bims-numges 2021-03-14 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2021–03–14
35 papers selected by
Sean Rudd, Karolinska Institutet

Quantitative imaging of RAD51 expression as a marker of platinum resistance in ovarian cancer.
The equilibrative nucleoside transporter 1 (ENT1) is critical for nucleotide homeostasis and optimal erythropoiesis.
Super-resolution mapping of cellular double-strand break resection complexes during homologous recombination.
Zinc finger protein E4F1 cooperates with PARP-1 and BRG1 to promote DNA double-strand break repair.
The phospho-dependent role of BRCA2 on the maintenance of chromosome integrity.
Mrc1-Dependent Chromatin Compaction Represses DNA Double-Stranded Break Repair by Homologous Recombination Upon Replication Stress.
CDK1 inhibitor controls G2/M phase transition and reverses DNA damage sensitivity.
Polθ promotes the repair of 5'-DNA-protein crosslinks by microhomology-mediated end-joining.
An extended APOBEC3A mutation signature in cancer.
DOCK7 protects against replication stress by promoting RPA stability on chromatin.
Folate metabolism: a re-emerging therapeutic target in haematological cancers.
Bi-allelic MCM10 variants associated with immune dysfunction and cardiomyopathy cause telomere shortening.
Synergistic effects of type I PRMT and PARP inhibitors against non-small cell lung cancer cells.
Discovery of MTR-106 as a highly potent G-quadruplex stabilizer for treating BRCA-deficient cancers.
β-TrCP1 facilitates cell cycle checkpoint activation, DNA repair and cell survival through ablation of β-TrCP2 in response to genotoxic stress.
aniFOUND: analysing the associated proteome and genomic landscape of the repaired nascent non-replicative chromatin.
FUS-dependent liquid-liquid phase separation is important for DNA repair initiation.
Mechanisms of damage tolerance and repair during DNA replication.
c-Src facilitates tumorigenesis by phosphorylating and activating G6PD.
Single-molecule view of coordination in a multi-functional DNA polymerase.
Who gets a license: DNA synthesis in quiescent cells re-entering the cell cycle.
An effective human uracil-DNA glycosylase inhibitor targets the open pre-catalytic active site conformation.
Improved Genome Editing through Inhibition of FANCM and Members of the BTR Dissolvase Complex.
In-depth characterization of ubiquitin turnover in mammalian cells by fluorescence tracking.
E2 Enzymes in Genome Stability: Pulling the Strings Behind the Scenes.
HPF1 and nucleosomes mediate a dramatic switch in activity of PARP1 from polymerase to hydrolase.
Targeting FBXO44/SUV39H1 elicits tumor cell-specific DNA replication stress and viral mimicry.
Regulation of DNA duplication by the mTOR signaling pathway.
Oligomeric complexes formed by Redβ single strand annealing protein in its different DNA bound states.
Ultraviolet-induced RNA:DNA hybrids interfere with chromosomal DNA synthesis.
RNF8-ubiquitinated KMT5A is required for RNF168-induced H2A ubiquitination in response to DNA damage.
ADAR1 RNA editing enzyme regulates R-loop formation and genome stability at telomeres in cancer cells.
Human ORC/MCM density is low in active genes and correlates with replication time but does not delimit initiation zones.
Frustrated folding of guanine quadruplexes in telomeric DNA.
Allele frequency of variants reported to cause adenine phosphoribosyltransferase deficiency.

EMBO Mol Med. 2021 Mar 11. e13366

Quantitative imaging of RAD51 expression as a marker of platinum resistance in ovarian cancer.

Michal M Hoppe, Patrick Jaynes, Joanna D Wardyn, Sai Srinivas Upadhyayula, Tuan Zea Tan, Stefanus Lie, Diana G Z Lim, Brendan N K Pang, Sherlly Lim, Joe P S Yeong, Anthony Karnezis, Derek S Chiu, Samuel Leung, David G Huntsman, Anna S Sedukhina, Ko Sato, Monique D Topp, Clare L Scott, Hyungwon Choi, Naina R Patel, Robert Brown, Stan B Kaye, Jason J Pitt, David S P Tan, Anand D Jeyasekharan.

  Early relapse after platinum chemotherapy in epithelial ovarian cancer (EOC) portends poor survival. A-priori identification of platinum resistance is therefore crucial to improve on standard first-line carboplatin-paclitaxel treatment. The DNA repair pathway homologous recombination (HR) repairs platinum-induced damage, and the HR recombinase RAD51 is overexpressed in cancer. We therefore designed a REMARK-compliant study of pre-treatment RAD51 expression in EOC, using fluorescent quantitative immunohistochemistry (qIHC) to overcome challenges in quantitation of protein expression in situ. In a discovery cohort (n = 284), RAD51-High tumours had shorter progression-free and overall survival compared to RAD51-Low cases in univariate and multivariate analyses. The association of RAD51 with relapse/survival was validated in a carboplatin monotherapy SCOTROC4 clinical trial cohort (n = 264) and was predominantly noted in HR-proficient cancers (Myriad HRDscore < 42). Interestingly, overexpression of RAD51 modified expression of immune-regulatory pathways in vitro, while RAD51-High tumours showed exclusion of cytotoxic T cells in situ. Our findings highlight RAD51 expression as a determinant of platinum resistance and suggest possible roles for therapy to overcome immune exclusion in RAD51-High EOC. The qIHC approach is generalizable to other proteins with a continuum instead of discrete/bimodal expression.

Keywords:  HRD; RAD51; immune exclusion; multiplexed IHC; ovarian cancer

DOI:  https://doi.org/10.15252/emmm.202013366
Blood. 2021 Mar 10. pii: blood.2020007281. [Epub ahead of print]

The equilibrative nucleoside transporter 1 (ENT1) is critical for nucleotide homeostasis and optimal erythropoiesis.

Mahmoud Mikdar, Pedro Gonzalez-Menendez, Xiaoli Cai, Yujin Zhang, Marion Serra, Abdoul Karim Dembélé, Anne-Claire Boschat, Sylvia Sanquer, Cerina Chhuon, Ida Chiara Guerrera, Marc Sitbon, Olivier Hermine, Yves Colin, Caroline Le Van Kim, Sandrina Kinet, Mohandas Narla, Yang Xia, Thierry Peyrard, Naomi Taylor, Slim Azouzi.

The tight regulation of intracellular nucleotides is critical for the self-renewal and lineage specification of hematopoietic stem cells (HSCs). Nucleosides are major metabolite precursors for nucleotide biosynthesis and their availability in HSCs is dependent on their transport through specific membrane transporters. However, the role of nucleoside transporters in the differentiation of HSCs to the erythroid lineage and in red cell biology remains to be fully defined. Here, we show that the absence of the equilibrative nucleoside transporter (ENT1) in human red blood cells (RBCs) with a rare Augustine-null blood type (AUG:-1) is associated with macrocytosis, anisopoikilocytosis, an abnormal nucleotide metabolome, and deregulated protein phosphorylation. A specific role for ENT1 in human erythropoiesis was demonstrated by a defective erythropoiesis of human CD34+ progenitors following shRNA-mediated knockdown of ENT1. Furthermore, genetic deletion of ENT1 in mice was associated with reduced erythroid progenitors in the bone marrow, anemia and macrocytosis. Mechanistically, we found that ENT1-mediated adenosine transport is critical for cAMP homeostasis and the regulation of erythroid transcription factors. Notably, genetic investigation of two ENT1null individuals demonstrated a compensation by a loss-of-function variant in the ABCC4 cyclic nucleotide exporter. Indeed, pharmacological inhibition of ABCC4 in Ent1-/- mice rescued erythropoiesis. Overall, our results highlight the importance of ENT1-mediated nucleotide metabolism in erythropoiesis.

DOI: https://doi.org/10.1182/blood.2020007281
Proc Natl Acad Sci U S A. 2021 Mar 16. pii: e2021963118. [Epub ahead of print]118(11):

Super-resolution mapping of cellular double-strand break resection complexes during homologous recombination.

Donna R Whelan, Eli Rothenberg.

  Homologous recombination (HR) is a major pathway for repair of DNA double-strand breaks (DSBs). The initial step that drives the HR process is resection of DNA at the DSB, during which a multitude of nucleases, mediators, and signaling proteins accumulates at the damage foci in a manner that remains elusive. Using single-molecule localization super-resolution (SR) imaging assays, we specifically visualize the spatiotemporal behavior of key mediator and nuclease proteins as they resect DNA at single-ended double-strand breaks (seDSBs) formed at collapsed replication forks. By characterizing these associations, we reveal the in vivo dynamics of resection complexes involved in generating the long single-stranded DNA (ssDNA) overhang prior to homology search. We show that 53BP1, a protein known to antagonize HR, is recruited to seDSB foci during early resection but is spatially separated from repair activities. Contemporaneously, CtBP-interacting protein (CtIP) and MRN (MRE11-RAD51-NBS1) associate with seDSBs, interacting with each other and BRCA1. The HR nucleases EXO1 and DNA2 are also recruited and colocalize with each other and with the repair helicase Bloom syndrome protein (BLM), demonstrating multiple simultaneous resection events. Quantification of replication protein A (RPA) accumulation and ssDNA generation shows that resection is completed 2 to 4 h after break induction. However, both BRCA1 and BLM persist later into HR, demonstrating potential roles in homology search and repair resolution. Furthermore, we show that initial recruitment of BRCA1 and removal of Ku are largely independent of MRE11 exonuclease activity but dependent on MRE11 endonuclease activity. Combined, our observations provide a detailed description of resection during HR repair.

Keywords:  BRCA1; DNA damage; DNA repair; homologous recombination; resection

DOI:  https://doi.org/10.1073/pnas.2021963118
Proc Natl Acad Sci U S A. 2021 Mar 16. pii: e2019408118. [Epub ahead of print]118(11):

Zinc finger protein E4F1 cooperates with PARP-1 and BRG1 to promote DNA double-strand break repair.

Céline Moison, Jalila Chagraoui, Marie-Christine Caron, Jean-Philippe Gagné, Yan Coulombe, Guy G Poirier, Jean-Yves Masson, Guy Sauvageau.

  Zinc finger (ZnF) proteins represent one of the largest families of human proteins, although most remain uncharacterized. Given that numerous ZnF proteins are able to interact with DNA and poly(ADP ribose), there is growing interest in understanding their mechanism of action in the maintenance of genome integrity. We now report that the ZnF protein E4F transcription factor 1 (E4F1) is an actor in DNA repair. Indeed, E4F1 is rapidly recruited, in a poly(ADP ribose) polymerase (PARP)-dependent manner, to DNA breaks and promotes ATR/CHK1 signaling, DNA-end resection, and subsequent homologous recombination. Moreover, we identify E4F1 as a regulator of the ATP-dependent chromatin remodeling SWI/SNF complex in DNA repair. E4F1 binds to the catalytic subunit BRG1/SMARCA4 and together with PARP-1 mediates its recruitment to DNA lesions. We also report that a proportion of human breast cancers show amplification and overexpression of E4F1 or BRG1 that are mutually exclusive with BRCA1/2 alterations. Together, these results reveal a function of E4F1 in the DNA damage response that orchestrates proper signaling and repair of double-strand breaks and document a molecular mechanism for its essential role in maintaining genome integrity and cell survival.

Keywords:  BRG1; E4F1; PARP-1; breast cancer; double-strand break

DOI:  https://doi.org/10.1073/pnas.2019408118
Cell Cycle. 2021 Mar 10. 1-12

The phospho-dependent role of BRCA2 on the maintenance of chromosome integrity.

Åsa Ehlén, Gaetana Sessa, Sophie Zinn-Justin, Aura Carreira.

  Chromosomal instability is a hallmark of cancer. The tumor suppressor protein BRCA2 performs an important role in the maintenance of genome integrity particularly in interphase; as a mediator of homologous recombination DNA repair pathway, it participates in the repair of DNA double-strand breaks, inter-strand crosslinks and replicative DNA lesions. BRCA2 also protects stalled replication forks from aberrant degradation. Defects in these functions lead to structural chromosomal aberrations. BRCA2 is a large protein containing highly disordered regions that are heavily phosphorylated particularly in mitosis. The functions of these modifications are getting elucidated and reveal emerging activities in chromosome alignment, chromosome segregation and abscission during cell division. Defects in these activities result in numerical chromosomal aberrations. In addition to BRCA2, other factors of the DNA damage response (DDR) participate in mitosis in close association with cell cycle kinases and phosphatases suggesting that the maintenance of genome integrity functions of these factors extends beyond DNA repair. Here we will discuss the regulation of BRCA2 functions through phosphorylation by cell cycle kinases particularly in mitosis, and illustrate with some examples how BRCA2 and other DDR proteins partially rewire their interactions, essentially via phosphorylation, to fulfill mitotic specific functions that ensure chromosome stability.

Keywords:  BRCA2; Phosphorylation; chromosomal instability

DOI:  https://doi.org/10.1080/15384101.2021.1892994
Front Cell Dev Biol. 2021 ;9 630777

Mrc1-Dependent Chromatin Compaction Represses DNA Double-Stranded Break Repair by Homologous Recombination Upon Replication Stress.

Poyuan Xing, Yang Dong, Jingyu Zhao, Zhou Zhou, Zhao Li, Yu Wang, Mengfei Li, Xinghua Zhang, Xuefeng Chen.

  The coordination of DNA replication and repair is critical for the maintenance of genome stability. It has been shown that the Mrc1-mediated S phase checkpoint inhibits DNA double-stranded break (DSB) repair through homologous recombination (HR). How the replication checkpoint inhibits HR remains only partially understood. Here we show that replication stress induces the suppression of both Sgs1/Dna2- and Exo1-mediated resection pathways in an Mrc1-dependent manner. As a result, the loading of the single-stranded DNA binding factor replication protein A (RPA) and Rad51 and DSB repair by HR were severely impaired under replication stress. Notably, the deletion of MRC1 partially restored the recruitment of resection enzymes, DSB end resection, and the loading of RPA and Rad51. The role of Mrc1 in inhibiting DSB end resection is independent of Csm3, Tof1, or Ctf4. Mechanistically, we reveal that replication stress induces global chromatin compaction in a manner partially dependent on Mrc1, and this chromatin compaction limits the access of chromatin remodeling factors and HR proteins, leading to the suppression of HR. Our study reveals a critical role of the Mrc1-dependent chromatin structure change in coordinating DNA replication and recombination under replication stress.

Keywords:  DNA double-stranded breaks; Mrc1; homologous recombination; replication checkpoint; replication stress

DOI:  https://doi.org/10.3389/fcell.2021.630777
Biochem Biophys Res Commun. 2021 Mar 05. pii: S0006-291X(21)00346-6. [Epub ahead of print]550 56-61

CDK1 inhibitor controls G2/M phase transition and reverses DNA damage sensitivity.

Shigeaki Sunada, Hiroko Saito, Doudou Zhang, Zeyu Xu, Yoshio Miki.

  CDK1 plays key roles in cell cycle progression through the G2/M phase transition and activation of homologous recombination (HR) DNA repair pathway. Accordingly, various CDK1 inhibitors have been developed for cancer therapy that induce prolonged G2 arrest and/or sensitize cells to DNA damaging agents in tumor cells, resulting in cell death. However, CDK1 inhibition can induce resistance to DNA damage in certain conditions. The mechanism of different DNA damage sensitivity is not completely understood. We performed immunofluorescence and flow cytometry analysis to investigate DNA damage responses in human tumor cells during low and high dose treatments with RO-3306, a selective CDK1 inhibitor. This comparative investigation demonstrated that RO-3306-induced G2 arrest prevented cells with DNA double-strand breaks from transitioning into the M-phase and that the cells maintained their DNA repair capacity in G2-phase, even under RO-3306 dose-dependent DNA repair inhibition. These findings reveal that CDK1 inhibitor-induced DNA repair inhibition and cell cycle control, which regulate each other during the G2/M phase transition determine the cellular sensitivity to DNA damage, providing insight useful for developing clinical strategies targeting CDK1 inhibition in tumor cells.

Keywords:  CDK inhibitor; DNA damage Response; DSB repair; G2 arrest; Homologous recombination

DOI:  https://doi.org/10.1016/j.bbrc.2021.02.117
Cell Rep. 2021 Mar 09. pii: S2211-1247(21)00134-0. [Epub ahead of print]34(10): 108820

Polθ promotes the repair of 5'-DNA-protein crosslinks by microhomology-mediated end-joining.

Gurushankar Chandramouly, Shuren Liao, Timur Rusanov, Nikita Borisonnik, Marissa L Calbert, Tatiana Kent, Katherine Sullivan-Reed, Umeshkumar Vekariya, Ekaterina Kashkina, Tomasz Skorski, Hong Yan, Richard T Pomerantz.

DNA polymerase θ (Polθ) confers resistance to chemotherapy agents that cause DNA-protein crosslinks (DPCs) at double-strand breaks (DSBs), such as topoisomerase inhibitors. This suggests Polθ might facilitate DPC repair by microhomology-mediated end-joining (MMEJ). Here, we investigate Polθ repair of DSBs carrying DPCs by monitoring MMEJ in Xenopus egg extracts. MMEJ in extracts is dependent on Polθ, exhibits the MMEJ repair signature, and efficiently repairs 5' terminal DPCs independently of non-homologous end-joining and the replisome. We demonstrate that Polθ promotes the repair of 5' terminal DPCs in mammalian cells by using an MMEJ reporter and find that Polθ confers resistance to formaldehyde in addition to topoisomerase inhibitors. Dual deficiency in Polθ and tyrosyl-DNA phosphodiesterase 2 (TDP2) causes severe cellular sensitivity to etoposide, which demonstrates MMEJ as an independent DPC repair pathway. These studies recapitulate MMEJ in vitro and elucidate how Polθ confers resistance to etoposide.

DOI: https://doi.org/10.1016/j.celrep.2021.108820
Nat Commun. 2021 03 11. 12(1): 1602

An extended APOBEC3A mutation signature in cancer.

Adam Langenbucher, Danae Bowen, Ramin Sakhtemani, Elodie Bournique, Jillian F Wise, Lee Zou, Ashok S Bhagwat, Rémi Buisson, Michael S Lawrence.

APOBEC mutagenesis, a major driver of cancer evolution, is known for targeting TpC sites in DNA. Recently, we showed that APOBEC3A (A3A) targets DNA hairpin loops. Here, we show that DNA secondary structure is in fact an orthogonal influence on A3A substrate optimality and, surprisingly, can override the TpC sequence preference. VpC (non-TpC) sites in optimal hairpins can outperform TpC sites as mutational hotspots. This expanded understanding of APOBEC mutagenesis illuminates the genomic Twin Paradox, a puzzling pattern of closely spaced mutation hotspots in cancer genomes, in which one is a canonical TpC site but the other is a VpC site, and double mutants are seen only in trans, suggesting a two-hit driver event. Our results clarify this paradox, revealing that both hotspots in these twins are optimal A3A substrates. Our findings reshape the notion of a mutation signature, highlighting the additive roles played by DNA sequence and DNA structure.

DOI: https://doi.org/10.1038/s41467-021-21891-0
Nucleic Acids Res. 2021 Mar 11. pii: gkab134. [Epub ahead of print]

DOCK7 protects against replication stress by promoting RPA stability on chromatin.

Ming Gao, Guijie Guo, Jinzhou Huang, Xiaonan Hou, Hyoungjun Ham, Wootae Kim, Fei Zhao, Xinyi Tu, Qin Zhou, Chao Zhang, Qian Zhu, Jiaqi Liu, Yuanliang Yan, Zhijie Xu, Ping Yin, Kuntian Luo, John Weroha, Min Deng, Daniel D Billadeau, Zhenkun Lou.

RPA is a critical factor for DNA replication and replication stress response. Surprisingly, we found that chromatin RPA stability is tightly regulated. We report that the GDP/GTP exchange factor DOCK7 acts as a critical replication stress regulator to promote RPA stability on chromatin. DOCK7 is phosphorylated by ATR and then recruited by MDC1 to the chromatin and replication fork during replication stress. DOCK7-mediated Rac1/Cdc42 activation leads to the activation of PAK1, which subsequently phosphorylates RPA1 at S135 and T180 to stabilize chromatin-loaded RPA1 and ensure proper replication stress response. Moreover, DOCK7 is overexpressed in ovarian cancer and depleting DOCK7 sensitizes cancer cells to camptothecin. Taken together, our results highlight a novel role for DOCK7 in regulation of the replication stress response and highlight potential therapeutic targets to overcome chemoresistance in cancer.

DOI: https://doi.org/10.1093/nar/gkab134
Leukemia. 2021 Mar 11.

Folate metabolism: a re-emerging therapeutic target in haematological cancers.

Martha M Zarou, Alexei Vazquez, G Vignir Helgason.

Folate-mediated one carbon (1C) metabolism supports a series of processes that are essential for the cell. Through a number of interlinked reactions happening in the cytosol and mitochondria of the cell, folate metabolism contributes to de novo purine and thymidylate synthesis, to the methionine cycle and redox defence. Targeting the folate metabolism gave rise to modern chemotherapy, through the introduction of antifolates to treat paediatric leukaemia. Since then, antifolates, such as methotrexate and pralatrexate have been used to treat a series of blood cancers in clinic. However, traditional antifolates have many deleterious side effects in normal proliferating tissue, highlighting the urgent need for novel strategies to more selectively target 1C metabolism. Notably, mitochondrial 1C enzymes have been shown to be significantly upregulated in various cancers, making them attractive targets for the development of new chemotherapeutic agents. In this article, we present a detailed overview of folate-mediated 1C metabolism, its importance on cellular level and discuss how targeting folate metabolism has been exploited in blood cancers. Additionally, we explore possible therapeutic strategies that could overcome the limitations of traditional antifolates.

DOI: https://doi.org/10.1038/s41375-021-01189-2
Nat Commun. 2021 Mar 12. 12(1): 1626

Bi-allelic MCM10 variants associated with immune dysfunction and cardiomyopathy cause telomere shortening.

Ryan M Baxley, Wendy Leung, Megan M Schmit, Jacob Peter Matson, Lulu Yin, Marissa K Oram, Liangjun Wang, John Taylor, Jack Hedberg, Colette B Rogers, Adam J Harvey, Debashree Basu, Jenny C Taylor, Alistair T Pagnamenta, Helene Dreau, Jude Craft, Elizabeth Ormondroyd, Hugh Watkins, Eric A Hendrickson, Emily M Mace, Jordan S Orange, Hideki Aihara, Grant S Stewart, Edward Blair, Jeanette Gowen Cook, Anja-Katrin Bielinsky.

Minichromosome maintenance protein 10 (MCM10) is essential for eukaryotic DNA replication. Here, we describe compound heterozygous MCM10 variants in patients with distinctive, but overlapping, clinical phenotypes: natural killer (NK) cell deficiency (NKD) and restrictive cardiomyopathy (RCM) with hypoplasia of the spleen and thymus. To understand the mechanism of MCM10-associated disease, we modeled these variants in human cell lines. MCM10 deficiency causes chronic replication stress that reduces cell viability due to increased genomic instability and telomere erosion. Our data suggest that loss of MCM10 function constrains telomerase activity by accumulating abnormal replication fork structures enriched with single-stranded DNA. Terminally-arrested replication forks in MCM10-deficient cells require endonucleolytic processing by MUS81, as MCM10:MUS81 double mutants display decreased viability and accelerated telomere shortening. We propose that these bi-allelic variants in MCM10 predispose specific cardiac and immune cell lineages to prematurely arrest during differentiation, causing the clinical phenotypes observed in both NKD and RCM patients.

DOI: https://doi.org/10.1038/s41467-021-21878-x
Clin Epigenetics. 2021 Mar 10. 13(1): 54

Synergistic effects of type I PRMT and PARP inhibitors against non-small cell lung cancer cells.

Claudia Dominici, Nicolas Sgarioto, Zhenbao Yu, Laura Sesma-Sanz, Jean-Yves Masson, Stéphane Richard, Noël J-M Raynal.

   BACKGROUND: Non-small cell lung carcinoma (NSCLC) is a leading cause of cancer-related death and represents a major health burden worldwide. Current therapies for NSCLC include chemotherapy, immunotherapy, and targeted molecular agents such as tyrosine kinase inhibitors and epigenetic drugs such as DNA methyltransferase inhibitors. However, survival rates remain low for patients with NSCLC, especially those with metastatic disease. A major cause for therapeutic failure is drug resistance, highlighting the need for novel therapies and combination strategies. Given that epigenetic modulators such as protein arginine methyltransferases (PRMTs) are frequently overexpressed in cancers, PRMT inhibitors are a promising class of cancer therapeutics. We screened a library of epigenetic and anticancer drugs to identify compounds that would synergize with MS023, a type I PRMT inhibitor, in decreasing the viability of methylthioadenosine phosphorylase (MTAP)-negative NSCLC cells.
RESULTS: Among 181 compounds, we identified PARP inhibitors (PARPi) as having a strong synergistic interaction with type I PRMT inhibition. The combination of MS023 and the PARP inhibitor BMN-673 (Talazoparib) demonstrated strong synergistic interaction at low nanomolar concentrations in MTAP-negative NSCLC cell lines A549, SK-LU-1 and HCC4006. The re-introduction of MTAP decreased the sensitivity of the combination therapy in A549. The combination therapy resulted in elevated γ-H2AX foci indicating increased DNA damage causing decreased cell viability. Lastly, the combination therapy was effective in PARPi resistant ovarian cancer cells, suggesting that type I PRMT inhibitors could mitigate PARPi resistance, thus potentially having an important clinical impact for cancer treatment.
CONCLUSIONS: These findings identify a novel cancer drug combination therapy, which is more potent than the separate single-agent therapies. Thus, combining PARP inhibitors and type I PRMT inhibitors represents a new therapeutic opportunity for MTAP-negative NSCLC and certain cancer cells resistant to PARP inhibitors.

Keywords:  And cytotoxic; DNA damage; Drug resistance; MTAP; Non-small cell lung cancer cells; PARP inhibitors; Synergy; Type I PRMT inhibitors

DOI:  https://doi.org/10.1186/s13148-021-01037-1
Invest New Drugs. 2021 Mar 12.

Discovery of MTR-106 as a highly potent G-quadruplex stabilizer for treating BRCA-deficient cancers.

Meng-Zhu Li, Tao Meng, Shan-Shan Song, Xu-Bin Bao, Lan-Ping Ma, Ning Zhang, Ting Yu, Yong-Liang Zhang, Bing Xiong, Jing-Kang Shen, Ze-Hong Miao, Jin-Xue He.

  G-quadruplexes (G4s) are DNA or RNA structures formed by guanine-rich repeating sequences. Recently, G4s have become a highly attractive therapeutic target for BRCA-deficient cancers. Here, we show that a substituted quinolone amide compound, MTR-106, stabilizes DNA G-quadruplexes in vitro. MTR-106 displayed significant antiproliferative activity in homologous recombination repair (HR)-deficient and PARP inhibitor (PARPi)-resistant cancer cells. Moreover, MTR-106 increased DNA damage and promoted cell cycle arrest and apoptosis to inhibit cell growth. Importantly, its oral and i.v. administration significantly impaired tumor growth in BRCA-deficient xenograft mouse models. However, MTR-106 showed modest activity against talazoparib-resistant xenograft models. In rats, the drug rapidly distributes to tissues within 5 min, and its average concentrations were 12-fold higher in the tissues than in the plasma. Overall, we identified MTR-106 as a novel G-quadruplex stabilizer with high tissue distribution, and it may serve as a potential anticancer agent.

Keywords:  BRCA-deficiency; DNA damage; G-quadruplex stabilizer; MTR-106; PARP inhibitor

DOI:  https://doi.org/10.1007/s10637-021-01096-4
J Biol Chem. 2021 Mar 04. pii: S0021-9258(21)00287-8. [Epub ahead of print] 100511

β-TrCP1 facilitates cell cycle checkpoint activation, DNA repair and cell survival through ablation of β-TrCP2 in response to genotoxic stress.

Sehbanul Islam, Parul Dutta, Osheen Sahay, Manas Kumar Santra.

  F-box proteins β-TrCP1 and β-TrCP2 are paralogs present in the human genome. They control several cellular processes including cell cycle and DNA damage signaling. Moreover, it is reported that they facilitate DNA damage-induced accumulation of p53 by directing proteasomal degradation of MDM2, a protein that promotes p53 degradation. However, the individual roles of β-TrCP1 and β-TrCP2 in the genotoxic stress-induced activation of cell cycle checkpoints and DNA damage repair remains largely unknown. Here, using biochemical, molecular biology, flow cytometric, and immunofluorescence techniques, we show that β-TrCP1 and β-TrCP2 communicate during genotoxic stress. We found that expression levels of β-TrCP1 are significantly increased while levels of β-TrCP2 are markedly decreased upon induction of genotoxic stress. Further, our results revealed that DNA damage-induced activation of ATM kinase plays an important role in maintaining the reciprocal expression levels of β-TrCP1 and β-TrCP2 via the phosphorylation of β-TrCP1 at Ser158. Phosphorylated β-TrCP1 potently promotes the proteasomal degradation of β-TrCP2 and MDM2, resulting in the activation of p53. Additionally, β-TrCP1 impedes MDM2 accumulation via abrogation of its lysine 63-linked polyubiquitination by β-TrCP2. Thus, β-TrCP1 helps to arrest cells at the G2/M phase of the cell cycle and promotes DNA repair upon DNA damage through attenuation of β-TrCP2. Collectively, our findings elucidate an intriguing post-translational regulatory mechanism of these two paralogs under genotoxic stress and revealed β-TrCP1 as a key player in maintaining the genome integrity through the attenuation of β-TrCP2 levels in response to genotoxic stress.

Keywords:  Cell cycle; DNA damage; Polyubiquitination; p53

DOI:  https://doi.org/10.1016/j.jbc.2021.100511
Nucleic Acids Res. 2021 Mar 08. pii: gkab144. [Epub ahead of print]

aniFOUND: analysing the associated proteome and genomic landscape of the repaired nascent non-replicative chromatin.

Georgios C Stefos, Eszter Szantai, Dimitris Konstantopoulos, Martina Samiotaki, Maria Fousteri.

Specific capture of chromatin fractions with distinct and well-defined features has emerged as both challenging and a key strategy towards a comprehensive understanding of genome biology. In this context, we developed aniFOUND (accelerated native isolation of factors on unscheduled nascent DNA), an antibody-free method, which can label, capture, map and characterise nascent chromatin fragments that are synthesized in response to specific cues outside S-phase. We used the 'unscheduled' DNA synthesis (UDS) that takes place during the repair of UV-induced DNA lesions and coupled the captured chromatin to high-throughput analytical technologies. By mass-spectrometry we identified several factors with no previously known role in UVC-DNA damage response (DDR) as well as known DDR proteins. We experimentally validated the repair-dependent recruitment of the chromatin remodeller RSF1 and the cohesin-loader NIPBL at sites of UVC-induced photolesions. Developing aniFOUND-seq, a protocol for mapping UDS activity with high resolution, allowed us to monitor the landscape of UVC repair-synthesis events genome wide. We further resolved repair efficacy of the rather unexplored repeated genome, in particular rDNA and telomeres. In summary, aniFOUND delineates the proteome composition and genomic landscape of chromatin loci with specific features by integrating state-of-the-art 'omics' technologies to promote a comprehensive view of their function.

DOI: https://doi.org/10.1093/nar/gkab144
J Cell Biol. 2021 May 03. pii: e202008030. [Epub ahead of print]220(5):

FUS-dependent liquid-liquid phase separation is important for DNA repair initiation.

Brunno R Levone, Silvia C Lenzken, Marco Antonaci, Andreas Maiser, Alexander Rapp, Francesca Conte, Stefan Reber, Jonas Mechtersheimer, Antonella E Ronchi, Oliver Mühlemann, Heinrich Leonhardt, M Cristina Cardoso, Marc-David Ruepp, Silvia M L Barabino.

RNA-binding proteins (RBPs) are emerging as important effectors of the cellular DNA damage response (DDR). The RBP FUS is implicated in RNA metabolism and DNA repair, and it undergoes reversible liquid-liquid phase separation (LLPS) in vitro. Here, we demonstrate that FUS-dependent LLPS is necessary for the initiation of the DDR. Using laser microirradiation in FUS-knockout cells, we show that FUS is required for the recruitment to DNA damage sites of the DDR factors KU80, NBS1, and 53BP1 and of SFPQ, another RBP implicated in the DDR. The relocation of KU80, NBS1, and SFPQ is similarly impaired by LLPS inhibitors, or LLPS-deficient FUS variants. We also show that LLPS is necessary for efficient γH2AX foci formation. Finally, using superresolution structured illumination microscopy, we demonstrate that the absence of FUS impairs the proper arrangement of γH2AX nanofoci into higher-order clusters. These findings demonstrate the early requirement for FUS-dependent LLPS in the activation of the DDR and the proper assembly of DSB repair complexes.

DOI: https://doi.org/10.1083/jcb.202008030
Nucleic Acids Res. 2021 Mar 08. pii: gkab101. [Epub ahead of print]

Mechanisms of damage tolerance and repair during DNA replication.

Mohamed Elsaid Ashour, Nima Mosammaparast.

Accurate duplication of chromosomal DNA is essential for the transmission of genetic information. The DNA replication fork encounters template lesions, physical barriers, transcriptional machinery, and topological barriers that challenge the faithful completion of the replication process. The flexibility of replisomes coupled with tolerance and repair mechanisms counteract these replication fork obstacles. The cell possesses several universal mechanisms that may be activated in response to various replication fork impediments, but it has also evolved ways to counter specific obstacles. In this review, we will discuss these general and specific strategies to counteract different forms of replication associated damage to maintain genomic stability.

DOI: https://doi.org/10.1093/nar/gkab101
Oncogene. 2021 Mar 08.

c-Src facilitates tumorigenesis by phosphorylating and activating G6PD.

Huanhuan Ma, Fengqiong Zhang, Lin Zhou, Tingyan Cao, Dachao Sun, Shixiong Wen, Jinpei Zhu, Zhaoqianyu Xiong, Ming-Tong Tsau, Mei-Ling Cheng, Li-Man Hung, Yanming Zhou, Qinxi Li.

Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limiting enzyme in pentose phosphate pathway (PPP), excessive activation of which has been considered to be involved in tumorigenesis. Here, we show that tyrosine kinase c-Src interacts with and phosphorylates G6PD at Tyr 112. This phosphorylation enhances catalytic activity of G6PD by dramatically decreasing its Km value and increasing its Kcat value for substrate glucose-6-phosphate. Activated G6PD therefore augments the PPP flux for NADPH and ribose-5-phosphate production which is required for detoxification of intracellular reactive oxygen species (ROS) and biosynthesis of cancer cells, and eventually contributes to tumorigenesis. Consistently, c-Src activation is closely correlated with tyrosine phosphorylation and activity of G6PD in clinical colorectal cancer samples. We thus uncover another aspect of c-Src in promoting cell proliferation and tumorigenesis, deepening our understanding of c-Src as a proto-oncogene.

DOI: https://doi.org/10.1038/s41388-021-01673-0
Elife. 2021 Mar 11. pii: e62046. [Epub ahead of print]10

Single-molecule view of coordination in a multi-functional DNA polymerase.

Raymond F Pauszek, Rajan Lamichhane, Arishma Rajkarnikar Singh, David P Millar.

  Replication and repair of genomic DNA requires the actions of multiple enzymatic functions that must be coordinated in order to ensure efficient and accurate product formation. Here, we have used single-molecule FRET microscopy to investigate the physical basis of functional coordination in DNA polymerase I (Pol I) from Escherichia coli, a key enzyme involved in lagging-strand replication and base excision repair. Pol I contains active sites for template-directed DNA polymerization and 5' flap processing in separate domains. We show that a DNA substrate can spontaneously transfer between polymerase and 5' nuclease domains during a single encounter with Pol I. Additionally, we show that the flexibly tethered 5' nuclease domain adopts different positions within Pol I-DNA complexes, depending on the nature of the DNA substrate. Our results reveal the structural dynamics that underlie functional coordination in Pol I and are likely relevant to other multi-functional DNA polymerases.

Keywords:  DNA polymerase; E. coli; functional coordination; molecular biophysics; single-molecule FRET; structural biology

DOI:  https://doi.org/10.7554/eLife.62046
Curr Genet. 2021 Mar 08.

Who gets a license: DNA synthesis in quiescent cells re-entering the cell cycle.

Po-Hsuen Lee, Mary Ann Osley.

  The precise regulation of the entry into S phase is critical for preventing genome instability. The first step in the initiation of eukaryotic DNA synthesis occurs in G1 phase cells and involves the loading of the conserved MCM helicase onto multiple origins of replication in a process known as origin licensing. In proliferating metazoan cells, an origin-licensing checkpoint delays initiation until high levels of MCM loading occur, with excess origins being licensed. One function of this checkpoint is to ensure that S phase can be completed in the face of replication stress by activation of dormant MCM bound origins. However, when both metazoan and yeast cells enter S phase from quiescence or G0 phase, a non-growing but reversible cell cycle state, origins are significantly under-licensed. In metazoan cells, under-licensing is the result of a compromised origin-licensing checkpoint. In budding yeast, our study has revealed that under-licensing can be attributed to the chromatin structure at a class of origins that is inhibitory to the binding of MCM. Thus, defects in multiple pathways may contribute to the failure to fully license origins in quiescent cells re-entering the cell cycle, thereby promoting a higher risk of genome instability.

Keywords:  Cell cycle; Chromatin structure; Origin licensing; Quiescent cells

DOI:  https://doi.org/10.1007/s00294-021-01170-7
Prog Biophys Mol Biol. 2021 Mar 03. pii: S0079-6107(21)00009-2. [Epub ahead of print]

An effective human uracil-DNA glycosylase inhibitor targets the open pre-catalytic active site conformation.

My T Nguyen, Davide Moiani, Zamal Ahmed, Andrew S Arvai, Sarita Namjoshi, Dave S Shin, Yuriy Fedorov, Edward J Selvik, Darin E Jones, John Pink, Yan Yan, Daniel J Laverty, Zachary D Nagel, John A Tainer, Stanton L Gerson.

  Human uracil DNA-glycosylase (UDG) is the prototypic and first identified DNA glycosylase with a vital role in removing deaminated cytosine and incorporated uracil and 5-fluorouracil (5-FU) from DNA. UDG depletion sensitizes cells to high APOBEC3B deaminase and to pemetrexed (PEM) and floxuridine (5-FdU), which are toxic to tumor cells through incorporation of uracil and 5-FU into DNA. To identify small-molecule UDG inhibitors for pre-clinical evaluation, we optimized biochemical screening of a selected diversity collection of >3,000 small-molecules. We found aurintricarboxylic acid (ATA) as an inhibitor of purified UDG at an initial calculated IC50 < 100 nM. Subsequent enzymatic assays confirmed effective ATA inhibition but with an IC50 of 700 nM and showed direct binding to the human UDG with a KD of <700 nM. ATA displays preferential, dose-dependent binding to purified human UDG compared to human 8-oxoguanine DNA glycosylase. ATA did not bind uracil-containing DNA at these concentrations. Yet, combined crystal structure and in silico docking results unveil ATA interactions with the DNA binding channel and uracil-binding pocket in an open, destabilized UDG conformation. Biologically relevant ATA inhibition of UDG was measured in cell lysates from human DLD1 colon cancer cells and in MCF-7 breast cancer cells using a host cell reactivation assay. Collective findings provide proof-of-principle for development of an ATA-based chemotype and "door stopper" strategy targeting inhibitor binding to a destabilized, open pre-catalytic glycosylase conformation that prevents active site closing for functional DNA binding and nucleotide flipping needed to excise altered bases in DNA.

Keywords:  Cancer; DNA repair; Ligand; Structures; UDG

DOI:  https://doi.org/10.1016/j.pbiomolbio.2021.02.004
Mol Ther. 2021 Mar 03. pii: S1525-0016(20)30554-2. [Epub ahead of print]29(3): 1016-1027

Improved Genome Editing through Inhibition of FANCM and Members of the BTR Dissolvase Complex.

Gustavo de Alencastro, Francesco Puzzo, Mara Pavel-Dinu, Feijie Zhang, Sirika Pillay, Karim Majzoub, Matthew Tiffany, Hagoon Jang, Adam Sheikali, M Kyle Cromer, Ruhikanta Meetei, Jan E Carette, Matthew H Porteus, Katja Pekrun, Mark A Kay.

  Recombinant adeno-associated virus (rAAV) vectors have the unique property of being able to perform genomic targeted integration (TI) without inducing a double-strand break (DSB). In order to improve our understanding of the mechanism behind TI mediated by AAV and improve its efficiency, we performed an unbiased genetic screen in human cells using a promoterless AAV-homologous recombination (AAV-HR) vector system. We identified that the inhibition of the Fanconi anemia complementation group M (FANCM) protein enhanced AAV-HR-mediated TI efficiencies in different cultured human cells by ∼6- to 9-fold. The combined knockdown of the FANCM and two proteins also associated with the FANCM complex, RecQ-mediated genome instability 1 (RMI1) and Bloom DNA helicase (BLM) from the BLM-topoisomerase IIIα (TOP3A)-RMI (BTR) dissolvase complex (RMI1, having also been identified in our screen), led to the enhancement of AAV-HR-mediated TI up to ∼17 times. AAV-HR-mediated TI in the presence of a nuclease (CRISPR-Cas9) was also increased by ∼1.5- to 2-fold in FANCM and RMI1 knockout cells, respectively. Furthermore, knockdown of FANCM in human CD34+ hematopoietic stem and progenitor cells (HSPCs) increased AAV-HR-mediated TI by ∼3.5-fold. This study expands our knowledge on the mechanisms related to AAV-mediated TI, and it highlights new pathways that might be manipulated for future improvements in AAV-HR-mediated TI.

Keywords:  AAV; AAV-HR; CRISPR/Cas9; GeneRide; genome editing; homologous recombination; nuclease-free; targeted integration

DOI:  https://doi.org/10.1016/j.ymthe.2020.10.020
Cell Chem Biol. 2021 Mar 02. pii: S2451-9456(21)00094-5. [Epub ahead of print]

In-depth characterization of ubiquitin turnover in mammalian cells by fluorescence tracking.

Anna A Kudriaeva, Ido Livneh, Mikhail S Baranov, Rustam H Ziganshin, Alexey E Tupikin, Snizhana O Zaitseva, Marsel R Kabilov, Aaron Ciechanover, Alexey A Belogurov.

  Despite almost 40 years having passed from the initial discovery of ubiquitin (Ub), fundamental questions related to its intracellular metabolism are still enigmatic. Here we utilized fluorescent tracking for monitoring ubiquitin turnover in mammalian cells, resulting in obtaining qualitatively new data. In the present study we report (1) short Ub half-life estimated as 4 h; (2) for a median of six Ub molecules per substrate as a dynamic equilibrium between Ub ligases and deubiquitinated enzymes (DUBs); (3) loss on average of one Ub molecule per four acts of engagement of polyubiquitinated substrate by the proteasome; (4) direct correlation between incorporation of Ub into the distinct type of chains and Ub half-life; and (5) critical influence of the single lysine residue K27 on the stability of the whole Ub molecule. Concluding, our data provide a comprehensive understanding of ubiquitin-proteasome system dynamics on the previously unreachable state of the art.

Keywords:  half-life; metabolism; polyubiquitin chain length; proteasome; resorufin ligase; stability; ubiquitin

DOI:  https://doi.org/10.1016/j.chembiol.2021.02.009
Trends Cell Biol. 2021 Mar 05. pii: S0962-8924(21)00024-6. [Epub ahead of print]

E2 Enzymes in Genome Stability: Pulling the Strings Behind the Scenes.

Hugh C Osborne, Elsa Irving, Josep V Forment, Christine K Schmidt.

  Ubiquitin and ubiquitin-like proteins (UBLs) function as critical post-translational modifiers in the maintenance of genome stability. Ubiquitin/UBL-conjugating enzymes (E2s) are responsible, as part of a wider enzymatic cascade, for transferring single moieties or polychains of ubiquitin/UBLs to one or multiple residues on substrate proteins. Recent advances in structural and mechanistic understanding of how ubiquitin/UBL substrate attachment is orchestrated indicate that E2s can exert control over chain topology, substrate-site specificity, and downstream physiological effects to help maintain genome stability. Drug discovery efforts have typically focussed on modulating other members of the ubiquitin/UBL cascades or the ubiquitin-proteasome system. Here, we review the current standing of E2s in genome stability and revisit their potential as pharmacological targets for developing novel anti-cancer therapies.

Keywords:  DNA repair; E2-conjugating enzymes (E2s); cell cycle; genome stability; telomeres; ubiquitin and ubiquitin-like proteins (UBLs)

DOI:  https://doi.org/10.1016/j.tcb.2021.01.009
Elife. 2021 Mar 08. pii: e65773. [Epub ahead of print]10

HPF1 and nucleosomes mediate a dramatic switch in activity of PARP1 from polymerase to hydrolase.

Johannes Rudolph, Genevieve Roberts, Uma M Muthurajan, Karolin Luger.

  Poly(ADP-ribose) polymerase 1 (PARP1) is an important player in the response to DNA damage. Recently, histone PARylation factor (HPF1) was shown to be a critical modulator of the activity of PARP1 by facilitating PARylation of histones and redirecting the target amino acid specificity from acidic to serine residues. Here we investigate the mechanism and specific consequences of HPF1-mediated PARylation using nucleosomes as both activators and substrates for PARP1. HPF1 provides that catalytic base Glu284 to substantially redirect PARylation by PARP1 such that the histones in nucleosomes become the primary recipients of PAR chains. Surprisingly, HPF1 partitions most of the reaction product to free ADPR, resulting in much shorter PAR chains compared to reactions in the absence of HPF1. This HPF1-mediated switch from polymerase to hydrolase has important implications for the PARP1-mediated response to DNA damage and raises interesting new questions about the role of intracellular ADPR and depletion of NAD+.

Keywords:  biochemistry; chemical biology; human

DOI:  https://doi.org/10.7554/eLife.65773
Cell Stress. 2021 Feb 18. 5(3): 37-39

Targeting FBXO44/SUV39H1 elicits tumor cell-specific DNA replication stress and viral mimicry.

Jia Z Shen, Charles Spruck.

  Repetitive elements (REs) are normally transcriptionally silenced in somatic cells by repressive epigenetic modifications, which are thought to include DNA methylation and histone modifications such as deacetylation, H3K9me3, and H4K20me3. Although, it is unclear how RE silencing is maintained through DNA replication cycles in rapidly growing cancer cells. On the other hand, the reactivation of endogenous retroelements beyond a threshold level of tolerance in cancer cells, such as by treatment with DNA demethylating agents or HDAC or LSD1 inhibitors, can induce viral mimicry responses that augment certain cancer therapies, including immunotherapy. However, these agents can also affect normal cells presenting obvious side effects. Therefore, uncovering cancer cell-specific RE silencing mechanisms could provide a basis for the development of a new generation of cancer immunotherapy drugs. In our study (Shen et al. (2020), Cell, doi: 10.1016/j.cell.2020.11.042), through a high-content RNAi screen we identified FBXO44 as a key regulator of H3K9me3-mediated transcriptional silencing of REs in cancer cells. Inhibition of FBXO44 or its co-factor SUV39H1 stimulated antiviral pathways and interferon (IFN) signaling and induced replication stress and DNA double-strand breaks (DSBs) in cancer cells, leading to restricted tumor growth and synergy with anti-PD-1 therapy (Figure 1). Figure 1FIGURE 1: Graphical representation of this study.FBXO44/SUV39H1 targeting activates REs that elicit DNA replication stress and viral mimicry in cancer cells, leading to tumor growth arrest and enhanced immunotherapy response.

Keywords:  FBXO44; SUV39H1; immunotherapy; replication stress; viral mimicry

DOI:  https://doi.org/10.15698/cst2021.03.245
Cell Cycle. 2021 Mar 10. 1-10

Regulation of DNA duplication by the mTOR signaling pathway.

Zhengfu He, Peter J Houghton, Terence M Williams, Changxian Shen.

  Accurate and complete DNA replication and separation are essential for genetic information inheritance and organism maintenance. Errors in DNA duplication are the main source of genetic instability. Understanding DNA duplication regulation is the key to elucidate the mechanisms and find treatment strategies for human genetic disorders, especially cancer. The mechanistic target of rapamycin (mTOR) is a central regulator of cell growth and proliferation by integrating and processing extracellular and intracellular signals to monitor the well-being of cell physiology. mTOR signaling dysregulation is associated with many human diseases including cancer and diabetes. Emerging evidence has demonstrated that mTOR signaling plays a key role in DNA duplication. We herein review the current knowledge of mTOR signaling in the regulation of DNA replication origin licensing, replication fork progression, and stabilization.

Keywords:  DNA repair; cell cycle; mTOR; DNA replication origin licensing; replication fork progression; replication fork stabilization

DOI:  https://doi.org/10.1080/15384101.2021.1897271
Nucleic Acids Res. 2021 Mar 08. pii: gkab125. [Epub ahead of print]

Oligomeric complexes formed by Redβ single strand annealing protein in its different DNA bound states.

Brian J Caldwell, Andrew Norris, Ekaterina Zakharova, Christopher E Smith, Carter T Wheat, Deepanshu Choudhary, Marcos Sotomayor, Vicki H Wysocki, Charles E Bell.

Redβ is a single strand annealing protein from bacteriophage λ that binds loosely to ssDNA, not at all to pre-formed dsDNA, but tightly to a duplex intermediate of annealing. As viewed by electron microscopy, Redβ forms oligomeric rings on ssDNA substrate, and helical filaments on the annealed duplex intermediate. However, it is not clear if these are the functional forms of the protein in vivo. We have used size-exclusion chromatography coupled with multi-angle light scattering, analytical ultracentrifugation and native mass spectrometry (nMS) to characterize the size of the oligomers formed by Redβ in its different DNA-bound states. The nMS data, which resolve species with the highest resolution, reveal that Redβ forms an oligomer of 12 subunits in the absence of DNA, complexes ranging from 4 to 14 subunits on 38-mer ssDNA, and a much more distinct and stable complex of 11 subunits on 38-mer annealed duplex. We also measure the concentration of Redβ in cells active for recombination and find it to range from 7 to 27 μM. Collectively, these data provide new insights into the dynamic nature of the complex on ssDNA, and the more stable and defined complex on annealed duplex.

DOI: https://doi.org/10.1093/nar/gkab125
Nucleic Acids Res. 2021 Mar 10. pii: gkab147. [Epub ahead of print]

Ultraviolet-induced RNA:DNA hybrids interfere with chromosomal DNA synthesis.

Elena A Kouzminova, Andrei Kuzminov.

Ultraviolet (UV) induces pyrimidine dimers (PDs) in DNA and replication-dependent fragmentation in chromosomes. The rnhAB mutants in Escherichia coli, accumulating R-loops and single DNA-rNs, are generally resistant to DNA damage, but are surprisingly UV-sensitive, even though they remove PDs normally, suggesting irreparable chromosome lesions. We show here that the RNase H defect does not cause additional chromosome fragmentation after UV, but inhibits DNA synthesis after replication restart. Genetic analysis implies formation of R-loop-anchored transcription elongation complexes (R-loop-aTECs) in UV-irradiated rnhAB mutants, predicting that their chromosomal DNA will accumulate: (i) RNA:DNA hybrids; (ii) a few slow-to-remove PDs. We confirm both features and also find that both, surprisingly, depend on replication restart. Finally, enriching for the UV-induced RNA:DNA hybrids in the rnhAB uvrA mutants also co-enriches for PDs, showing their co-residence in the same structures. We propose that PD-triggered R-loop-aTECs block head-on replication in RNase H-deficient mutants.

DOI: https://doi.org/10.1093/nar/gkab147
FASEB J. 2021 Apr;35(4): e21326

RNF8-ubiquitinated KMT5A is required for RNF168-induced H2A ubiquitination in response to DNA damage.

Xiaopeng Lu, Min Xu, Qian Zhu, Jun Zhang, Ge Liu, Yantao Bao, Luo Gu, Yuan Tian, He Wen, Wei-Guo Zhu.

  Histone modifications play critical roles in DNA damage repair to safeguard genome integrity. However, how different histone modifiers coordinate to build appropriate chromatin context for DNA damage repair is largely unknown. Here, we report a novel interplay between the histone methyltransferase KMT5A and two E3 ligases RNF8 and RNF168 in establishing the histone modification status for DNA damage repair. KMT5A is a newly identified substrate of RNF8 in vitro and in vivo. In response to DNA double-strand breaks (DSBs), RNF8 promotes KMT5A recruitment onto damaged chromatin in a ubiquitination-dependent manner. RNF8-induced KMT5A ubiquitination increases the binding capacity of KMT5A to RNF168. Interestingly, KMT5A not only drives a local increase in H4K20 monomethylation at DSBs, but also promotes RNF168's activity in catalyzing H2A ubiquitination. We proved that the interaction between the H2A acidic patch and KMT5A R188/R189 residues is critical for KMT5A-mediated regulation of H2A ubiquitination. Taken together, our results highlight a new role for KMT5A in linking H4K20 methylation and H2A ubiquitination and provide insight into the histone modification network during DNA damage repair.

Keywords:  DNA double-strand breaks; KMT5A; RNF168; RNF8; histone ubiquitination

DOI:  https://doi.org/10.1096/fj.202002234R
Nat Commun. 2021 Mar 12. 12(1): 1654

ADAR1 RNA editing enzyme regulates R-loop formation and genome stability at telomeres in cancer cells.

Yusuke Shiromoto, Masayuki Sakurai, Moeko Minakuchi, Kentaro Ariyoshi, Kazuko Nishikura.

ADAR1 is involved in adenosine-to-inosine RNA editing. The cytoplasmic ADAR1p150 edits 3'UTR double-stranded RNAs and thereby suppresses induction of interferons. Loss of this ADAR1p150 function underlies the embryonic lethality of Adar1 null mice, pathogenesis of the severe autoimmune disease Aicardi-Goutières syndrome, and the resistance developed in cancers to immune checkpoint blockade. In contrast, the biological functions of the nuclear-localized ADAR1p110 remain largely unknown. Here, we report that ADAR1p110 regulates R-loop formation and genome stability at telomeres in cancer cells carrying non-canonical variants of telomeric repeats. ADAR1p110 edits the A-C mismatches within RNA:DNA hybrids formed between canonical and non-canonical variant repeats. Editing of A-C mismatches to I:C matched pairs facilitates resolution of telomeric R-loops by RNase H2. This ADAR1p110-dependent control of telomeric R-loops is required for continued proliferation of telomerase-reactivated cancer cells, revealing the pro-oncogenic nature of ADAR1p110 and identifying ADAR1 as a promising therapeutic target of telomerase positive cancers.

DOI: https://doi.org/10.1038/s41467-021-21921-x
Elife. 2021 Mar 08. pii: e62161. [Epub ahead of print]10

Human ORC/MCM density is low in active genes and correlates with replication time but does not delimit initiation zones.

Nina Kirstein, Alexander Buschle, Xia Wu, Stefan Krebs, Helmut Blum, Elisabeth Kremmer, Ina M Vorberg, Wolfgang Hammerschmidt, Laurent Lacroix, Olivier Hyrien, Benjamin Audit, Aloys Schepers.

  Eukaryotic DNA replication initiates during S phase from origins that have been licensed in the preceding G1 phase. Here, we compare ChIP-seq profiles of the licensing factors Orc2, Orc3, Mcm3, and Mcm7 with gene expression, replication timing and fork directionality profiles obtained by RNA-seq, Repli-seq and OK-seq. ORC and MCM are significantly and homogeneously depleted from transcribed genes, enriched at gene promoters, and more abundant in early- than in late-replicating domains. Surprisingly, after controlling these variables, no difference in ORC/MCM density is detected between initiation zones, termination zones, unidirectionally replicating and randomly replicating regions. Therefore, ORC/MCM density correlates with replication timing but does not solely regulate the probability of replication initiation. Interestingly, H4K20me3, a histone modification proposed to facilitate late origin licensing, was enriched in late replicating initiation zones and gene deserts of stochastic replication fork direction. We discuss potential mechanisms specifying when and where replication initiates in human cells.

Keywords:  chromosomes; gene expression; human; mouse

DOI:  https://doi.org/10.7554/eLife.62161
Nucleic Acids Res. 2021 Mar 08. pii: gkab140. [Epub ahead of print]

Frustrated folding of guanine quadruplexes in telomeric DNA.

Simone Carrino, Christopher D Hennecker, Ana C Murrieta, Anthony Mittermaier.

Human chromosomes terminate in long, single-stranded, DNA overhangs of the repetitive sequence (TTAGGG)n. Sets of four adjacent TTAGGG repeats can fold into guanine quadruplexes (GQ), four-stranded structures that are implicated in telomere maintenance and cell immortalization and are targets in cancer therapy. Isolated GQs have been studied in detail, however much less is known about folding in long repeat sequences. Such chains adopt an enormous number of configurations containing various arrangements of GQs and unfolded gaps, leading to a highly frustrated energy landscape. To better understand this phenomenon, we used mutagenesis, thermal melting, and global analysis to determine stability, kinetic, and cooperativity parameters for GQ folding within chains containing 8-12 TTAGGG repeats. We then used these parameters to simulate the folding of 32-repeat chains, more representative of intact telomeres. We found that a combination of folding frustration and negative cooperativity between adjacent GQs increases TTAGGG unfolding by up to 40-fold, providing an abundance of unfolded gaps that are potential binding sites for telomeric proteins. This effect was most pronounced at the chain termini, which could promote telomere extension by telomerase. We conclude that folding frustration is an important and largely overlooked factor controlling the structure of telomeric DNA.

DOI: https://doi.org/10.1093/nar/gkab140
Eur J Hum Genet. 2021 Mar 11.

Allele frequency of variants reported to cause adenine phosphoribosyltransferase deficiency.

Hrafnhildur L Runolfsdottir, John A Sayer, Olafur S Indridason, Vidar O Edvardsson, Brynjar O Jensson, Gudny A Arnadottir, Sigurjon A Gudjonsson, Run Fridriksdottir, Hildigunnur Katrinardottir, Daniel Gudbjartsson, Unnur Thorsteinsdottir, Patrick Sulem, Kari Stefansson, Runolfur Palsson.

Adenine phosphoribosyltransferase deficiency is a rare, autosomal recessive disorder of purine metabolism that causes nephrolithiasis and progressive chronic kidney disease. The small number of reported cases indicates an extremely low prevalence, although it has been suggested that missed diagnoses may play a role. We assessed the prevalence of APRT deficiency based on the frequency of causally-related APRT sequence variants in a diverse set of large genomic databases. A thorough search was carried out for all APRT variants that have been confirmed as pathogenic under recessive mode of inheritance, and the frequency of the identified variants examined in six population genomic databases: the deCODE genetics database, the UK Biobank, the 100,000 Genomes Project, the Genome Aggregation Database, the Human Genetic Variation Database and the Korean Variant Archive. The estimated frequency of homozygous genotypes was calculated using the Hardy-Weinberg equation. Sixty-two pathogenic APRT variants were identified, including six novel variants. Most common were the missense variants c.407T>C (p.(Met136Thr)) in Japan and c.194A>T (p.(Asp65Val)) in Iceland, as well as the splice-site variant c.400 + 2dup (p.(Ala108Glufs*3)) in the European population. Twenty-nine variants were detected in at least one of the six genomic databases. The highest cumulative minor allele frequency (cMAF) of pathogenic variants outside of Japan and Iceland was observed in the Irish population (0.2%), though no APRT deficiency cases have been reported in Ireland. The large number of cases in Japan and Iceland is consistent with a founder effect in these populations. There is no evidence for widespread underdiagnosis based on the current analysis.

DOI: https://doi.org/10.1038/s41431-020-00805-6