types. This review enlists the molecular players which interact with BACH1 to regulate DNA metabolic functions, thereby revealing its potential for cancer therapeutics. We have identified the most mutated functional domain of BACH1, the hot spot for tumorigenesis, justifying it as a target molecule in different cancer types for therapeutics. BACH1 has high potentials of transforming a normal cell into a tumor cell if compromised under certain circumstances. Thus, through this review, we justify BACH1 as an oncogene along with the existing role of being a tumor suppressant.
    Keywords:  BACH1/BRIP1; Chl1p; Chromatin remodeling; genomic stability; tumorigenesis
    DOI:  https://doi.org/10.4081/oncol.2021.519
  30. ACS Chem Biol. 2021 Jul 30.
    A Minimalistic Coumarin Turn-On Probe for Selective Recognition of Parallel G-Quadruplex DNA Structures.
    Marco Deiana, Ikenna Obi, Måns Andreasson, Shanmugam Tamilselvi, Karam Chand, Erik Chorell, Nasim Sabouri.
      G-quadruplex (G4) DNA structures are widespread in the human genome and are implicated in biologically important processes such as telomere maintenance, gene regulation, and DNA replication. Guanine-rich sequences with potential to form G4 structures are prevalent in the promoter regions of oncogenes, and G4 sites are now considered as attractive targets for anticancer therapies. However, there are very few reports of small "druglike" optical G4 reporters that are easily accessible through one-step synthesis and that are capable of discriminating between different G4 topologies. Here, we present a small water-soluble light-up fluorescent probe that features a minimalistic amidinocoumarin-based molecular scaffold that selectively targets parallel G4 structures over antiparallel and non-G4 structures. We showed that this biocompatible ligand is able to selectively stabilize the G4 template resulting in slower DNA synthesis. By tracking individual DNA molecules, we demonstrated that the G4-stabilizing ligand perturbs DNA replication in cancer cells, resulting in decreased cell viability. Moreover, the fast-cellular entry of the probe enabled detection of nucleolar G4 structures in living cells. Finally, insights gained from the structure-activity relationships of the probe suggest the basis for the recognition of parallel G4s, opening up new avenues for the design of new biocompatible G4-specific small molecules for G4-driven theranostic applications.
    DOI:  https://doi.org/10.1021/acschembio.1c00134
  31. Proc Natl Acad Sci U S A. 2021 Aug 03. pii: e2100178118. [Epub ahead of print]118(31):
    Replication-dependent histone biosynthesis is coupled to cell-cycle commitment.
    Claire Armstrong, Sabrina L Spencer.
      The current model of replication-dependent (RD) histone biosynthesis posits that RD histone gene expression is coupled to DNA replication, occurring only in S phase of the cell cycle once DNA synthesis has begun. However, several key factors in the RD histone biosynthesis pathway are up-regulated by E2F or phosphorylated by CDK2, suggesting these processes may instead begin much earlier, at the point of cell-cycle commitment. In this study, we use both fixed- and live-cell imaging of human cells to address this question, revealing a hybrid model in which RD histone biosynthesis is first initiated in G1, followed by a strong increase in histone production in S phase of the cell cycle. This suggests a mechanism by which cells that have committed to the cell cycle build up an initial small pool of RD histones to be available for the start of DNA replication, before producing most of the necessary histones required in S phase. Thus, a clear distinction exists at completion of mitosis between cells that are born with the intention of proceeding through the cell cycle and replicating their DNA and cells that have chosen to exit the cell cycle and have no immediate need for histone synthesis.
    Keywords:  NPAT; SLBP; histone locus body; replication-dependent histone; restriction point
    DOI:  https://doi.org/10.1073/pnas.2100178118
  32. DNA Repair (Amst). 2021 Jul 17. pii: S1568-7864(21)00138-5. [Epub ahead of print]106 103182
    Defining R-loop classes and their contributions to genome instability.
    Daisy Castillo-Guzman, Frédéric Chédin.
      R-loops are non-B DNA structures that form during transcription when the nascent RNA anneals to the template DNA strand forming a RNA:DNA hybrid. Understanding the genomic distribution and function of R-loops is an important goal, since R-loops have been implicated in a number of adaptive and maladaptive processes under physiological and pathological conditions. Based on R-loop mapping datasets, we propose the existence of two main classes of R-loops, each associated with unique characteristics. Promoter-paused R-loops (Class I) are short R-loops that form at high frequency during promoter-proximal pausing by RNA polymerase II. Elongation-associated R-loops (Class II) are long structures that occur throughout gene bodies at modest frequencies. We further discuss the relationships between each R-loop class with instances of genome instability and suggest that increased class I R-loops, resulting from enhanced promoter-proximal pausing, represent the main culprits for R-loop mediated genome instability under pathological conditions.
    Keywords:  DNA damage; Elongation; Genome instability; Promoter-pausing; R-loops; Transcription
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103182
  33. Eur J Hum Genet. 2021 Jul 26.
    IMPDH2: a new gene associated with dominant juvenile-onset dystonia-tremor disorder.
    Anna Kuukasjärvi, Juan C Landoni, Jyrki Kaukonen, Mika Juhakoski, Mari Auranen, Tommi Torkkeli, Vidya Velagapudi, Anu Suomalainen.
      The aetiology of dystonia disorders is complex, and next-generation sequencing has become a useful tool in elucidating the variable genetic background of these diseases. Here we report a deleterious heterozygous truncating variant in the inosine monophosphate dehydrogenase gene (IMPDH2) by whole-exome sequencing, co-segregating with a dominantly inherited dystonia-tremor disease in a large Finnish family. We show that the defect results in degradation of the gene product, causing IMPDH2 deficiency in patient cells. IMPDH2 is the first and rate-limiting enzyme in the de novo biosynthesis of guanine nucleotides, a dopamine synthetic pathway previously linked to childhood or adolescence-onset dystonia disorders. We report IMPDH2 as a new gene to the dystonia disease entity. The evidence underlines the important link between guanine metabolism, dopamine biosynthesis and dystonia.
    DOI:  https://doi.org/10.1038/s41431-021-00939-1
  34. Sci Adv. 2021 Jul;pii: eabi6508. [Epub ahead of print]7(31):
    The major mechanism of melanoma mutations is based on deamination of cytosine in pyrimidine dimers as determined by circle damage sequencing.
    Seung-Gi Jin, Dean Pettinga, Jennifer Johnson, Peipei Li, Gerd P Pfeifer.
      Sunlight-associated melanomas carry a unique C-to-T mutation signature. UVB radiation induces cyclobutane pyrimidine dimers (CPDs) as the major form of DNA damage, but the mechanism of how CPDs cause mutations is unclear. To map CPDs at single-base resolution genome wide, we developed the circle damage sequencing (circle-damage-seq) method. In human cells, CPDs form preferentially in a tetranucleotide sequence context (5'-Py-T<>Py-T/A), but this alone does not explain the tumor mutation patterns. To test whether mutations arise at CPDs by cytosine deamination, we specifically mapped UVB-induced cytosine-deaminated CPDs. Transcription start sites (TSSs) were protected from CPDs and deaminated CPDs, but both lesions were enriched immediately upstream of the TSS, suggesting a mutation-promoting role of bound transcription factors. Most importantly, the genomic dinucleotide and trinucleotide sequence specificity of deaminated CPDs matched the prominent mutation signature of melanomas. Our data identify the cytosine-deaminated CPD as the leading premutagenic lesion responsible for mutations in melanomas.
    DOI:  https://doi.org/10.1126/sciadv.abi6508
  35. Nat Commun. 2021 07 28. 12(1): 4582
    SUMOylation of SAMHD1 at Lysine 595 is required for HIV-1 restriction in non-cycling cells.
    Charlotte Martinat, Arthur Cormier, Joëlle Tobaly-Tapiero, Noé Palmic, Nicoletta Casartelli, Bijan Mahboubi, Si'Ana A Coggins, Julian Buchrieser, Mirjana Persaud, Felipe Diaz-Griffero, Lucile Espert, Guillaume Bossis, Pascale Lesage, Olivier Schwartz, Baek Kim, Florence Margottin-Goguet, Ali Saïb, Alessia Zamborlini.
      SAMHD1 is a cellular triphosphohydrolase (dNTPase) proposed to inhibit HIV-1 reverse transcription in non-cycling immune cells by limiting the supply of the dNTP substrates. Yet, phosphorylation of T592 downregulates SAMHD1 antiviral activity, but not its dNTPase function, implying that additional mechanisms contribute to viral restriction. Here, we show that SAMHD1 is SUMOylated on residue K595, a modification that relies on the presence of a proximal SUMO-interacting motif (SIM). Loss of K595 SUMOylation suppresses the restriction activity of SAMHD1, even in the context of the constitutively active phospho-ablative T592A mutant but has no impact on dNTP depletion. Conversely, the artificial fusion of SUMO2 to a non-SUMOylatable inactive SAMHD1 variant restores its antiviral function, a phenotype that is reversed by the phosphomimetic T592E mutation. Collectively, our observations clearly establish that lack of T592 phosphorylation cannot fully account for the restriction activity of SAMHD1. We find that SUMOylation of K595 is required to stimulate a dNTPase-independent antiviral activity in non-cycling immune cells, an effect that is antagonized by cyclin/CDK-dependent phosphorylation of T592 in cycling cells.
    DOI:  https://doi.org/10.1038/s41467-021-24802-5
  36. Nucleic Acids Res. 2021 Jul 30. pii: gkab642. [Epub ahead of print]
    TDRD3 promotes DHX9 chromatin recruitment and R-loop resolution.
    Wei Yuan, Qais Al-Hadid, Zhihao Wang, Lei Shen, Hyejin Cho, Xiwei Wu, Yanzhong Yang.
      R-loops, which consist of a DNA/RNA hybrid and a displaced single-stranded DNA (ssDNA), are increasingly recognized as critical regulators of chromatin biology. R-loops are particularly enriched at gene promoters, where they play important roles in regulating gene expression. However, the molecular mechanisms that control promoter-associated R-loops remain unclear. The epigenetic 'reader' Tudor domain-containing protein 3 (TDRD3), which recognizes methylarginine marks on histones and on the C-terminal domain of RNA polymerase II, was previously shown to recruit DNA topoisomerase 3B (TOP3B) to relax negatively supercoiled DNA and prevent R-loop formation. Here, we further characterize the function of TDRD3 in R-loop metabolism and introduce the DExH-box helicase 9 (DHX9) as a novel interaction partner of the TDRD3/TOP3B complex. TDRD3 directly interacts with DHX9 via its Tudor domain. This interaction is important for recruiting DHX9 to target gene promoters, where it resolves R-loops in a helicase activity-dependent manner to facilitate gene expression. Additionally, TDRD3 also stimulates the helicase activity of DHX9. This stimulation relies on the OB-fold of TDRD3, which likely binds the ssDNA in the R-loop structure. Thus, DHX9 functions together with TOP3B to suppress promoter-associated R-loops. Collectively, these findings reveal new functions of TDRD3 and provide important mechanistic insights into the regulation of R-loop metabolism.
    DOI:  https://doi.org/10.1093/nar/gkab642
  37. Nat Commun. 2021 07 28. 12(1): 4581
    Mechanistic insights into the three steps of poly(ADP-ribosylation) reversal.
    Johannes Gregor Matthias Rack, Qiang Liu, Valentina Zorzini, Jim Voorneveld, Antonio Ariza, Kourosh Honarmand Ebrahimi, Julia M Reber, Sarah C Krassnig, Dragana Ahel, Gijsbert A van der Marel, Aswin Mangerich, James S O McCullagh, Dmitri V Filippov, Ivan Ahel.
      Poly(ADP-ribosyl)ation (PAR) is a versatile and complex posttranslational modification composed of repeating units of ADP-ribose arranged into linear or branched polymers. This scaffold is linked to the regulation of many of cellular processes including the DNA damage response, alteration of chromatin structure and Wnt signalling. Despite decades of research, the principles and mechanisms underlying all steps of PAR removal remain actively studied. In this work, we synthesise well-defined PAR branch point molecules and demonstrate that PARG, but not ARH3, can resolve this distinct PAR architecture. Structural analysis of ARH3 in complex with dimeric ADP-ribose as well as an ADP-ribosylated peptide reveal the molecular basis for the hydrolysis of linear and terminal ADP-ribose linkages. We find that ARH3-dependent hydrolysis requires both rearrangement of a catalytic glutamate and induction of an unusual, square-pyramidal magnesium coordination geometry.
    DOI:  https://doi.org/10.1038/s41467-021-24723-3
  38. NAR Cancer. 2021 Jun;3(2): zcab021
    Tumor-selective, antigen-independent delivery of a pH sensitive peptide-topoisomerase inhibitor conjugate suppresses tumor growth without systemic toxicity.
    Sophia Gayle, Robert Aiello, Nalin Leelatian, Jason M Beckta, Jane Bechtold, Patricia Bourassa, Johanna Csengery, Robert J Maguire, Dan Marshall, Ranjini K Sundaram, Jinny Van Doorn, Kelli Jones, Hunter Moore, Lori Lopresti-Morrow, Timothy Paradis, Laurie Tylaska, Qing Zhang, Hannah Visca, Yana K Reshetnyak, Oleg A Andreev, Donald M Engelman, Peter M Glazer, Ranjit S Bindra, Vishwas M Paralkar.
      Topoisomerase inhibitors are potent DNA damaging agents which are widely used in oncology, and they demonstrate robust synergistic tumor cell killing in combination with DNA repair inhibitors, including poly(ADP)-ribose polymerase (PARP) inhibitors. However, their use has been severely limited by the inability to achieve a favorable therapeutic index due to severe systemic toxicities. Antibody-drug conjugates address this issue via antigen-dependent targeting and delivery of their payloads, but this approach requires specific antigens and yet still suffers from off-target toxicities. There is a high unmet need for a more universal tumor targeting technology to broaden the application of cytotoxic payloads. Acidification of the extracellular milieu arises from metabolic adaptions associated with the Warburg effect in cancer. Here we report the development of a pH-sensitive peptide-drug conjugate to deliver the topoisomerase inhibitor, exatecan, selectively to tumors in an antigen-independent manner. Using this approach, we demonstrate potent in vivo cytotoxicity, complete suppression of tumor growth across multiple human tumor models, and synergistic interactions with a PARP inhibitor. These data highlight the identification of a peptide-topoisomerase inhibitor conjugate for cancer therapy that provides a high therapeutic index, and is applicable to all types of human solid tumors in an antigen-independent manner.
    DOI:  https://doi.org/10.1093/narcan/zcab021
  39. Nat Commun. 2021 Jul 30. 12(1): 4626
    PRMT1-dependent regulation of RNA metabolism and DNA damage response sustains pancreatic ductal adenocarcinoma.
    Virginia Giuliani, Meredith A Miller, Chiu-Yi Liu, Stella R Hartono, Caleb A Class, Christopher A Bristow, Erika Suzuki, Lionel A Sanz, Guang Gao, Jason P Gay, Ningping Feng, Johnathon L Rose, Hideo Tomihara, Joseph R Daniele, Michael D Peoples, Jennifer P Bardenhagen, Mary K Geck Do, Qing E Chang, Bhavatarini Vangamudi, Christopher Vellano, Haoqiang Ying, Angela K Deem, Kim-Anh Do, Giannicola Genovese, Joseph R Marszalek, Jeffrey J Kovacs, Michael Kim, Jason B Fleming, Ernesto Guccione, Andrea Viale, Anirban Maitra, M Emilia Di Francesco, Timothy A Yap, Philip Jones, Giulio Draetta, Alessandro Carugo, Frederic Chedin, Timothy P Heffernan.
      Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer that has remained clinically challenging to manage. Here we employ an RNAi-based in vivo functional genomics platform to determine epigenetic vulnerabilities across a panel of patient-derived PDAC models. Through this, we identify protein arginine methyltransferase 1 (PRMT1) as a critical dependency required for PDAC maintenance. Genetic and pharmacological studies validate the role of PRMT1 in maintaining PDAC growth. Mechanistically, using proteomic and transcriptomic analyses, we demonstrate that global inhibition of asymmetric arginine methylation impairs RNA metabolism, which includes RNA splicing, alternative polyadenylation, and transcription termination. This triggers a robust downregulation of multiple pathways involved in the DNA damage response, thereby promoting genomic instability and inhibiting tumor growth. Taken together, our data support PRMT1 as a compelling target in PDAC and informs a mechanism-based translational strategy for future therapeutic development.Statement of significancePDAC is a highly lethal cancer with limited therapeutic options. This study identified and characterized PRMT1-dependent regulation of RNA metabolism and coordination of key cellular processes required for PDAC tumor growth, defining a mechanism-based translational hypothesis for PRMT1 inhibitors.
    DOI:  https://doi.org/10.1038/s41467-021-24798-y
  40. Nat Commun. 2021 07 27. 12(1): 4542
    An enzymatic activation of formaldehyde for nucleotide methylation.
    Charles Bou-Nader, Frederick W Stull, Ludovic Pecqueur, Philippe Simon, Vincent Guérineau, Antoine Royant, Marc Fontecave, Murielle Lombard, Bruce A Palfey, Djemel Hamdane.
      Folate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH2O-shunt may be relevant in several other important folate-dependent reactions.
    DOI:  https://doi.org/10.1038/s41467-021-24756-8
  41. J Biol Chem. 2021 Jul 24. pii: S0021-9258(21)00810-3. [Epub ahead of print] 101008
    Structural determinants and distribution of phosphate specificity in ribonucleotide reductases.
    Eugen Schell, Ghada Nouairia, Elisabeth Steiner, Niclas Weber, Daniel Lundin, Christoph Loderer.
      Ribonucleotide reductases (RNRs) catalyse the reduction of ribonucleotides to the corresponding deoxyribonucleotides, the building blocks of DNA. RNRs are specific for either ribonucleoside di- or triphosphates as substrates. As far as is known, oxygen-dependent class I RNRs (NrdAB) all reduce ribonucleoside diphosphates and oxygen-sensitive class III RNRs (NrdD) are all ribonucleoside triphosphate reducers, whereas the adenosylcobalamin-dependent class II (NrdJ) contains both ribonucleoside di- and triphosphate reducers. However, it is unknown how this specificity is conveyed by the active site of the enzymes and how this feature developed in RNR evolution. By structural comparison of the active sites in different RNRs, we identified the apical loop of the phosphate-binding site as a potential structural determinant of substrate specificity. Grafting two residues from this loop from a diphosphate- to a triphosphate-specific RNR caused a change in preference from ribonucleoside tri- to diphosphate substrates in a class II model enzyme, confirming them as the structural determinants of phosphate specificity. The investigation of the phylogenetic distribution of this motif in class II RNRs yielded a likely monophyletic clade with the diphosphate-defining motif. This indicates a single evolutionary split event early in NrdJ evolution in which diphosphate specificity developed from the earlier triphosphate specificity. For those interesting cases where organisms contain more than one nrdJ gene, we observed a preference for encoding enzymes with diverse phosphate specificities, suggesting that this varying phosphate specificity confers a selective advantage.
    Keywords:  enzyme catalysis; enzyme kinetics; nucleic acid enzymology; nucleoside/nucleotide biosynthesis; nucleoside/nucleotide metabolism; phosphate specificity; ribonucleotide reductases; site directed mutagenesis
    DOI:  https://doi.org/10.1016/j.jbc.2021.101008
  42. Cancer Res. 2021 Jul 28. pii: canres.CAN-21-0463-A.2021. [Epub ahead of print]
    Long noncoding RNA NIHCOLE promotes ligation efficiency of DNA double-strand breaks in hepatocellular carcinoma.
    Juan P Unfried, Mikel Marín-Baquero, Ángel Rivera-Calzada, Nerea Razquin, Eva M Martín-Cuevas, Sara de Bragança, Clara Aicart-Ramos, Christopher McCoy, Laura Prats-Mari, Raquel Arribas-Bosacoma, Linda Lee, Stefano Caruso, Jessica Zucman-Rossi, Bruno Sangro, Gareth Williams, Fernando Moreno-Herrero, Oscar Llorca, Susan P Lees-Miller, Puri Fortes.
      Long noncoding RNAs (lncRNAs) are emerging as key players in cancer as parts of poorly understood molecular mechanisms. Here, we investigated lncRNAs that play a role in hepatocellular carcinoma (HCC) and identified NIHCOLE, a novel lncRNA induced in HCC with oncogenic potential and a role in the ligation efficiency of DNA double-stranded breaks (DSB). NIHCOLE expression was associated with poor prognosis and survival of HCC patients. Depletion of NIHCOLE from HCC cells led to impaired proliferation and increased apoptosis. NIHCOLE deficiency led to accumulation of DNA damage due to a specific decrease in the activity of the non-homologous end-joining (NHEJ) pathway of DSB repair. DNA damage induction in NIHCOLE-depleted cells further decreased HCC cell growth. NIHCOLE was associated with DSB markers and recruited several molecules of the Ku70/Ku80 heterodimer. Further, NIHCOLE putative structural domains supported stable multimeric complexes formed by several NHEJ factors including Ku70/80, APLF, XRCC4, and DNA Ligase IV. NHEJ reconstitution assays showed that NIHCOLE promoted the ligation efficiency of blunt-ended DSBs. Collectively, these data show that NIHCOLE serves as a scaffold and facilitator of NHEJ machinery and confers an advantage to HCC cells, which could be exploited as a targetable vulnerability.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-21-0463
  43. FEBS J. 2021 Jul 29.
    ADP-ribosyltransferases, an update on function and nomenclature.
    Bernhard Lüscher, Ivan Ahel, Matthias Altmeyer, Alan Ashworth, Peter Bai, Paul Chang, Michael Cohen, Daniela Corda, Françoise Dantzer, Matthew D Daugherty, Ted M Dawson, Valina L Dawson, Sebastian Deindl, Anthony R Fehr, Karla L H Feijs, Dmitri V Filippov, Jean-Philippe Gagné, Giovanna Grimaldi, Sebastian Guettler, Nicolas C Hoch, Michael O Hottiger, Patricia Korn, W Lee Kraus, Andreas Ladurner, Lari Lehtiö, Anthony K L Leung, Christopher J Lord, Aswin Mangerich, Ivan Matic, Jason Matthews, George-Lucian Moldovan, Joel Moss, Gioacchino Natoli, Michael L Nielsen, Mario Niepel, Friedrich Nolte, John Pascal, Bryce M Paschal, Krzysztof Pawłowski, Guy G Poirier, Susan Smith, Gyula Timinszky, Zhao-Qi Wang, José Yélamos, Xiaochun Yu, Roko Zaja, Mathias Ziegler.
      ADP-ribosylation, a modification of proteins, nucleic acids and metabolites, confers broad functions, including roles in stress responses elicited for example by DNA damage and viral infection and is involved in intra- and extracellular signaling, chromatin and transcriptional regulation, protein biosynthesis and cell death. ADP-ribosylation is catalyzed by ADP-ribosyltransferases, which transfer ADP-ribose from NAD+ onto substrates. The modification, which occurs as mono- or poly-ADP-ribosylation, is reversible due to the action of different ADP-ribosylhydrolases. Importantly, inhibitors of ADP-ribosyltransferases are approved or are being developed for clinical use. Moreover, ADP-ribosylhydrolases are being assessed as therapeutic targets, foremost as anti-viral drugs and for oncological indications. Due to the development of novel reagents and major technological advances that allow the study of ADP-ribosylation in unprecedented detail, an increasing number of cellular processes and pathways are being identified that are regulated by ADP-ribosylation. In addition, characterization of biochemical and structural aspects of the ADP-ribosyltransferases and their catalytic activities have expanded our understanding of this protein family. This increased knowledge requires that a common nomenclature be used to describe the relevant enzymes. Therefore, in this viewpoint, we propose an updated and broadly supported nomenclature for mammalian ADP-ribosyltransferases that will facilitate future discussions when addressing the biochemistry and biology of ADP-ribosylation. This is combined with a brief description of the main functions of mammalian ADP-ribosyltransferases to illustrate the increasing diversity of mono- and poly-ADP-ribose mediated cellular processes.
    Keywords:  ADP-ribosylation; MARylation; PARP; PARylation; posttranslational modification
    DOI:  https://doi.org/10.1111/febs.16142
  44. Nat Commun. 2021 07 29. 12(1): 4605
    BRCA2 binding through a cryptic repeated motif to HSF2BP oligomers does not impact meiotic recombination.
    Rania Ghouil, Simona Miron, Lieke Koornneef, Jasper Veerman, Maarten W Paul, Marie-Hélène Le Du, Esther Sleddens-Linkels, Sari E van Rossum-Fikkert, Yvette van Loon, Natalia Felipe-Medina, Alberto M Pendas, Alex Maas, Jeroen Essers, Pierre Legrand, Willy M Baarends, Roland Kanaar, Sophie Zinn-Justin, Alex N Zelensky.
      BRCA2 and its interactors are required for meiotic homologous recombination (HR) and fertility. Loss of HSF2BP, a BRCA2 interactor, disrupts HR during spermatogenesis. We test the model postulating that HSF2BP localizes BRCA2 to meiotic HR sites, by solving the crystal structure of the BRCA2 fragment in complex with dimeric armadillo domain (ARM) of HSF2BP and disrupting this interaction in a mouse model. This reveals a repeated 23 amino acid motif in BRCA2, each binding the same conserved surface of one ARM domain. In the complex, two BRCA2 fragments hold together two ARM dimers, through a large interface responsible for the nanomolar affinity - the strongest interaction involving BRCA2 measured so far. Deleting exon 12, encoding the first repeat, from mBrca2 disrupts BRCA2 binding to HSF2BP, but does not phenocopy HSF2BP loss. Thus, results herein suggest that the high-affinity oligomerization-inducing BRCA2-HSF2BP interaction is not required for RAD51 and DMC1 recombinase localization in meiotic HR.
    DOI:  https://doi.org/10.1038/s41467-021-24871-6
  45. Mol Metab. 2021 Jul 22. pii: S2212-8778(21)00156-3. [Epub ahead of print] 101309
    Hepatic mTORC1 signaling activates ATF4 as part of its metabolic response to feeding and insulin.
    Vanessa Byles, Yann Cormerais, Krystle Kalafut, Victor Barrera, James E Hughes Hallett, Shannan Ho Sui, John M Asara, Christopher M Adams, Gerta Hoxhaj, Issam Ben-Sahra, Brendan D Manning.
       OBJECTIVE: The mechanistic target of rapamycin complex 1 (mTORC1) is dynamically regulated by fasting and feeding cycles in the liver to promote protein and lipid synthesis while suppressing autophagy. However, beyond these functions, the metabolic response of the liver to feeding and insulin signaling orchestrated by mTORC1 remains poorly defined. Here, we determine whether ATF4, a stress responsive transcription factor recently found to be independently regulated by mTORC1 signaling in proliferating cells, is responsive to hepatic mTORC1 signaling to alter hepatocyte metabolism.
    METHODS: ATF4 protein levels and expression of canonical gene targets were analyzed in the liver following fasting and physiological feeding in the presence or absence of the mTORC1 inhibitor rapamycin. Primary hepatocytes from wild-type or liver-specific Atf4 knockout (LAtf4KO) mice were used to characterize the effects of insulin-stimulated mTORC1-ATF4 function on hepatocyte gene expression and metabolism. Both unbiased steady-state metabolomics and stable-isotope tracing methods were employed to define mTORC1 and ATF4-dependent metabolic changes. RNA-sequencing was used to determine global changes in feeding-induced transcripts in the livers of wild-type versus LAtf4KO mice.
    RESULTS: We demonstrate that ATF4 and its metabolic gene targets are stimulated by mTORC1 signaling in the liver in response to feeding and in a hepatocyte-intrinsic manner by insulin. While we demonstrate that de novo purine and pyrimidine synthesis is stimulated by insulin through mTORC1 signaling in primary hepatocytes, this regulation was independent of ATF4. Metabolomics and metabolite tracing studies revealed that insulin-mTORC1-ATF4 signaling stimulates pathways of non-essential amino acid synthesis in primary hepatocytes, including those of alanine, aspartate, methionine, and cysteine, but not serine.
    CONCLUSION: The results demonstrate that ATF4 is a novel metabolic effector of mTORC1 in liver, extending the molecular consequences of feeding and insulin-induced mTORC1 signaling in this key metabolic tissue to the control of amino acid metabolism.
    Keywords:  ATF4; feeding; insulin; liver; mTORC1; methionine metabolism
    DOI:  https://doi.org/10.1016/j.molmet.2021.101309
  46. Biochem Biophys Res Commun. 2021 Jul 27. pii: S0006-291X(21)01118-9. [Epub ahead of print]571 181-187
    Sirt3 increases CNPase enzymatic activity through deacetylation and facilitating substrate accessibility.
    Dongfang Wang, Keai Sinn Tan, Xabier Arias-Moreno, Wen Tan, Guohua Cheng.
      Myocardial 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) metabolizes a nucleoside 2',3'-cyclic phosphate to a nucleoside 2'-phosphate. Recently, the roles of CNPase in the pathophysiological processes of heart failure have emerged. The mitochondrial acylome subjected to SIRT3 regulation give us comprehensive understanding of acylation modifications to a vast array of protein targets, and the list of acetylated mitochondrial proteins is still growing. However, it remains elusive whether CNPase is subjected to the regulation of acetylation and deacetylation, and the effects of which on CNPase enzymatic activity are still unknown. In this study, the mitochondrial distribution of CNPase was identified by immunofluorescence and cytosol/mitochondria fractioning. The immunofluorescence staining pattern of CNPase and Sirt3 overlapped on the same focal plane. Moreover, Sirt3 associates directly with CNPase, and the CNPase enzymatic activity was subjected to Sirt3 activity. Then biochemical methods using acetic anhydride was employed to acetylate the CNPase proteins, the enzymatic activity of CNPase decreased. Furthermore, co-immunoprecipitation coupled mass spectrometry identifies K196, K379, K128 as the main acetylation sites. Molecular dynamic simulation shows that acetylation modification suppressed the CNPase enzymatic activity through decreasing the opening probability of the binding pocket and restricting substrate accessibility. Together with these findings, this study reveals a molecular mechanism underlying Sirt3 regulating CNPase enzymatic activity, and suggests that targeting CNPase's post-translational modifications represents a promising therapeutic strategy.
    Keywords:  2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase); Enzymatic activity; Molecular dynamic simulations; Protein conformation; SIRT3; Substrate accessibility
    DOI:  https://doi.org/10.1016/j.bbrc.2021.07.079
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