bims-numges 2021-11-07 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2021‒11‒07
thirty-nine papers selected by
Sean Rudd
Karolinska Institutet

Crosstalk between CST and RPA regulates RAD51 activity during replication stress.
Nucleotide excision repair leaves a mark on chromatin: DNA damage detection in nucleosomes.
Arginine methylation and ubiquitylation crosstalk controls DNA end-resection and homologous recombination repair.
MicroDNA levels are dependent on MMEJ, repressed by c-NHEJ pathway, and stimulated by DNA damage.
Two redundant ubiquitin-dependent pathways of BRCA1 localization to DNA damage sites.
Caspase-2 regulates S-phase cell cycle events to protect from DNA damage accumulation independent of apoptosis.
Homologous recombination-mediated irreversible genome damage underlies telomere-induced senescence.
Timely termination of repair DNA synthesis by ATAD5 is important in oxidative DNA damage-induced single-strand break repair.
The base excision repair process: comparison between higher and lower eukaryotes.
Targeting DNA Damage Response Pathway in Ovarian Clear Cell Carcinoma.
When the RAP (80) fades out, you can hear BRCA1 RING.
ID3 promotes homologous recombination via non-transcriptional and transcriptional mechanisms and its loss confers sensitivity to PARP inhibition.
Phospho-Ku70 induced by DNA damage interacts with RNA Pol II and promotes the formation of phospho-53BP1 foci to ensure optimal cNHEJ.
RBM6 splicing factor promotes homologous recombination repair of double-strand breaks and modulates sensitivity to chemotherapeutic drugs.
DNA damage triggers an interplay between wtp53 and c-Myc affecting lymphoma cell proliferation and KSHV replication.
Temporal dynamics of base excision/single-strand break repair protein complex assembly/disassembly are modulated by the PARP/NAD+/SIRT6 axis.
Microscopy analysis of the smallest subunit of the RPA complex, Rfa3p, prompts consideration of how RPA subunits gather at single-stranded DNA sites.
Genetic and physical interactions between Polη and Rev1 in response to UV-induced DNA damage in mammalian cells.
CST does not evict elongating telomerase but prevents initiation by ssDNA binding.
SPINDOC binds PARP1 to facilitate PARylation.
Involvement of classic and alternative non-homologous end joining pathways in hematologic malignancies: targeting strategies for treatment.
Alterations of DNA damage response pathway: Biomarker and therapeutic strategy for cancer immunotherapy.
Structural insights into the role of DNA-PK as a master regulator in NHEJ.
Poly(ADP-ribose) polymerase inhibitors in prostate cancer: a cornerstone in precision oncology.
The Shu complex prevents mutagenesis and cytotoxicity of single-strand specific alkylation lesions.
Loss of FBXW7 correlates with increased IDH1 expression in glioma and enhances IDH1-mutant cancer cell sensitivity to radiation.
Aryloxy Pivaloyloxymethyl Prodrugs as Nucleoside Monophosphate Prodrugs.
Intrinsic S phase checkpoint enforced by an antiproliferative oncosuppressor cytokine.
Identification of Dihydroorotate Dehydrogenase Inhibitors─Indoluidins─That Inhibit Cancer Cell Growth.
Clinical and biological impact of SAMHD1 expression in mantle cell lymphoma.
Cul4A-DDB1-mediated monoubiquitination of phosphoglycerate dehydrogenase promotes colorectal cancer metastasis via increased S-adenosylmethionine.
RNA-directed DNA repair and antibody somatic hypermutation.
Small Molecule Targeting Topoisomerase 3β for Cancer Therapy.
Dual function of HPF1 in the modulation of PARP1 and PARP2 activities.
BEAR reveals that increased fidelity variants can successfully reduce the mismatch tolerance of adenine but not cytosine base editors.
Mammalian dihydropyrimidine dehydrogenase.
FAN1's protection against CGG repeat expansion requires its nuclease activity and is FANCD2-independent.
Deficiency of replication-independent DNA mismatch repair drives a 5-methylcytosine deamination mutational signature in cancer.
Inferring primase-DNA specific recognition using a data driven approach.

Nat Commun. 2021 Nov 05. 12(1): 6412

Crosstalk between CST and RPA regulates RAD51 activity during replication stress.

Kai-Hang Lei, Han-Lin Yang, Hao-Yen Chang, Hsin-Yi Yeh, Dinh Duc Nguyen, Tzu-Yu Lee, Xinxing Lyu, Megan Chastain, Weihang Chai, Hung-Wen Li, Peter Chi.

Replication stress causes replication fork stalling, resulting in an accumulation of single-stranded DNA (ssDNA). Replication protein A (RPA) and CTC1-STN1-TEN1 (CST) complex bind ssDNA and are found at stalled forks, where they regulate RAD51 recruitment and foci formation in vivo. Here, we investigate crosstalk between RPA, CST, and RAD51. We show that CST and RPA localize in close proximity in cells. Although CST stably binds to ssDNA with a high affinity at low ionic strength, the interaction becomes more dynamic and enables facilitated dissociation at high ionic strength. CST can coexist with RPA on the same ssDNA and target RAD51 to RPA-coated ssDNA. Notably, whereas RPA-coated ssDNA inhibits RAD51 activity, RAD51 can assemble a functional filament and exhibit strand-exchange activity on CST-coated ssDNA at high ionic strength. Our findings provide mechanistic insights into how CST targets and tethers RAD51 to RPA-coated ssDNA in response to replication stress.

DOI: https://doi.org/10.1038/s41467-021-26624-x
Cell Mol Life Sci. 2021 Nov 03.

Nucleotide excision repair leaves a mark on chromatin: DNA damage detection in nucleosomes.

Katja Apelt, Hannes Lans, Orlando D Schärer, Martijn S Luijsterburg.

  Global genome nucleotide excision repair (GG-NER) eliminates a broad spectrum of DNA lesions from genomic DNA. Genomic DNA is tightly wrapped around histones creating a barrier for DNA repair proteins to access DNA lesions buried in nucleosomal DNA. The DNA-damage sensors XPC and DDB2 recognize DNA lesions in nucleosomal DNA and initiate repair. The emerging view is that a tight interplay between XPC and DDB2 is regulated by post-translational modifications on the damage sensors themselves as well as on chromatin containing DNA lesions. The choreography between XPC and DDB2, their interconnection with post-translational modifications such as ubiquitylation, SUMOylation, methylation, poly(ADP-ribos)ylation, acetylation, and the functional links with chromatin remodelling activities regulate not only the initial recognition of DNA lesions in nucleosomes, but also the downstream recruitment and necessary displacement of GG-NER factors as repair progresses. In this review, we highlight how nucleotide excision repair leaves a mark on chromatin to enable DNA damage detection in nucleosomes.

Keywords:  Chromatin; DDB2; Nucleotide excision repair; PTM; Post-translational modification; XPC

DOI:  https://doi.org/10.1007/s00018-021-03984-7
Nat Commun. 2021 Nov 02. 12(1): 6313

Arginine methylation and ubiquitylation crosstalk controls DNA end-resection and homologous recombination repair.

Maria Pilar Sanchez-Bailon, Soo-Youn Choi, Elizabeth R Dufficy, Karan Sharma, Gavin S McNee, Emma Gunnell, Kelly Chiang, Debashish Sahay, Sarah Maslen, Grant S Stewart, J Mark Skehel, Ingrid Dreveny, Clare C Davies.

Cross-talk between distinct protein post-translational modifications is critical for an effective DNA damage response. Arginine methylation plays an important role in maintaining genome stability, but how this modification integrates with other enzymatic activities is largely unknown. Here, we identify the deubiquitylating enzyme USP11 as a previously uncharacterised PRMT1 substrate, and demonstrate that the methylation of USP11 promotes DNA end-resection and the repair of DNA double strand breaks (DSB) by homologous recombination (HR), an event that is independent from another USP11-HR activity, the deubiquitylation of PALB2. We also show that PRMT1 is a ubiquitylated protein that it is targeted for deubiquitylation by USP11, which regulates the ability of PRMT1 to bind to and methylate MRE11. Taken together, our findings reveal a specific role for USP11 during the early stages of DSB repair, which is mediated through its ability to regulate the activity of the PRMT1-MRE11 pathway.

DOI: https://doi.org/10.1038/s41467-021-26413-6
Nucleic Acids Res. 2021 Oct 30. pii: gkab984. [Epub ahead of print]

MicroDNA levels are dependent on MMEJ, repressed by c-NHEJ pathway, and stimulated by DNA damage.

Teressa Paulsen, Pumoli Malapati, Yoshiyuki Shibata, Briana Wilson, Rebeka Eki, Mouadh Benamar, Tarek Abbas, Anindya Dutta.

Extrachromosomal circular DNA (eccDNA) are present within all eukaryotic organisms and actively contribute to gene expression changes. MicroDNA (200-1000bp) are the most abundant type of eccDNA and can amplify tRNA, microRNA, and novel si-like RNA sequences. Due to the heterogeneity of microDNA and the limited technology to directly quantify circular DNA molecules, the specific DNA repair pathways that contribute to microDNA formation have not been fully elucidated. Using a sensitive and quantitative assay that quantifies eight known abundant microDNA, we report that microDNA levels are dependent on resection after double-strand DNA break (DSB) and repair by Microhomology Mediated End Joining (MMEJ). Further, repair of DSB without resection by canonical Non-Homologous End Joining (c-NHEJ) diminishes microDNA formation. MicroDNA levels are induced locally even by a single site-directed DSB, suggesting that excision of genomic DNA by two closely spaced DSB is not necessary for microDNA formation. Consistent with all this, microDNA levels accumulate as cells undergo replication in S-phase, when DNA breaks and repair are elevated, and microDNA levels are decreased if DNA synthesis is prevented. Thus, formation of microDNA occurs during the repair of endogenous or induced DNA breaks by resection-based DNA repair pathways.

DOI: https://doi.org/10.1093/nar/gkab984
EMBO Rep. 2021 Nov 02. e53679

Two redundant ubiquitin-dependent pathways of BRCA1 localization to DNA damage sites.

Alana Sherker, Natasha Chaudhary, Salomé Adam, Anne Margriet Heijink, Sylvie M Noordermeer, Amélie Fradet-Turcotte, Daniel Durocher.

  The tumor suppressor BRCA1 accumulates at sites of DNA damage in a ubiquitin-dependent manner. In this work, we revisit the role of RAP80 in promoting BRCA1 recruitment to damaged chromatin. We find that RAP80 acts redundantly with the BRCA1 RING domain to promote BRCA1 recruitment to DNA damage sites. We show that that RNF8 E3 ligase acts upstream of both the RAP80- and RING-dependent activities, whereas RNF168 acts uniquely upstream of the RING domain. BRCA1 RING mutations that do not impact BARD1 interaction, such as the E2 binding-deficient I26A mutation, render BRCA1 unable to accumulate at DNA damage sites in the absence of RAP80. Cells that combine BRCA1 I26A and mutations that disable the RAP80-BRCA1 interaction are hypersensitive to PARP inhibition and are unable to form RAD51 foci. Our results suggest that in the absence of RAP80, the BRCA1 E3 ligase activity is necessary for recognition of histone H2A Lys13/Lys15 ubiquitylation by BARD1, although we cannot rule out the possibility that the BRCA1 RING facilitates ubiquitylated nucleosome recognition in other ways.

Keywords:  BARD1; BRCA1; DNA damage; RAP80; ubiquitin

DOI:  https://doi.org/10.15252/embr.202153679
Oncogene. 2021 Oct 30.

Caspase-2 regulates S-phase cell cycle events to protect from DNA damage accumulation independent of apoptosis.

Ashley G Boice, Karla E Lopez, Raj K Pandita, Melissa J Parsons, Chloe I Charendoff, Vijay Charaka, Alexandre F Carisey, Tej K Pandita, Lisa Bouchier-Hayes.

In addition to its classical role in apoptosis, accumulating evidence suggests that caspase-2 has non-apoptotic functions, including regulation of cell division. Loss of caspase-2 is known to increase proliferation rates but how caspase-2 is regulating this process is currently unclear. We show that caspase-2 is activated in dividing cells in G1-phase of the cell cycle. In the absence of caspase-2, cells exhibit numerous S-phase defects including delayed exit from S-phase, defects in repair of chromosomal aberrations during S-phase, and increased DNA damage following S-phase arrest. In addition, caspase-2-deficient cells have a higher frequency of stalled replication forks, decreased DNA fiber length, and impeded progression of DNA replication tracts. This indicates that caspase-2 protects from replication stress and promotes replication fork protection to maintain genomic stability. These functions are independent of the pro-apoptotic function of caspase-2 because blocking caspase-2-induced cell death had no effect on cell division, DNA damage-induced cell cycle arrest, or DNA damage. Thus, our data supports a model where caspase-2 regulates cell cycle and DNA repair events to protect from the accumulation of DNA damage independently of its pro-apoptotic function.

DOI: https://doi.org/10.1038/s41388-021-02085-w
Nucleic Acids Res. 2021 Nov 02. pii: gkab965. [Epub ahead of print]

Homologous recombination-mediated irreversible genome damage underlies telomere-induced senescence.

Sabrina Ghadaouia, Marc-Alexandre Olivier, Aurélie Martinez, Tibila Kientega, Jian Qin, Patrick Lambert-Lanteigne, Guillaume B Cardin, Chantal Autexier, Nicolas Malaquin, Francis Rodier.

Loss of telomeric DNA leads to telomere uncapping, which triggers a persistent, p53-centric DNA damage response that sustains a stable senescence-associated proliferation arrest. Here, we show that in normal cells telomere uncapping triggers a focal telomeric DNA damage response accompanied by a transient cell cycle arrest. Subsequent cell division with dysfunctional telomeres resulted in sporadic telomeric sister chromatid fusions that gave rise to next-mitosis genome instability, including non-telomeric DNA lesions responsible for a stable, p53-mediated, senescence-associated proliferation arrest. Unexpectedly, the blocking of Rad51/RPA-mediated homologous recombination, but not non-homologous end joining (NHEJ), prevented senescence despite multiple dysfunctional telomeres. When cells approached natural replicative senescence, interphase senescent cells displayed genome instability, whereas near-senescent cells that underwent mitosis despite the presence of uncapped telomeres did not. This suggests that these near-senescent cells had not yet acquired irreversible telomeric fusions. We propose a new model for telomere-initiated senescence where tolerance of telomere uncapping eventually results in irreversible non-telomeric DNA lesions leading to stable senescence. Paradoxically, our work reveals that senescence-associated tumor suppression from telomere shortening requires irreversible genome instability at the single-cell level, which suggests that interventions to repair telomeres in the pre-senescent state could prevent senescence and genome instability.

DOI: https://doi.org/10.1093/nar/gkab965
Nucleic Acids Res. 2021 Oct 30. pii: gkab999. [Epub ahead of print]

Timely termination of repair DNA synthesis by ATAD5 is important in oxidative DNA damage-induced single-strand break repair.

Su Hyung Park, Youyoung Kim, Jae Sun Ra, Min Woo Wie, Mi-Sun Kang, Sukhyun Kang, Kyungjae Myung, Kyoo-Young Lee.

Reactive oxygen species (ROS) generate oxidized bases and single-strand breaks (SSBs), which are fixed by base excision repair (BER) and SSB repair (SSBR), respectively. Although excision and repair of damaged bases have been extensively studied, the function of the sliding clamp, proliferating cell nuclear antigen (PCNA), including loading/unloading, remains unclear. We report that, in addition to PCNA loading by replication factor complex C (RFC), timely PCNA unloading by the ATPase family AAA domain-containing protein 5 (ATAD5)-RFC-like complex is important for the repair of ROS-induced SSBs. We found that PCNA was loaded at hydrogen peroxide (H2O2)-generated direct SSBs after the 3'-terminus was converted to the hydroxyl moiety by end-processing enzymes. However, PCNA loading rarely occurred during BER of oxidized or alkylated bases. ATAD5-depleted cells were sensitive to acute H2O2 treatment but not methyl methanesulfonate treatment. Unexpectedly, when PCNA remained on DNA as a result of ATAD5 depletion, H2O2-induced repair DNA synthesis increased in cancerous and normal cells. Based on higher H2O2-induced DNA breakage and SSBR protein enrichment by ATAD5 depletion, we propose that extended repair DNA synthesis increases the likelihood of DNA polymerase stalling, shown by increased PCNA monoubiquitination, and consequently, harmful nick structures are more frequent.

DOI: https://doi.org/10.1093/nar/gkab999
Cell Mol Life Sci. 2021 Nov 03.

The base excision repair process: comparison between higher and lower eukaryotes.

Nagham Nafiz Hindi, Noha Elsakrmy, Dindial Ramotar.

  The base excision repair (BER) pathway is essential for maintaining the stability of DNA in all organisms and defects in this process are associated with life-threatening diseases. It is involved in removing specific types of DNA lesions that are induced by both exogenous and endogenous genotoxic substances. BER is a multi-step mechanism that is often initiated by the removal of a damaged base leading to a genotoxic intermediate that is further processed before the reinsertion of the correct nucleotide and the restoration of the genome to a stable structure. Studies in human and yeast cells, as well as fruit fly and nematode worms, have played important roles in identifying the components of this conserved DNA repair pathway that maintains the integrity of the eukaryotic genome. This review will focus on the components of base excision repair, namely, the DNA glycosylases, the apurinic/apyrimidinic endonucleases, the DNA polymerase, and the ligases, as well as other protein cofactors. Functional insights into these conserved proteins will be provided from humans, Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans, and the implications of genetic polymorphisms and knockouts of the corresponding genes.

Keywords:  Cancers; Genome instability; Neurodegenerative diseases; Organismal differences; Oxidative DNA damage and repair; Sub-pathways

DOI:  https://doi.org/10.1007/s00018-021-03990-9
Front Oncol. 2021 ;11 666815

Targeting DNA Damage Response Pathway in Ovarian Clear Cell Carcinoma.

Oscar G W Wong, Jing Li, Annie N Y Cheung.

  Ovarian clear cell carcinoma (OCCC) is one of the major types of ovarian cancer and is of higher relative prevalence in Asians. It also shows higher possibility of resistance to cisplatin-based chemotherapy leading to poor prognosis. This may be attributed to the relative lack of mutations and aberrations in homologous recombination-associated genes, which are crucial in DNA damage response (DDR), such as BRCA1, BRCA2, p53, RAD51, and genes in the Fanconi anemia pathway. On the other hand, OCCC is characterized by a number of genetic defects rendering it vulnerable to DDR-targeting therapy, which is emerging as a potent treatment strategy for various cancer types. Mutations of ARID1A, PIK3CA, PTEN, and catenin beta 1 (CTNNB1), as well as overexpression of transcription factor hepatocyte nuclear factor-1β (HNF-1β), and microsatellite instability are common in OCCC. Of particular note is the loss-of-function mutations in ARID1A, which is found in approximately 50% of OCCC. ARID1A is crucial for processing of DNA double-strand break (DSB) and for sustaining DNA damage signaling, rendering ARID1A-deficient cells prone to impaired DNA damage checkpoint regulation and hence sensitive to poly ADP ribose polymerase (PARP) inhibitors. However, while preclinical studies have demonstrated the possibility to exploit DDR deficiency in OCCC for therapeutic purpose, progress in clinical application is lagging. In this review, we will recapitulate the preclinical studies supporting the potential of DDR targeting in OCCC treatment, with emphasis on the role of ARID1A in DDR. Companion diagnostic tests (CDx) for predicting susceptibility to PARP inhibitors are rapidly being developed for solid tumors including ovarian cancers and may readily be applicable on OCCC. The potential of various available DDR-targeting drugs for treating OCCC by drawing analogies with other solid tumors sharing similar genetic characteristics with OCCC will also be discussed.

Keywords:  ARID1A; DNA damage response (DDR); ovarian cancer (OC); ovarian clear cell carcinoma; targeted therapy

DOI:  https://doi.org/10.3389/fonc.2021.666815
EMBO Rep. 2021 Nov 02. e54116

When the RAP (80) fades out, you can hear BRCA1 RING.

Andreas Panagopoulos, Matthias Altmeyer.

The tumor suppressor protein BRCA1 plays an important role in DNA repair by homologous recombination. Despite being encoded by the first familial breast and ovarian cancer gene identified, how BRCA1 is recruited to sites of DNA damage to execute its repair functions has remained poorly understood. Several recent studies highlight the role of its constitutive interaction partner BARD1 in this process. In this issue, parallel work by Sherker et al (2021) focused on a second route of BRCA1 recruitment, connected to the BRCA1-A complex protein RAP80. Studying BRCA1 recruitment in RAP80-deficient cells exposed a critical role for the BRCA1 RING domain and its associated ubiquitin ligase activity. Given that tumors expressing RING-less BRCA1 isoforms can become resistant to therapy, targeting the RAP80 recruitment axis in such tumors might restore effective treatment.

DOI: https://doi.org/10.15252/embr.202154116
Nucleic Acids Res. 2021 Oct 28. pii: gkab964. [Epub ahead of print]

ID3 promotes homologous recombination via non-transcriptional and transcriptional mechanisms and its loss confers sensitivity to PARP inhibition.

Ali Bakr, Joschka Hey, Gianluca Sigismondo, Chun-Shan Liu, Ahmed Sadik, Ashish Goyal, Alice Cross, Ramya Lakshmana Iyer, Patrick Müller, Max Trauernicht, Kersten Breuer, Pavlo Lutsik, Christiane A Opitz, Jeroen Krijgsveld, Dieter Weichenhan, Christoph Plass, Odilia Popanda, Peter Schmezer.

The inhibitor of DNA-binding 3 (ID3) is a transcriptional regulator that limits interaction of basic helix-loop-helix transcription factors with their target DNA sequences. We previously reported that ID3 loss is associated with mutational signatures linked to DNA repair defects. Here we demonstrate that ID3 exhibits a dual role to promote DNA double-strand break (DSB) repair, particularly homologous recombination (HR). ID3 interacts with the MRN complex and RECQL helicase to activate DSB repair and it facilitates RAD51 loading and downstream steps of HR. In addition, ID3 promotes the expression of HR genes in response to ionizing radiation by regulating both chromatin accessibility and activity of the transcription factor E2F1. Consistently, analyses of TCGA cancer patient data demonstrate that low ID3 expression is associated with impaired HR. The loss of ID3 leads to sensitivity of tumor cells to PARP inhibition, offering new therapeutic opportunities in ID3-deficient tumors.

DOI: https://doi.org/10.1093/nar/gkab964
Nucleic Acids Res. 2021 Oct 30. pii: gkab980. [Epub ahead of print]

Phospho-Ku70 induced by DNA damage interacts with RNA Pol II and promotes the formation of phospho-53BP1 foci to ensure optimal cNHEJ.

Amelie Schellenbauer, Marie-Noelle Guilly, Romain Grall, Romain Le Bars, Vincent Paget, Thierry Kortulewski, Haser Sutcu, Cécile Mathé, Marie Hullo, Denis Biard, François Leteurtre, Vilma Barroca, Youenn Corre, Lamya Irbah, Emilie Rass, Benoit Theze, Pascale Bertrand, Jeroen A A Demmers, Josée Guirouilh-Barbat, Bernard S Lopez, Sylvie Chevillard, Jozo Delic.

Canonical non-homologous end-joining (cNHEJ) is the prominent mammalian DNA double-strand breaks (DSBs) repair pathway operative throughout the cell cycle. Phosphorylation of Ku70 at ser27-ser33 (pKu70) is induced by DNA DSBs and has been shown to regulate cNHEJ activity, but the underlying mechanism remained unknown. Here, we established that following DNA damage induction, Ku70 moves from nucleoli to the sites of damage, and once linked to DNA, it is phosphorylated. Notably, the novel emanating functions of pKu70 are evidenced through the recruitment of RNA Pol II and concomitant formation of phospho-53BP1 foci. Phosphorylation is also a prerequisite for the dynamic release of Ku70 from the repair complex through neddylation-dependent ubiquitylation. Although the non-phosphorylable ala-Ku70 form does not compromise the formation of the NHEJ core complex per se, cells expressing this form displayed constitutive and stress-inducible chromosomal instability. Consistently, upon targeted induction of DSBs by the I-SceI meganuclease into an intrachromosomal reporter substrate, cells expressing pKu70, rather than ala-Ku70, are protected against the joining of distal DNA ends. Collectively, our results underpin the essential role of pKu70 in the orchestration of DNA repair execution in living cells and substantiated the way it paves the maintenance of genome stability.

DOI: https://doi.org/10.1093/nar/gkab980
Nucleic Acids Res. 2021 Oct 28. pii: gkab976. [Epub ahead of print]

RBM6 splicing factor promotes homologous recombination repair of double-strand breaks and modulates sensitivity to chemotherapeutic drugs.

Feras E Machour, Enas R Abu-Zhayia, Samah W Awwad, Tirza Bidany-Mizrahi, Stefan Meinke, Laila A Bishara, Florian Heyd, Rami I Aqeilan, Nabieh Ayoub.

RNA-binding proteins regulate mRNA processing and translation and are often aberrantly expressed in cancer. The RNA-binding motif protein 6, RBM6, is a known alternative splicing factor that harbors tumor suppressor activity and is frequently mutated in human cancer. Here, we identify RBM6 as a novel regulator of homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Mechanistically, we show that RBM6 regulates alternative splicing-coupled nonstop-decay of a positive HR regulator, Fe65/APBB1. RBM6 knockdown leads to a severe reduction in Fe65 protein levels and consequently impairs HR of DSBs. Accordingly, RBM6-deficient cancer cells are vulnerable to ATM and PARP inhibition and show remarkable sensitivity to cisplatin. Concordantly, cisplatin administration inhibits the growth of breast tumor devoid of RBM6 in mouse xenograft model. Furthermore, we observe that RBM6 protein is significantly lost in metastatic breast tumors compared with primary tumors, thus suggesting RBM6 as a potential therapeutic target of advanced breast cancer. Collectively, our results elucidate the link between the multifaceted roles of RBM6 in regulating alternative splicing and HR of DSBs that may contribute to tumorigenesis, and pave the way for new avenues of therapy for RBM6-deficient tumors.

DOI: https://doi.org/10.1093/nar/gkab976
Biochim Biophys Acta Mol Cell Res. 2021 Oct 30. pii: S0167-4889(21)00222-6. [Epub ahead of print] 119168

DNA damage triggers an interplay between wtp53 and c-Myc affecting lymphoma cell proliferation and KSHV replication.

Andrea Arena, Maria Saveria Gilardini Montani, Maria Anele Romeo, Rossella Benedetti, Aurelia Gaeta, Mara Cirone.

  The induction of DNA damage together with the interference with DNA repair represents a promising strategy in cancer treatment. Here we show that the PARP-1/2/3 inhibitor AZD2461 in combination with the CHK1 inhibitor UCN-01 altered the DNA damage response and reduced cell proliferation in PEL cells, an aggressive B cell lymphoma highly resistant to chemotherapies. AZD2461/UCN-01 combination activated p53/p21 and downregulated c-Myc in these cells, leading to a reduced expression level of RAD51, molecule involved in DNA repair. The effect of AZD2461/UCN-01 on c-Myc and p53/p21 was inter-dependent and, besides impairing cell proliferation, contributed to the activation of the replicative cycle of KSHV, carried in a latent state in PEL cells. Finally, we found that the pharmacological or genetic inhibition of p21 counteracted the viral lytic cycle activation and further reduced PEL cell proliferation, suggesting that it could induce a double beneficial effect in this setting. This study unveils that, therapeutic approaches, based on the induction of DNA damage and the reduction of DNA repair, could be used to successfully treat this malignant lymphoma.

Keywords:  AZD2461; DDR; KSHV; PEL; RAD51; UCN-01; c-Myc; p53

DOI:  https://doi.org/10.1016/j.bbamcr.2021.119168
Cell Rep. 2021 Nov 02. pii: S2211-1247(21)01390-5. [Epub ahead of print]37(5): 109917

Temporal dynamics of base excision/single-strand break repair protein complex assembly/disassembly are modulated by the PARP/NAD+/SIRT6 axis.

Christopher A Koczor, Kate M Saville, Joel F Andrews, Jennifer Clark, Qingming Fang, Jianfeng Li, Rasha Q Al-Rahahleh, Md Ibrahim, Steven McClellan, Mikhail V Makarov, Marie E Migaud, Robert W Sobol.

  Assembly and disassembly of DNA repair protein complexes at DNA damage sites are essential for maintaining genomic integrity. Investigating factors coordinating assembly of the base excision repair (BER) proteins DNA polymerase β (Polβ) and XRCC1 to DNA lesion sites identifies a role for Polβ in regulating XRCC1 disassembly from DNA repair complexes and, conversely, demonstrates Polβ's dependence on XRCC1 for complex assembly. LivePAR, a genetically encoded probe for live-cell imaging of poly(ADP-ribose) (PAR), reveals that Polβ and XRCC1 require PAR for repair-complex assembly, with PARP1 and PARP2 playing unique roles in complex dynamics. Further, BER complex assembly is modulated by attenuation/augmentation of NAD+ biosynthesis. Finally, SIRT6 does not modulate PARP1 or PARP2 activation but does regulate XRCC1 recruitment, leading to diminished Polβ abundance at sites of DNA damage. These findings highlight coordinated yet independent roles for PARP1, PARP2, and SIRT6 and their regulation by NAD+ bioavailability to facilitate BER.

Keywords:  BER; DNA polymerase β; LivePAR; NAD(+); NRH; PAR; SIRT6; SSBR; XRCC1; poly(ADP-ribose)

DOI:  https://doi.org/10.1016/j.celrep.2021.109917
MicroPubl Biol. 2021 ;2021

Microscopy analysis of the smallest subunit of the RPA complex, Rfa3p, prompts consideration of how RPA subunits gather at single-stranded DNA sites.

Agnès Ramonatxo, María Moriel-Carretero.

The heterotrimeric Replication Protein A (RPA) complex preserves genome integrity by protecting the single-stranded DNA that becomes exposed during repair, replication, and recombination. Its two biggest subunits, Rfa1p and Rfa2p (as named in S. cerevisiae) contact DNA and interact with other partners, while the smallest Rfa3p subunit is considered to fulfill a structural role. Perhaps because of this, mostly Rfa1p and eventually Rfa2p are used for microscopy studies upon tagging them with fluorophores. In this work, we explore the behavior of GFP-tagged Rfa3p basally and in response to DNA damage conditions and compare it with tagged Rfa1p. We find that fluorescent Rfa3p yields signals that are (or are detected) significantly more frequent(ly). By making a careful comparison with our own and with previously published data, we propose that Rfa3p, by virtue of its scaffolding role, may reach single-stranded DNA sites first thus serving to nucleate the full RPA complex.

DOI: https://doi.org/10.17912/micropub.biology.000493
Sci Rep. 2021 Nov 01. 11(1): 21364

Genetic and physical interactions between Polη and Rev1 in response to UV-induced DNA damage in mammalian cells.

Tonghui Bi, Xiaohong Niu, Chunping Qin, Wei Xiao.

In response to UV irradiation, translesion DNA synthesis (TLS) utilizes specialized DNA polymerases to bypass replication-blocking lesions. In a well-established polymerase switch model, Polη is thought to be a preferred TLS polymerase to insert correct nucleotides across from the thymine dimer, and Rev1 plays a scaffold role through physical interaction with Polη and the Rev7 subunit of Polζ for continual DNA synthesis. Defective Polη causes a variant form of xeroderma pigmentosum (XPV), a disease with predisposition to sunlight-induced skin cancer. Previous studies revealed that expression of Rev1 alone is sufficient to confer enhanced UV damage tolerance in mammalian cells, which depends on its physical interaction with Polζ but is independent of Polη, a conclusion that appears to contradict current literature on the critical roles of Polη in TLS. To test a hypothesis that the Rev1 catalytic activity is required to backup Polη in TLS, we found that the Rev1 polymerase-dead mutation is synergistic with either Polη mutation or the Polη-interaction mutation in response to UV-induced DNA damage. On the other hand, functional complementation of polH cells by Polη relies on its physical interaction with Rev1. Hence, our studies reveal critical interactions between Rev1 and Polη in response to UV damage.

DOI: https://doi.org/10.1038/s41598-021-00878-3
Nucleic Acids Res. 2021 Oct 28. pii: gkab942. [Epub ahead of print]

CST does not evict elongating telomerase but prevents initiation by ssDNA binding.

Arthur J Zaug, Ci Ji Lim, Conner L Olson, Maria T Carilli, Karen J Goodrich, Deborah S Wuttke, Thomas R Cech.

The CST complex (CTC1-STN1-TEN1) has been shown to inhibit telomerase extension of the G-strand of telomeres and facilitate the switch to C-strand synthesis by DNA polymerase alpha-primase (pol α-primase). Recently the structure of human CST was solved by cryo-EM, allowing the design of mutant proteins defective in telomeric ssDNA binding and prompting the reexamination of CST inhibition of telomerase. The previous proposal that human CST inhibits telomerase by sequestration of the DNA primer was tested with a series of DNA-binding mutants of CST and modeled by a competitive binding simulation. The DNA-binding mutants had substantially reduced ability to inhibit telomerase, as predicted from their reduced affinity for telomeric DNA. These results provide strong support for the previous primer sequestration model. We then tested whether addition of CST to an ongoing processive telomerase reaction would terminate DNA extension. Pulse-chase telomerase reactions with addition of either wild-type CST or DNA-binding mutants showed that CST has no detectable ability to terminate ongoing telomerase extension in vitro. The same lack of inhibition was observed with or without pol α-primase bound to CST. These results suggest how the switch from telomerase extension to C-strand synthesis may occur.

DOI: https://doi.org/10.1093/nar/gkab942
Nat Commun. 2021 Nov 04. 12(1): 6362

SPINDOC binds PARP1 to facilitate PARylation.

Fen Yang, Jianji Chen, Bin Liu, Guozhen Gao, Manu Sebastian, Collene Jeter, Jianjun Shen, Maria D Person, Mark T Bedford.

SPINDOC is tightly associated with the histone H3K4me3 effector protein SPIN1. To gain a better understanding of the biological roles of SPINDOC, we identified its interacting proteins. Unexpectedly, SPINDOC forms two mutually exclusive protein complexes, one with SPIN1 and the other with PARP1. Consistent with its ability to directly interact with PARP1, SPINDOC expression is induced by DNA damage, likely by KLF4, and recruited to DNA lesions with dynamics that follows PARP1. In SPINDOC knockout cells, the levels of PARylation are reduced, in both the absence and presence of DNA damage. The SPINDOC/PARP1 interaction promotes the clearance of PARP1 from damaged DNA, and also impacts the expression of known transcriptional targets of PARP1. To address the in vivo roles of SPINDOC in PARP1 regulation, we generate SPINDOC knockout mice, which are viable, but slightly smaller than their wildtype counterparts. The KO mice display reduced levels of PARylation and, like PARP1 KO mice, are hypersensitive to IR-induced DNA damage. The findings identify a SPIN1-independent role for SPINDOC in the regulation of PARP1-mediated PARylation and the DNA damage response.

DOI: https://doi.org/10.1038/s41467-021-26588-y
Exp Hematol Oncol. 2021 Nov 03. 10(1): 51

Involvement of classic and alternative non-homologous end joining pathways in hematologic malignancies: targeting strategies for treatment.

Mohsen Valikhani, Elahe Rahimian, Seyed Esmaeil Ahmadi, Rouzbeh Chegeni, Majid Safa.

  Chromosomal translocations are the main etiological factor of hematologic malignancies. These translocations are generally the consequence of aberrant DNA double-strand break (DSB) repair. DSBs arise either exogenously or endogenously in cells and are repaired by major pathways, including non-homologous end-joining (NHEJ), homologous recombination (HR), and other minor pathways such as alternative end-joining (A-EJ). Therefore, defective NHEJ, HR, or A-EJ pathways force hematopoietic cells toward tumorigenesis. As some components of these repair pathways are overactivated in various tumor entities, targeting these pathways in cancer cells can sensitize them, especially resistant clones, to radiation or chemotherapy agents. However, targeted therapy-based studies are currently underway in this area, and furtherly there are some biological pitfalls, clinical issues, and limitations related to these targeted therapies, which need to be considered. This review aimed to investigate the alteration of DNA repair elements of C-NHEJ and A-EJ in hematologic malignancies and evaluate the potential targeted therapies against these pathways.

Keywords:  Alternative end-joining pathways; Double-strand break; Double-strand break repair; Hematologic malignancies; Non-homologous end-joining; Targeted therapy

DOI:  https://doi.org/10.1186/s40164-021-00242-1
Acta Pharm Sin B. 2021 Oct;11(10): 2983-2994

Alterations of DNA damage response pathway: Biomarker and therapeutic strategy for cancer immunotherapy.

Minlin Jiang, Keyi Jia, Lei Wang, Wei Li, Bin Chen, Yu Liu, Hao Wang, Sha Zhao, Yayi He, Caicun Zhou.

  Genomic instability remains an enabling feature of cancer and promotes malignant transformation. Alterations of DNA damage response (DDR) pathways allow genomic instability, generate neoantigens, upregulate the expression of programmed death ligand 1 (PD-L1) and interact with signaling such as cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signaling. Here, we review the basic knowledge of DDR pathways, mechanisms of genomic instability induced by DDR alterations, impacts of DDR alterations on immune system, and the potential applications of DDR alterations as biomarkers and therapeutic targets in cancer immunotherapy.

Keywords:  ATM, ataxia-telangiectasia mutated; ATR, ataxia telangiectasia and Rad3 related; BAP1, BRCA1-associated protein 1; BER, base excision repair; BRAF, v-RAF murine sarcoma viral oncogene homologue B; BRCA, breast cancer susceptibility gene; CHEK, cell-cycle checkpoint kinase; CHK1, checkpoint kinase 1; DAMP, damage-associated molecular patterns; DDR, DNA damage response; DNA damage response; DNA repair; DR, direct repair; DSBs, double-strand breaks; FDA, United State Food and Drug Administration; GSK3β, glycogen synthase kinase 3β; Genomic instability; HMGB1, high mobility group box-1; HRR, homologous recombination repair; ICI, immune checkpoint inhibitor; IFNγ, interferon gamma; IHC, immunohistochemistry; IRF1, interferon regulatory factor 1; Immunotherapy; JAK, Janus kinase; MAD1, mitotic arrest deficient-like 1; MGMT, O6-methylguanine methyltransferase; MLH1, MutL homolog 1; MMR, mismatch repair; MNT, MAX network transcriptional repressor; MSH2/6, MutS protein homologue-2/6; MSI, microsatellite instability; MUTYH, MutY homolog; MyD88, myeloid differentiation factor 88; NEK1, NIMA-related kinase 1; NER, nucleotide excision repair; NGS, next generation sequencing; NHEJ, nonhomologous end-joining; NIMA, never-in-mitosis A; NSCLC, non-small cell lung cancer; ORR, objective response rate; OS, overall survival; PALB2, partner and localizer of BRCA2; PARP, poly-ADP ribose polymerase; PCR, polymerase chain reaction; PD-1; PD-1, programmed death 1; PD-L1; PD-L1, programmed death ligand 1; PFS, progression-free survival; RAD51C, RAD51 homolog C; RB1, retinoblastoma 1; RPA, replication protein A; RSR, replication stress response; SCNAs, somatic copy number alterations; STAT, signal transducer and activator of transcription; STING, stimulator of interferon genes; TBK1, TANK-binding kinase 1; TILs, tumor-infiltrating lymphocytes; TLR4, Toll-like receptor 4; TMB, tumor mutational burden; TME, tumor microenvironment; TP53, tumor protein P53; TRIF, Toll-interleukin 1 receptor domain-containing adaptor inducing INF-β; Tumor microenvironment; XRCC4, X-ray repair cross complementing protein 4; cGAS, cyclic GMP–AMP synthase; cGAS–STING; ssDNA, single-stranded DNA

DOI:  https://doi.org/10.1016/j.apsb.2021.01.003
Genome Instab Dis. 2021 ;2(4): 195-210

Structural insights into the role of DNA-PK as a master regulator in NHEJ.

Siyu Chen, James P Lees-Miller, Yuan He, Susan P Lees-Miller.

  DNA-dependent protein kinase catalytic subunit DNA-PKcs/PRKDC is the largest serine/threonine protein kinase of the phosphatidyl inositol 3-kinase-like protein kinase (PIKK) family and is the most highly expressed PIKK in human cells. With its DNA-binding partner Ku70/80, DNA-PKcs is required for regulated and efficient repair of ionizing radiation-induced DNA double-strand breaks via the non-homologous end joining (NHEJ) pathway. Loss of DNA-PKcs or other NHEJ factors leads to radiation sensitivity and unrepaired DNA double-strand breaks (DSBs), as well as defects in V(D)J recombination and immune defects. In this review, we highlight the contributions of the late Dr. Carl W. Anderson to the discovery and early characterization of DNA-PK. We furthermore build upon his foundational work to provide recent insights into the structure of NHEJ synaptic complexes, an evolutionarily conserved and functionally important YRPD motif, and the role of DNA-PKcs and its phosphorylation in NHEJ. The combined results identify DNA-PKcs as a master regulator that is activated by its detection of two double-strand DNA ends for a cascade of phosphorylation events that provide specificity and efficiency in assembling the synaptic complex for NHEJ.

Keywords:  Cryo-EM; DNA-PKcs; Non-homologous end joining; Phosphorylation; Structural biology

DOI:  https://doi.org/10.1007/s42764-021-00047-w
Pharmacogenomics. 2021 Nov 03.

Poly(ADP-ribose) polymerase inhibitors in prostate cancer: a cornerstone in precision oncology.

Talal Ziadeh, Hampig Raphael Kourie.

  Poly-(ADP-ribose) polymerase (PARP) inhibitors act in cells with defects in homologous recombination DNA repair (HRR) caused by genomic aberrations such as BRCA mutations. This phenomenon called synthetic lethality is known now to be more common in prostate cancer than previously thought. Olaparib and rucaparib, two PARP inhibitors, were successfully tested in clinical trials for HRR-deficient metastatic castration-resistant prostate cancer. They received a breakthrough US FDA approval in HRR altered metastatic castration-resistant prostate cancer in May 2020. Consequently, the combination of PARP inhibitors with other agents such as androgen receptor pathway inhibitors, immune checkpoint inhibitors or DNA damage inducing chemotherapy are being currently largely studied. In our review, we aim to summarize the key PARP inhibitors published and ongoing trials in prostate cancer.

Keywords:  PARP inhibitors; homologous recombination repair; niraparib; olaparib; prostate cancer; rucaparib; talazoparib; targeted therapy; veliparib

DOI:  https://doi.org/10.2217/pgs-2021-0119
Elife. 2021 Nov 01. pii: e68080. [Epub ahead of print]10

The Shu complex prevents mutagenesis and cytotoxicity of single-strand specific alkylation lesions.

Braulio Bonilla, Alexander I Brown, Sarah R Hengel, Kyle S Rapchak, Debra Mitchell, Catherine A Pressimone, Adeola A Fagunloye, Thong T Luong, Reagan A Russell, Rudri K Vyas, Tony M Mertz, Hani S Zaher, Nima Mosammaparast, Ewa P Malc, Piotr A Mieczkowski, Steven Roberts, Kara A Bernstein.

  Three-methyl cytosine (3meC) are toxic DNA lesions, blocking base pairing. Bacteria and humans, express members of the AlkB enzymes family, which directly remove 3meC. However, other organisms, including budding yeast, lack this class of enzymes. It remains an unanswered evolutionary question as to how yeast repairs 3meC, particularly in single-stranded DNA. The yeast Shu complex, a conserved homologous recombination factor, aids in preventing replication-associated mutagenesis from DNA base damaging agents such as methyl methanesulfonate (MMS). We found that MMS-treated Shu complex-deficient cells, exhibit a genome-wide increase in A:T and G:C substitutions mutations. The G:C substitutions displayed transcriptional and replicational asymmetries consistent with mutations resulting from 3meC. Ectopic expression of a human AlkB homolog in Shu-deficient yeast rescues MMS-induced growth defects and increased mutagenesis. Thus, our work identifies a novel homologous recombination-based mechanism mediated by the Shu complex for coping with alkylation adducts.

Keywords:  S. cerevisiae; biochemistry; cell biology; chemical biology

DOI:  https://doi.org/10.7554/eLife.68080
Cancer Res. 2021 Nov 04. pii: canres.0384.2021. [Epub ahead of print]

Loss of FBXW7 correlates with increased IDH1 expression in glioma and enhances IDH1-mutant cancer cell sensitivity to radiation.

Zhuo Yang, Nan Hu, Weiwei Wang, Weihua Hu, Shaolong Zhou, Jianxiang Shi, Minghe Li, Zhou Jing, Chao Chen, Xuyang Zhang, Ruyi Yang, Xudong Fu, Xinjun Wang.

F-box and WD repeat domain containing 7 (FBXW7) is a substrate receptor of the ubiquitin ligase SKP1-Cullin1-F-box complex and a potent tumor suppressor that prevents unregulated cell growth and tumorigenesis. However, little is known about FBXW7-mediated control of cell metabolism and related functions in cancer therapy. Here, we report that FBXW7 expression inversely correlates with the expression levels of the key metabolic enzyme isocitrate dehydrogenase 1 (IDH1) in glioma patients and public glioma datasets. Deletion of FBXW7 significantly increased both wild type (WT) and mutant IDH1 expression, which was mediated by blocking degradation of sterol regulatory element binding protein 1 (SREBP1). The upregulation of neomorphic mutant IDH1 by FBXW7 deletion stimulated production of the oncometabolite 2-hydroxyglutarate (2-HG) at the expense of increasing pentose phosphate pathway (PPP) activity and NADPH consumption, limiting the buffering ability against radiation-induced oxidative stress. Additionally, FBXW7 knockout and IDH1 mutations induced non-homologous end joining (NHEJ) and homologous recombination (HR) defects, respectively. In vitro and in vivo, loss of FBXW7 dramatically enhanced the efficacy of radiation treatment in IDH1 mutant cancer cells. Taken together, this work identifies FBXW7 deficiency as a potential biomarker representing both DNA repair and metabolic vulnerabilities that sensitizes IDH1 mutant cancers to radiotherapy.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-0384
J Med Chem. 2021 Nov 04.

Aryloxy Pivaloyloxymethyl Prodrugs as Nucleoside Monophosphate Prodrugs.

Ashwag S Alanazi, Ageo Miccoli, Youcef Mehellou.

Intracellular phosphorylation of therapeutic nucleoside analogues into their active triphosphate metabolites is a prerequisite for their pharmacological activity. However, the initial phosphorylation of these unnatural nucleosides into their monophosphate derivatives can be a rate-limiting step in their activation. To address this, we herein report the development of the aryloxy pivaloyloxymethyl prodrugs (POMtides) as a novel and effective nucleoside monophosphate prodrug technology and its successful application to the anticancer nucleoside analogue 5-fluoro-2'-deoxyuridine (FdUR).

DOI: https://doi.org/10.1021/acs.jmedchem.1c01490
Cancer Gene Ther. 2021 Nov 04.

Intrinsic S phase checkpoint enforced by an antiproliferative oncosuppressor cytokine.

Livio Mallucci, Valerie Wells.

The cell cycle is strictly programmed with control mechanisms that dictate order in cell cycle progression to ensure faithful DNA replication, whose deviance may lead to cancer. Checkpoint control at the G1/S, S/G2 and G2/M portals have been defined but no statutory time-programmed control for securing orderly transition through S phase has so far been identified. Here we report that in normal cells DNA synthesis is controlled by a checkpoint sited within the early part of S phase, enforced by the βGBP cytokine an antiproliferative molecule otherwise known for its oncosuppressor properties that normal cells constitutively produce for self-regulation. Suppression of active Ras and active MAPK, block of cyclin A gene expression and suppression of CDK2-cyclin A activity are events which while specific to the control of a cell cycle phase in normal cells are part of the apoptotic network in cancer cells.

DOI: https://doi.org/10.1038/s41417-021-00397-3
ACS Chem Biol. 2021 Nov 03.

Identification of Dihydroorotate Dehydrogenase Inhibitors─Indoluidins─That Inhibit Cancer Cell Growth.

Makoto Kawatani, Harumi Aono, Takeshi Shimizu, Shouta Ohkura, Sayoko Hiranuma, Makoto Muroi, Naoko Ogawa, Tomokazu Ohishi, Shun-Ichi Ohba, Manabu Kawada, Kanami Yamazaki, Shingo Dan, Hiroyuki Osada.

Dihydroorotate dehydrogenase (DHODH) catalyzes the rate-limiting step in de novo pyrimidine biosynthesis and is a promising cancer treatment target. This study reports the identification of indoluidin D and its derivatives as inhibitors of DHODH. Cell-based phenotypic screening revealed that indoluidin D promoted myeloid differentiation and inhibited the proliferation of acute promyelocytic leukemia HL-60 cells. Indoluidin D also suppressed cell growth in various other types of cancer cells. Cancer cell sensitivity profiling with JFCR39 and proteomic profiling with ChemProteoBase revealed that indoluidin D is a DHODH inhibitor. Indoluidin D inhibited human DHODH activity in vitro; the DHODH reaction product orotic acid rescued indoluidin D-induced cell differentiation. We synthesized several indoluidin D diastereomer derivatives and demonstrated that stereochemistry was vital to their molecular activity. The indoluidin D derivative indoluidin E showed similar activity to its parent compound and suppressed tumor growth in a murine lung cancer xenograft model. Hence, indoluidin D and its derivatives selectively inhibit DHODH and suppress cancer cell growth.

DOI: https://doi.org/10.1021/acschembio.1c00625
Virchows Arch. 2021 Nov 04.

Clinical and biological impact of SAMHD1 expression in mantle cell lymphoma.

Magali Merrien, Agata M Wasik, Elin Ljung, Mohammad H A Morsy, Joana de Matos Rodrigues, Mattias Carlsten, Georgios Z Rassidakis, Birger Christensson, Arne Kolstad, Mats Jerkeman, Sara Ek, Nikolas Herold, Björn E Wahlin, Birgitta Sander.

  SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that restricts viral replication in infected cells and limits the sensitivity to cytarabine by hydrolysing its active metabolite, as recently shown in acute myeloid leukemia. Cytarabine is an essential component in the Nordic mantle cell lymphoma protocols (MCL2 and MCL3) for induction and high-dose chemotherapy treatment before autologous stem cell transplantation for younger patients with mantle cell lymphoma (MCL). We here investigated the expression of SAMHD1 in a population-based cohort of MCL (N = 150). SAMHD1 was highly variably expressed in MCL (range, 0.4% to 100% of positive tumor cells). Cases with blastoid/pleomorphic morphology had higher SAMHD1 expression (P = 0.028) and SAMHD1 was also correlated to tumor cell proliferation (P = 0.016). SAMHD1 expression showed moderate correlation to the expression of the transcriptional regulator SOX11 (P = 0.036) but genetic silencing of SOX11 and SAMHD1 by siRNA in MCL cell lines did not suggest mutual regulation. We hypothesized that expression of SAMHD1 could predict short time to progression in patients treated with Cytarabine as part of high-dose chemotherapy. Despite the correlation with known biological adverse prognostic factors, neither low or high SAMHD1 expression correlated to PFS or OS in patients treated according to the Nordic MCL2 or MCL3 protocols (N = 158).

Keywords:  Cytarabine; Mantle cell lymphoma; Prognostic factors; SAMHD1

DOI:  https://doi.org/10.1007/s00428-021-03228-w
J Clin Invest. 2021 Nov 01. pii: e146187. [Epub ahead of print]131(21):

Cul4A-DDB1-mediated monoubiquitination of phosphoglycerate dehydrogenase promotes colorectal cancer metastasis via increased S-adenosylmethionine.

Yajuan Zhang, Hua Yu, Jie Zhang, Hong Gao, Siyao Wang, Shuxian Li, Ping Wei, Ji Liang, Guanzhen Yu, Xiongjun Wang, Xinxiang Li, Dawei Li, Weiwei Yang.

  Although serine metabolism plays a crucial role in the proliferation and survival of tumor cells, how it supports tumor cell migration remains poorly understood. Phosphoglycerate dehydrogenase (PHGDH) catalyzes the oxidation of 3-phosphoglycerate to 3-phosphonooxypyruvate, the first committed step in de novo serine biosynthesis. Here we show that PHGDH was monoubiquitinated by cullin 4A-based E3 ligase complex at lysine 146 in colorectal cancer (CRC) cells, which enhanced PHGDH activity by recruiting a chaperone protein, DnaJ homolog subfamily A member 1, to promote its tetrameric formation, thereby increasing the levels of serine, glycine, and S-adenosylmethionine (SAM). Increased levels of SAM upregulated the expression of cell adhesion genes (laminin subunit gamma 2 and cysteine rich angiogenic inducer 61) by initiating SET domain containing 1A-mediated trimethylation of histone H3K4, thereby promoting tumor cell migration and CRC metastasis. Intriguingly, SAM levels in tumors or blood samples correlated with the metastatic recurrence of patients with CRC. Our finding not only reveals a potentially new role and mechanism of SAM-promoted tumor metastasis but also demonstrates a regulatory mechanism of PHGDH activity by monoubiquitination.

Keywords:  Amino acid metabolism; Cell migration/adhesion; Colorectal cancer; Metabolism; Oncology

DOI:  https://doi.org/10.1172/JCI146187
Trends Genet. 2021 Nov 02. pii: S0168-9525(21)00289-4. [Epub ahead of print]

RNA-directed DNA repair and antibody somatic hypermutation.

Andrew Franklin, Edward J Steele.

  Somatic hypermutation at antibody loci affects both deoxyadenosine-deoxythymidine (A/T) and deoxycytidine-deoxyguanosine (C/G) pairs. Deamination of C to deoxyuridine (U) by activation-induced deaminase (AID) explains how mutation at C/G pairs is potentiated. Mutation at A/T pairs is triggered during the initial stages of repair of AID-generated U lesions and occurs through an as yet unknown mechanism in which polymerase η has a major role. Recent evidence confirms that human polymerase η can act as a reverse transcriptase. Here, we compare the popular suggestion of mutation at A/T pairs through nucleotide mispairing (owing to polymerase error) during short-patch repair synthesis with the alternative proposal of mutation at A/T pairs through RNA editing and RNA-directed DNA repair.

Keywords:  antibody; polymerase error; polymerase η; reverse transcriptase; somatic hypermutation

DOI:  https://doi.org/10.1016/j.tig.2021.10.005
Pharmacol Res. 2021 Nov 02. pii: S1043-6618(21)00511-9. [Epub ahead of print] 105927

Small Molecule Targeting Topoisomerase 3β for Cancer Therapy.

Xue Zhang, Lei Wang, Qi Zhang, Song Lyu, Darong Zhu, Mengzhen Shen, Xisong Ke, Yi Qu.

  DNA topoisomerases are proved cancer therapeutic targets with clinically successful anticancer drugs for decades. However, the role of RNA topoisomerase (TOP3β) remained mysterious especially in cancer, and no targeted agent has been reported yet. In a target identification assay of anti-cancer compound using a modified DrugTargetSeqR strategy, mutation of TOP3B was detected in cancer cells acquired resistance to cinobufagin (CBG), a key compound of Huachansu that has been approved for cancer therapy in China. We demonstrated that CBG directly engaged with TOP3β, and promoted TOP3β depletion in wildtype but not mutant cancer cells. Notably, knockout of TOP3β in cancer cells significantly reduced tumor enlargement but not initiation, and inhibited colony formation upon nutrient deprivation. We also demonstrated that CBG induced formation of stress granule, RNA-loop and asymmetric DNA damages in cancer cells, and all these phenotypes were significantly attenuated in TOP3B knockout cells. Of note, examination of a panel of cancer cell lines revealed associations among cell growth inhibition and induction of DNA damage as well as TOP3B depletion upon CBG treatment. Our findings not only highlighted TOP3β as a promising therapeutic target of cancer, but also identified CBG as a lead chemical inhibitor of TOP3β for cancer therapy.

Keywords:  Cancer; Cinobufagin; DNA damage; Stress granule; TOP3β

DOI:  https://doi.org/10.1016/j.phrs.2021.105927
Commun Biol. 2021 Nov 03. 4(1): 1259

Dual function of HPF1 in the modulation of PARP1 and PARP2 activities.

Tatyana A Kurgina, Nina A Moor, Mikhail M Kutuzov, Konstantin N Naumenko, Alexander A Ukraintsev, Olga I Lavrik.

Poly(ADP-ribosyl)ation catalyzed by poly(ADP-ribose) polymerases (PARPs) is one of the immediate cellular responses to DNA damage. The histone PARylation factor 1 (HPF1) discovered recently to form a joint active site with PARP1 and PARP2 was shown to limit the PARylation activity of PARPs and stimulate their NAD+-hydrolase activity. Here we demonstrate that HPF1 can stimulate the DNA-dependent and DNA-independent autoPARylation of PARP1 and PARP2 as well as the heteroPARylation of histones in the complex with nucleosome. The stimulatory action is detected in a defined range of HPF1 and NAD+ concentrations at which no HPF1-dependent enhancement in the hydrolytic NAD+ consumption occurs. PARP2, comparing with PARP1, is more efficiently stimulated by HPF1 in the autoPARylation reaction and is more active in the heteroPARylation of histones than in the automodification, suggesting a specific role of PARP2 in the ADP-ribosylation-dependent modulation of chromatin structure. Possible role of the dual function of HPF1 in the maintaining PARP activity is discussed.

DOI: https://doi.org/10.1038/s42003-021-02780-0
Nat Commun. 2021 Nov 03. 12(1): 6353

BEAR reveals that increased fidelity variants can successfully reduce the mismatch tolerance of adenine but not cytosine base editors.

András Tálas, Dorottya A Simon, Péter I Kulcsár, Éva Varga, Sarah L Krausz, Ervin Welker.

Adenine and cytosine base editors (ABE, CBE) allow for precision genome engineering. Here, Base Editor Activity Reporter (BEAR), a plasmid-based fluorescent tool is introduced, which can be applied to report on ABE and CBE editing in a virtually unrestricted sequence context or to label base edited cells for enrichment. Using BEAR-enrichment, we increase the yield of base editing performed by nuclease inactive base editors to the level of the nickase versions while maintaining significantly lower indel background. Furthermore, by exploiting the semi-high-throughput potential of BEAR, we examine whether increased fidelity SpCas9 variants can be used to decrease SpCas9-dependent off-target effects of ABE and CBE. Comparing them on the same target sets reveals that CBE remains active on sequences, where increased fidelity mutations and/or mismatches decrease the activity of ABE. Our results suggest that the deaminase domain of ABE is less effective to act on rather transiently separated target DNA strands, than that of CBE explaining its lower mismatch tolerance.

DOI: https://doi.org/10.1038/s41467-021-26461-y
Arch Biochem Biophys. 2021 Oct 27. pii: S0003-9861(21)00315-5. [Epub ahead of print]714 109066

Mammalian dihydropyrimidine dehydrogenase.

Dariush C Forouzesh, Graham R Moran.

  Dihydropyrimidine dehydrogenase (DPD) catalyzes the two-electron reduction of pyrimidine bases uracil and thymine as the first step in pyrimidine catabolism. The enzyme achieves this simple chemistry using a complex cofactor set including two flavins and four Fe4S4 centers. The flavins, FAD and FMN, interact with respective NADPH and pyrimidine substrates and the iron-sulfur centers form an electron transfer wire that links the two active sites that are separated by 56 Å. DPD accepts the common antineoplastic agent 5-fluorouracil as a substrate and so undermines the establishment of efficacious toxicity. Though studied for multiple decades, a precise description of the behavior of the enzyme had remained elusive. It was recently shown that the active form of DPD has the cofactor set of FAD-4(Fe4S4)-FMNH2. This two-electron reduced state is consistent with fewer mechanistic possibilities and data suggests that the instigating and rate determining step in the catalytic cycle is reduction of the pyrimidine substrate that is followed by relatively rapid oxidation of NADPH at the FAD that, via the electron conduit of the 4(Fe4S4) centers, reinstates the FMNH2 cofactor for subsequent catalytic turnover.

Keywords:  Dehydrogenase; Flavin; Hydride; Iron-sulfur; Oxidoreductase; Pyrimidine

DOI:  https://doi.org/10.1016/j.abb.2021.109066
Nucleic Acids Res. 2021 Oct 28. pii: gkab899. [Epub ahead of print]

FAN1's protection against CGG repeat expansion requires its nuclease activity and is FANCD2-independent.

Xiaonan Zhao, Huiyan Lu, Karen Usdin.

The Repeat Expansion Diseases, a large group of human diseases that includes the fragile X-related disorders (FXDs) and Huntington's disease (HD), all result from expansion of a disease-specific microsatellite via a mechanism that is not fully understood. We have previously shown that mismatch repair (MMR) proteins are required for expansion in a mouse model of the FXDs, but that the FANCD2 and FANCI associated nuclease 1 (FAN1), a component of the Fanconi anemia (FA) DNA repair pathway, is protective. FAN1's nuclease activity has been reported to be dispensable for protection against expansion in an HD cell model. However, we show here that in a FXD mouse model a point mutation in the nuclease domain of FAN1 has the same effect on expansion as a null mutation. Furthermore, we show that FAN1 and another nuclease, EXO1, have an additive effect in protecting against MSH3-dependent expansions. Lastly, we show that the loss of FANCD2, a vital component of the Fanconi anemia DNA repair pathway, has no effect on expansions. Thus, FAN1 protects against MSH3-dependent expansions without diverting the expansion intermediates into the canonical FA pathway and this protection depends on FAN1 having an intact nuclease domain.

DOI: https://doi.org/10.1093/nar/gkab899
Sci Adv. 2021 Nov 05. 7(45): eabg4398

Deficiency of replication-independent DNA mismatch repair drives a 5-methylcytosine deamination mutational signature in cancer.

Hu Fang, Xiaoqiang Zhu, Haocheng Yang, Jieun Oh, Jayne A Barbour, Jason W H Wong.

[Figure: see text].

DOI: https://doi.org/10.1126/sciadv.abg4398
Nucleic Acids Res. 2021 Oct 29. pii: gkab956. [Epub ahead of print]

Inferring primase-DNA specific recognition using a data driven approach.

Adam Soffer, Sarah A Eisdorfer, Morya Ifrach, Stefan Ilic, Ariel Afek, Hallel Schussheim, Dan Vilenchik, Barak Akabayov.

DNA-protein interactions play essential roles in all living cells. Understanding of how features embedded in the DNA sequence affect specific interactions with proteins is both challenging and important, since it may contribute to finding the means to regulate metabolic pathways involving DNA-protein interactions. Using a massive experimental benchmark dataset of binding scores for DNA sequences and a machine learning workflow, we describe the binding to DNA of T7 primase, as a model system for specific DNA-protein interactions. Effective binding of T7 primase to its specific DNA recognition sequences triggers the formation of RNA primers that serve as Okazaki fragment start sites during DNA replication.

DOI: https://doi.org/10.1093/nar/gkab956