bims-numges 2021-09-19 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2021–09–19
27 papers selected by
Sean Rudd, Karolinska Institutet

REV1-Polζ maintains the viability of homologous recombination-deficient cancer cells through mutagenic repair of PRIMPOL-dependent ssDNA gaps.
DNA polymerase zeta contributes to heterochromatin replication to prevent genome instability.
Replication of the Mammalian Genome by Replisomes Specific for Euchromatin and Heterochromatin.
The deubiquitinating enzyme USP37 enhances CHK1 activity to promote the cellular response to replication stress.
Mechanism, cellular functions and cancer roles of polymerase-theta-mediated DNA end joining.
A mechanism for Rad53 to couple leading- and lagging-strand DNA synthesis under replication stress in budding yeast.
ADAR-mediated RNA editing of DNA:RNA hybrids is required for DNA double strand break repair.
Molecular mechanisms involved in DNA repair in human cancers: An overview of PI3k/Akt signaling and PIKKs crosstalk.
The chromatin remodeler Chd1 supports MRX and Exo1 functions in resection of DNA double-strand breaks.
The CRL4DTL E3 ligase induces degradation of the DNA replication initiation factor TICRR/TRESLIN specifically during S phase.
MAD2L2 dimerization and TRIP13 control shieldin activity in DNA repair.
DNA polymerase delta Exo domain stabilizes mononucleotide microsatellites in human cells.
Mild phenotype of knockouts of the major apurinic/apyrimidinic endonuclease APEX1 in a non-cancer human cell line.
DNA damage-induced phosphorylation of CtIP at a conserved ATM/ATR site T855 promotes lymphomagenesis in mice.
Epigenetic regulation of Fanconi anemia genes implicates PRMT5 blockage as a strategy for tumor chemosensitization.
Versatile cell-based assay for measuring DNA alkylation damage and its repair.
APOBEC3C, a nucleolar protein induced by genotoxins, is excluded from DNA damage sites.
Structural basis of human transcription-DNA repair coupling.
Poly (ADP-ribose) polymerase-1 (PARP1) as a therapeutic target in acute myeloid leukemia and myelodysplastic syndrome.
SLC5A3-dependent myo-inositol auxotrophy in acute myeloid leukemia.
Single-Molecule Insights Into the Dynamics of Replicative Helicases.
Exploring the ATR-CHK1 pathway in the response of doxorubicin-induced DNA damages in acute lymphoblastic leukemia cells.
SAMHD1 Phosphorylation at T592 Regulates Cellular Localization and S-phase Progression.
Nucleotide Metabolism Behind Epigenetics.
EMP3 negatively modulates breast cancer cell DNA replication, DNA damage repair, and stem-like properties.
SMARCA4/2 loss inhibits chemotherapy-induced apoptosis by restricting IP3R3-mediated Ca2+ flux to mitochondria.
Targeting DNA repair pathways to overcome cancer drug resistance.

Mol Cell. 2021 Sep 07. pii: S1097-2765(21)00683-3. [Epub ahead of print]

REV1-Polζ maintains the viability of homologous recombination-deficient cancer cells through mutagenic repair of PRIMPOL-dependent ssDNA gaps.

Angelo Taglialatela, Giuseppe Leuzzi, Vincenzo Sannino, Raquel Cuella-Martin, Jen-Wei Huang, Foon Wu-Baer, Richard Baer, Vincenzo Costanzo, Alberto Ciccia.

  BRCA1/2 mutant tumor cells display an elevated mutation burden, the etiology of which remains unclear. Here, we report that these cells accumulate ssDNA gaps and spontaneous mutations during unperturbed DNA replication due to repriming by the DNA primase-polymerase PRIMPOL. Gap accumulation requires the DNA glycosylase SMUG1 and is exacerbated by depletion of the translesion synthesis (TLS) factor RAD18 or inhibition of the error-prone TLS polymerase complex REV1-Polζ by the small molecule JH-RE-06. JH-RE-06 treatment of BRCA1/2-deficient cells results in reduced mutation rates and PRIMPOL- and SMUG1-dependent loss of viability. Through cellular and animal studies, we demonstrate that JH-RE-06 is preferentially toxic toward HR-deficient cancer cells. Furthermore, JH-RE-06 remains effective toward PARP inhibitor (PARPi)-resistant BRCA1 mutant cells and displays additive toxicity with crosslinking agents or PARPi. Collectively, these studies identify a protective and mutagenic role for REV1-Polζ in BRCA1/2 mutant cells and provide the rationale for using REV1-Polζ inhibitors to treat BRCA1/2 mutant tumors.

Keywords:  BRCA1 and BRCA2; DNA repriming; PRIMPOL; RAD18; REV1 and Polζ; breast and ovarian cancer; homologous recombination; ssDNA gaps; synthetic lethality; translesion synthesis

DOI:  https://doi.org/10.1016/j.molcel.2021.08.016
EMBO J. 2021 Sep 17. e104543

DNA polymerase zeta contributes to heterochromatin replication to prevent genome instability.

Barbara Ben Yamin, Sana Ahmed-Seghir, Junya Tomida, Emmanuelle Despras, Caroline Pouvelle, Andrey Yurchenko, Jordane Goulas, Raphael Corre, Quentin Delacour, Nathalie Droin, Philippe Dessen, Didier Goidin, Sabine S Lange, Sarita Bhetawal, Maria Teresa Mitjavila-Garcia, Giuseppe Baldacci, Sergey Nikolaev, Jean Charles Cadoret, Richard D Wood, Patricia L Kannouche.

  The DNA polymerase zeta (Polζ) plays a critical role in bypassing DNA damage. REV3L, the catalytic subunit of Polζ, is also essential in mouse embryonic development and cell proliferation for reasons that remain incompletely understood. In this study, we reveal that REV3L protein interacts with heterochromatin components including repressive histone marks and localizes in pericentromeric regions through direct interaction with HP1 dimer. We demonstrate that Polζ/REV3L ensures progression of replication forks through difficult-to-replicate pericentromeric heterochromatin, thereby preventing spontaneous chromosome break formation. We also find that Rev3l-deficient cells are compromised in the repair of heterochromatin-associated double-stranded breaks, eliciting deletions in late-replicating regions. Lack of REV3L leads to further consequences that may be ascribed to heterochromatin replication and repair-associated functions of Polζ, with a disruption of the temporal replication program at specific loci. This is correlated with changes in epigenetic landscape and transcriptional control of developmentally regulated genes. These results reveal a new function of Polζ in preventing chromosome instability during replication of heterochromatic regions.

Keywords:  DNA replication; REV3L; TLS polymerase; heterochromatin; replication timing

DOI:  https://doi.org/10.15252/embj.2020104543
Front Cell Dev Biol. 2021 ;9 729265

Replication of the Mammalian Genome by Replisomes Specific for Euchromatin and Heterochromatin.

Jing Zhang, Marina A Bellani, Jing Huang, Ryan C James, Durga Pokharel, Julia Gichimu, Himabindu Gali, Grant Stewart, Michael M Seidman.

  Replisomes follow a schedule in which replication of DNA in euchromatin is early in S phase while sequences in heterochromatin replicate late. Impediments to DNA replication, referred to as replication stress, can stall replication forks triggering activation of the ATR kinase and downstream pathways. While there is substantial literature on the local consequences of replisome stalling-double strand breaks, reversed forks, or genomic rearrangements-there is limited understanding of the determinants of replisome stalling vs. continued progression. Although many proteins are recruited to stalled replisomes, current models assume a single species of "stressed" replisome, independent of genomic location. Here we describe our approach to visualizing replication fork encounters with the potent block imposed by a DNA interstrand crosslink (ICL) and our discovery of an unexpected pathway of replication restart (traverse) past an intact ICL. Additionally, we found two biochemically distinct replisomes distinguished by activity in different stages of S phase and chromatin environment. Each contains different proteins that contribute to ICL traverse.

Keywords:  CMG; DONSON; FANCM; GINS; replication stress; replisome

DOI:  https://doi.org/10.3389/fcell.2021.729265
J Biol Chem. 2021 Sep 09. pii: S0021-9258(21)00986-8. [Epub ahead of print] 101184

The deubiquitinating enzyme USP37 enhances CHK1 activity to promote the cellular response to replication stress.

Benjamin R Stromberg, Mayank Singh, Adrian E Torres, Amy C Burrows, Debjani Pal, Christine Insinna, Yosup Rhee, Andrew S Dickson, Christopher J Westlake, Matthew K Summers.

  The deubiquitinating enzyme USP37 is known to contribute to timely onset of S-phase and progression of mitosis. However, it is not clear if USP37 is required beyond S-phase entry despite expression and activity of USP37 peaking within S-phase. We have utilized flow cytometry and microscopy to analyze populations of replicating cells labeled with thymidine analogs and monitored mitotic entry in synchronized cells to determine that USP37-depleted cells exhibited altered S-phase kinetics. Further analysis revealed that cells depleted of USP37 harbored increased levels of the replication stress and DNA damage markers γH2AX and 53BP1 in response to perturbed replication. Depletion of USP37 also reduced cellular proliferation and led to increased sensitivity to agents that induce replication stress. Underlying the increased sensitivity, we found that the checkpoint kinase CHK1 is destabilized in the absence of USP37, attenuating its function. We further demonstrated that USP37 deubiquitinates CHK1, promoting its stability. Together our results establish that USP37 is required beyond S-phase entry to promote the efficiency and fidelity of replication. These data further define the role of USP37 in the regulation of cell proliferation and contribute to an evolving understanding of USP37 as a multifaceted regulator of genome stability.

Keywords:  CHK1; DNA damage response; DNA replication; USP37; cell cycle; checkpoint control; deubiquitination; ubiquitination

DOI:  https://doi.org/10.1016/j.jbc.2021.101184
Nat Rev Mol Cell Biol. 2021 Sep 14.

Mechanism, cellular functions and cancer roles of polymerase-theta-mediated DNA end joining.

Dale A Ramsden, Juan Carvajal-Garcia, Gaorav P Gupta.

Cellular pathways that repair chromosomal double-strand breaks (DSBs) have pivotal roles in cell growth, development and cancer. These DSB repair pathways have been the target of intensive investigation, but one pathway - alternative end joining (a-EJ) - has long resisted elucidation. In this Review, we highlight recent progress in our understanding of a-EJ, especially the assignment of DNA polymerase theta (Polθ) as the predominant mediator of a-EJ in most eukaryotes, and discuss a potential molecular mechanism by which Polθ-mediated end joining (TMEJ) occurs. We address possible cellular functions of TMEJ in resolving DSBs that are refractory to repair by non-homologous end joining (NHEJ), DSBs generated following replication fork collapse and DSBs present owing to stalling of repair by homologous recombination. We also discuss how these context-dependent cellular roles explain how TMEJ can both protect against and cause genome instability, and the emerging potential of Polθ as a therapeutic target in cancer.

DOI: https://doi.org/10.1038/s41580-021-00405-2
Proc Natl Acad Sci U S A. 2021 Sep 21. pii: e2109334118. [Epub ahead of print]118(38):

A mechanism for Rad53 to couple leading- and lagging-strand DNA synthesis under replication stress in budding yeast.

Albert Serra-Cardona, Chuanhe Yu, Xinmin Zhang, Xu Hua, Yuan Yao, Jiaqi Zhou, Haiyun Gan, Zhiguo Zhang.

  In response to DNA replication stress, DNA replication checkpoint kinase Mec1 phosphorylates Mrc1, which in turn activates Rad53 to prevent the generation of deleterious single-stranded DNA, a process that remains poorly understood. We previously reported that lagging-strand DNA synthesis proceeds farther than leading strand in rad53-1 mutant cells defective in replication checkpoint under replication stress, resulting in the exposure of long stretches of the leading-strand templates. Here, we show that asymmetric DNA synthesis is also observed in mec1-100 and mrc1-AQ cells defective in replication checkpoint but, surprisingly, not in mrc1∆ cells in which both DNA replication and checkpoint functions of Mrc1 are missing. Furthermore, depletion of either Mrc1 or its partner, Tof1, suppresses the asymmetric DNA synthesis in rad53-1 mutant cells. Thus, the DNA replication checkpoint pathway couples leading- and lagging-strand DNA synthesis by attenuating the replication function of Mrc1-Tof1 under replication stress.

Keywords:  Mrc1; Rad53; asymmetric DNA synthesis; deleterious ssDNA; replication stress

DOI:  https://doi.org/10.1073/pnas.2109334118
Nat Commun. 2021 Sep 17. 12(1): 5512

ADAR-mediated RNA editing of DNA:RNA hybrids is required for DNA double strand break repair.

Sonia Jimeno, Rosario Prados-Carvajal, María Jesús Fernández-Ávila, Sonia Silva, Domenico Alessandro Silvestris, Martín Endara-Coll, Guillermo Rodríguez-Real, Judit Domingo-Prim, Fernando Mejías-Navarro, Amador Romero-Franco, Silvia Jimeno-González, Sonia Barroso, Valeriana Cesarini, Andrés Aguilera, Angela Gallo, Neus Visa, Pablo Huertas.

The maintenance of genomic stability requires the coordination of multiple cellular tasks upon the appearance of DNA lesions. RNA editing, the post-transcriptional sequence alteration of RNA, has a profound effect on cell homeostasis, but its implication in the response to DNA damage was not previously explored. Here we show that, in response to DNA breaks, an overall change of the Adenosine-to-Inosine RNA editing is observed, a phenomenon we call the RNA Editing DAmage Response (REDAR). REDAR relies on the checkpoint kinase ATR and the recombination factor CtIP. Moreover, depletion of the RNA editing enzyme ADAR2 renders cells hypersensitive to genotoxic agents, increases genomic instability and hampers homologous recombination by impairing DNA resection. Such a role of ADAR2 in DNA repair goes beyond the recoding of specific transcripts, but depends on ADAR2 editing DNA:RNA hybrids to ease their dissolution.

DOI: https://doi.org/10.1038/s41467-021-25790-2
J Cell Physiol. 2021 Sep 13.

Molecular mechanisms involved in DNA repair in human cancers: An overview of PI3k/Akt signaling and PIKKs crosstalk.

Forough Alemi, Aydin Raei Sadigh, Faezeh Malakoti, Yusuf Elhaei, Seyed Hamed Ghaffari, Masomeh Maleki, Zatollah Asemi, Bahman Yousefi, Niloufar Targhazeh, Maryam Majidinia.

  The cellular genome is frequently subjected to abundant endogenous and exogenous factors that induce DNA damage. Most of the Phosphatidylinositol 3-kinase-related kinases (PIKKs) family members are activated in response to DNA damage and are the most important DNA damage response (DDR) proteins. The DDR system protects the cells against the wrecking effects of these genotoxicants and repairs the DNA damage caused by them. If the DNA damage is severe, such as when DNA is the goal of chemo-radiotherapy, the DDR drives cells toward cell cycle arrest and apoptosis. Some intracellular pathways, such as PI3K/Akt, which is overactivated in most cancers, could stimulate the DDR process and failure of chemo-radiotherapy with the increasing repair of damaged DNA. This signaling pathway induces DNA repair through the regulation of proteins that are involved in DDR like BRCA1, HMGB1, and P53. In this review, we will focus on the crosstalk of the PI3K/Akt and PIKKs involved in DDR and then discuss current achievements in the sensitization of cancer cells to chemo-radiotherapy by PI3K/Akt inhibitors.

Keywords:  DNA damage response; PI3K/Akt; malignancy; signaling pathway

DOI:  https://doi.org/10.1002/jcp.30573
PLoS Genet. 2021 Sep 14. 17(9): e1009807

The chromatin remodeler Chd1 supports MRX and Exo1 functions in resection of DNA double-strand breaks.

Marco Gnugnoli, Erika Casari, Maria Pia Longhese.

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) requires that the 5'-terminated DNA strands are resected to generate single-stranded DNA overhangs. This process is initiated by a short-range resection catalyzed by the MRX (Mre11-Rad50-Xrs2) complex, which is followed by a long-range step involving the nuclease Exo1 or Dna2. Here we show that the Saccharomyces cerevisiae ATP-dependent chromatin-remodeling protein Chd1 participates in both short- and long-range resection by promoting MRX and Exo1 association with the DSB ends. Furthermore, Chd1 reduces histone occupancy near the DSB ends and promotes DSB repair by HR. All these functions require Chd1 ATPase activity, supporting a role for Chd1 in the opening of chromatin at the DSB site to facilitate MRX and Exo1 processing activities.

DOI: https://doi.org/10.1371/journal.pgen.1009807
Nucleic Acids Res. 2021 Sep 17. pii: gkab805. [Epub ahead of print]

The CRL4DTL E3 ligase induces degradation of the DNA replication initiation factor TICRR/TRESLIN specifically during S phase.

Kimberlie A Wittig, Courtney G Sansam, Tyler D Noble, Duane Goins, Christopher L Sansam.

A DNA replication program, which ensures that the genome is accurately and wholly replicated, is established during G1, before the onset of S phase. In G1, replication origins are licensed, and upon S phase entry, a subset of these will form active replisomes. Tight regulation of the number of active replisomes is crucial to prevent replication stress-induced DNA damage. TICRR/TRESLIN is essential for DNA replication initiation, and the level of TICRR and its phosphorylation determine the number of origins that initiate during S phase. However, the mechanisms regulating TICRR protein levels are unknown. Therefore, we set out to define the TICRR/TRESLIN protein dynamics throughout the cell cycle. Here, we show that TICRR levels are high during G1 and dramatically decrease as cells enter S phase and begin DNA replication. We show that degradation of TICRR occurs specifically during S phase and depends on ubiquitin ligases and proteasomal degradation. Using two targeted siRNA screens, we identify CRL4DTL as a cullin complex necessary for TICRR degradation. We propose that this mechanism moderates the level of TICRR protein available for replication initiation, ensuring the proper number of active origins as cells progress through S phase.

DOI: https://doi.org/10.1093/nar/gkab805
Nat Commun. 2021 Sep 14. 12(1): 5421

MAD2L2 dimerization and TRIP13 control shieldin activity in DNA repair.

Inge de Krijger, Bastian Föhr, Santiago Hernández Pérez, Estelle Vincendeau, Judit Serrat, Alexander Marc Thouin, Vivek Susvirkar, Chloé Lescale, Inés Paniagua, Liesbeth Hoekman, Simranjeet Kaur, Maarten Altelaar, Ludovic Deriano, Alex C Faesen, Jacqueline J L Jacobs.

MAD2L2 (REV7) plays an important role in DNA double-strand break repair. As a member of the shieldin complex, consisting of MAD2L2, SHLD1, SHLD2 and SHLD3, it controls DNA repair pathway choice by counteracting DNA end-resection. Here we investigated the requirements for shieldin complex assembly and activity. Besides a dimerization-surface, HORMA-domain protein MAD2L2 has the extraordinary ability to wrap its C-terminus around SHLD3, likely creating a very stable complex. We show that appropriate function of MAD2L2 within shieldin requires its dimerization, mediated by SHLD2 and accelerating MAD2L2-SHLD3 interaction. Dimerization-defective MAD2L2 impairs shieldin assembly and fails to promote NHEJ. Moreover, MAD2L2 dimerization, along with the presence of SHLD3, allows shieldin to interact with the TRIP13 ATPase, known to drive topological switches in HORMA-domain proteins. We find that appropriate levels of TRIP13 are important for proper shieldin (dis)assembly and activity in DNA repair. Together our data provide important insights in the dependencies for shieldin activity.

DOI: https://doi.org/10.1038/s41467-021-25724-y
DNA Repair (Amst). 2021 Sep 03. pii: S1568-7864(21)00172-5. [Epub ahead of print]108 103216

DNA polymerase delta Exo domain stabilizes mononucleotide microsatellites in human cells.

Seijiro Shioi, Akiyoshi Shimamoto, Yingxia Song, Kyoko Hidaka, Maki Nakamura, Ayumi Take, Namiko Hayashi, Soichi Takiguchi, Ryosuke Fujikane, Masumi Hidaka, Shinya Oda, Yoshimichi Nakatsu.

  In prokaryotes and yeasts, DNA polymerase proofreading (PPR) and DNA mismatch repair (MMR) cooperatively counteracts replication errors leading to repeat sequence destabilization (i.e. insertions/deletions of repeat units). However, PPR has not thus far been regarded as a mechanism stabilizing repeat sequences in higher eukaryotic cells. In a human cancer cell line, DLD-1, which carries mutations in both MSH6 and the Exo domain of POLD1, we previously observed that mononucleotide microsatellites were markedly destabilized whereas being stable in the simple MMR-defective backgrounds. In this study, we introduced the Exo domain mutation found in DLD-1 cells into MSH2-null HeLa cell clones, using CRISPR/Cas9 system. In the established Exo-/MMR-mutated HeLa clones, mononucleotide repeat sequences were remarkably destabilized as in DLD-1 cells. In contrast, dinucleotide microsatellites were readily destabilized in the parental MMR-deficient backgrounds, and the instability was not notably increased in the genome-edited HeLa clones. Here, we show an involvement of the Exo domain functions of DNA polymerase delta in mononucleotide repeat stabilization in human cells, which also suggests a possible role division between DNA polymerase and MMR in repeat maintenance in the human genome.

Keywords:  DNA mismatch repair; Microsatellites; Polymerase proofreading

DOI:  https://doi.org/10.1016/j.dnarep.2021.103216
PLoS One. 2021 ;16(9): e0257473

Mild phenotype of knockouts of the major apurinic/apyrimidinic endonuclease APEX1 in a non-cancer human cell line.

Daria V Kim, Liliya M Kulishova, Natalia A Torgasheva, Vasily S Melentyev, Grigory L Dianov, Sergey P Medvedev, Suren M Zakian, Dmitry O Zharkov.

The major human apurinic/apyrimidinic (AP) site endonuclease, APEX1, is a central player in the base excision DNA repair (BER) pathway and has a role in the regulation of DNA binding by transcription factors. In vertebrates, APEX1 knockouts are embryonic lethal, and only a handful of knockout cell lines are known. To facilitate studies of multiple functions of this protein in human cells, we have used the CRISPR/Cas9 system to knock out the APEX1 gene in a widely used non-cancer hypotriploid HEK 293FT cell line. Two stable knockout lines were obtained, one carrying two single-base deletion alleles and one single-base insertion allele in exon 3, another homozygous in the single-base insertion allele. Both mutations cause a frameshift that leads to premature translation termination before the start of the protein's catalytic domain. Both cell lines totally lacked the APEX1 protein and AP site-cleaving activity, and showed significantly lower levels of the APEX1 transcript. The APEX1-null cells were unable to support BER on uracil- or AP site-containing substrates. Phenotypically, they showed a moderately increased sensitivity to methyl methanesulfonate (MMS; ~2-fold lower EC50 compared with wild-type cells), and their background level of natural AP sites detected by the aldehyde-reactive probe was elevated ~1.5-2-fold. However, the knockout lines retained a nearly wild-type sensitivity to oxidizing agents hydrogen peroxide and potassium bromate. Interestingly, despite the increased MMS cytotoxicity, we observed no additional increase in AP sites in knockout cells upon MMS treatment, which could indicate their conversion into more toxic products in the absence of repair. Overall, the relatively mild cell phenotype in the absence of APEX1-dependent BER suggests that mammalian cells possess mechanisms of tolerance or alternative repair of AP sites. The knockout derivatives of the extensively characterized HEK 293FT cell line may provide a valuable tool for studies of APEX1 in DNA repair and beyond.

DOI: https://doi.org/10.1371/journal.pone.0257473
Proc Natl Acad Sci U S A. 2021 Sep 21. pii: e2105440118. [Epub ahead of print]118(38):

DNA damage-induced phosphorylation of CtIP at a conserved ATM/ATR site T855 promotes lymphomagenesis in mice.

Xiaobin S Wang, Demis Menolfi, Foon Wu-Baer, Marco Fangazio, Stefanie N Meyer, Zhengping Shao, Yunyue Wang, Yimeng Zhu, Brian J Lee, Verna M Estes, Olivia M Cupo, Jean Gautier, Laura Pasqualucci, Riccardo Dalla-Favera, Richard Baer, Shan Zha.

  CtIP is a DNA end resection factor widely implicated in alternative end-joining (A-EJ)-mediated translocations in cell-based reporter systems. To address the physiological role of CtIP, an essential gene, in translocation-mediated lymphomagenesis, we introduced the T855A mutation at murine CtIP to nonhomologous end-joining and Tp53 double-deficient mice that routinely succumbed to lymphomas carrying A-EJ-mediated IgH-Myc translocations. T855 of CtIP is phosphorylated by ATM or ATR kinases upon DNA damage to promote end resection. Here, we reported that the T855A mutation of CtIP compromised the neonatal development of Xrcc4 -/- Tp53 -/- mice and the IgH-Myc translocation-driven lymphomagenesis in DNA-PKcs -/- Tp53 -/- mice. Mechanistically, the T855A mutation limits DNA end resection length without affecting hairpin opening, translocation frequency, or fork stability. Meanwhile, after radiation, CtIP-T855A mutant cells showed a consistent decreased Chk1 phosphorylation and defects in the G2/M cell cycle checkpoint. Consistent with the role of T855A mutation in lymphomagenesis beyond translocation, the CtIP-T855A mutation also delays splenomegaly in λ-Myc mice. Collectively, our study revealed a role of CtIP-T855 phosphorylation in lymphomagenesis beyond A-EJ-mediated chromosomal translocation.

Keywords:  CtIP; DNA damage response; alternative end-joining; checkpoint; lymphomagenesis

DOI:  https://doi.org/10.1073/pnas.2105440118
Mol Cancer Res. 2021 Sep 14. pii: molcanres.MCR-21-0093-A.2021. [Epub ahead of print]

Epigenetic regulation of Fanconi anemia genes implicates PRMT5 blockage as a strategy for tumor chemosensitization.

Changzheng Du, Steven W Li, Simranjit X Singh, Kristen Roso, Michael A Sun, Christopher J Pirozzi, Rui Yang, Jian-Liang Li, Yiping He.

Strengthened DNA repair pathways in tumor cells contribute to the development of resistance to DNA-damaging agents. Consequently, targeting proteins in these pathways is a promising strategy for tumor chemosensitization. Here, we show that the expression of a subset of Fanconi anemia (FA) genes is attenuated in glioblastoma tumor cells deficient in Methylthioadenosine Phosphorylase (MTAP), a common genetic alteration in a variety of cancers. Subsequent experiments in cell line models of different cancer types illustrate that this reduced transcription of FA genes can be recapitulated by blockage of Protein Arginine Methyltransferase 5 (PRMT5), a promising therapeutically targetable epigenetic regulator whose enzymatic activity is compromised in MTAP-deficient cells. Further analyses provide evidence to support that PRMT5 can function as an epigenetic regulator that contributes to the increased expression of FA genes in cancer cells. Most notably and consistent with the essential roles of FA proteins in resolving DNA damage elicited by interstrand crosslinking (ICL) agents, PRMT5 blockage, as well as MTAP loss, sensitizes tumor cells to ICL agents both in vitro and in xenografts. Collectively, these findings reveal a novel epigenetic mechanism underlying the upregulated expression of FA genes in cancer cells and suggest that therapeutically targeting PRMT5 can have an additional benefit of chemosensitizing tumor cells to ICL agents. Implications: PRMT5 positively regulates the expression of FA genes. Inhibition of PRMT5 attenuates FA-dependent DNA repair pathway and sensitizes tumor cells to ICL agents.

DOI: https://doi.org/10.1158/1541-7786.MCR-21-0093
Sci Rep. 2021 Sep 15. 11(1): 18393

Versatile cell-based assay for measuring DNA alkylation damage and its repair.

Yong Li, Peng Mao, Evelina Y Basenko, Zachary Lewis, Michael J Smerdon, Wioletta Czaja.

DNA alkylation damage induced by environmental carcinogens, chemotherapy drugs, or endogenous metabolites plays a central role in mutagenesis, carcinogenesis, and cancer therapy. Base excision repair (BER) is a conserved, front line DNA repair pathway that removes alkylation damage from DNA. The capacity of BER to repair DNA alkylation varies markedly between different cell types and tissues, which correlates with cancer risk and cellular responses to alkylation chemotherapy. The ability to measure cellular rates of alkylation damage repair by the BER pathway is critically important for better understanding of the fundamental processes involved in carcinogenesis, and also to advance development of new therapeutic strategies. Methods for assessing the rates of alkylation damage and repair, especially in human cells, are limited, prone to significant variability due to the unstable nature of some of the alkyl adducts, and often rely on indirect measurements of BER activity. Here, we report a highly reproducible and quantitative, cell-based assay, named alk-BER (alkylation Base Excision Repair) for measuring rates of BER following alkylation DNA damage. The alk-BER assay involves specific detection of methyl DNA adducts (7-methyl guanine and 3-methyl adenine) directly in genomic DNA. The assay has been developed and adapted to measure the activity of BER in fungal model systems and human cell lines. Considering the specificity and conserved nature of BER enzymes, the assay can be adapted to virtually any type of cultured cells. Alk-BER offers a cost efficient and reliable method that can effectively complement existing approaches to advance integrative research on mechanisms of alkylation DNA damage and repair.

DOI: https://doi.org/10.1038/s41598-021-97523-w
FEBS J. 2021 Sep 16.

APOBEC3C, a nucleolar protein induced by genotoxins, is excluded from DNA damage sites.

Daniel Constantin, Gilles Dubuis, María Del Carmen Conde-Rubio, Christian Widmann.

  The human genome contains 11 APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like) cytidine deaminases classified into four families. These proteins function mainly in innate antiviral immunity and can also restrict endogenous retrotransposable element multiplication. The present study focuses on APOBEC3C (A3C), a member of the APOBEC3 sub-family. Some APOBEC3 proteins use their enzymatic activity on genomic DNA, inducing mutations and DNA damage, while other members facilitate DNA repair. Our results show that A3C is highly expressed in cells treated with DNA damaging agents. Its expression is regulated by p53. Depletion of A3C slightly decreases proliferation and does not affect DNA repair via homologous recombination or non-homologous end joining. The A3C interactomes obtained from control cells and cells exposed to the genotoxin etoposide indicated that A3C is a nucleolar protein. This was confirmed by the detection of either endogenous or ectopic A3C in nucleoli. Interestingly, we show that A3C is excluded from areas of DNA breaks in live cells. Our data also indicate that the C-terminal part of A3C is responsible for its nucleolar localization and exclusion from DNA damage sites.

Keywords:  APOBEC3C; DNA repair; anticancer drug; cancer biology; etoposide; nucleolus; p53

DOI:  https://doi.org/10.1111/febs.16202
Nature. 2021 Sep 15.

Structural basis of human transcription-DNA repair coupling.

Goran Kokic, Felix R Wagner, Aleksandar Chernev, Henning Urlaub, Patrick Cramer.

Transcription-coupled DNA repair removes bulky DNA lesions from the genome1,2 and protects cells against ultraviolet (UV) irradiation3. Transcription-coupled DNA repair begins when RNA polymerase II (Pol II) stalls at a DNA lesion and recruits the Cockayne syndrome protein CSB, the E3 ubiquitin ligase, CRL4CSA and UV-stimulated scaffold protein A (UVSSA)3. Here we provide five high-resolution structures of Pol II transcription complexes containing human transcription-coupled DNA repair factors and the elongation factors PAF1 complex (PAF) and SPT6. Together with biochemical and published3,4 data, the structures provide a model for transcription-repair coupling. Stalling of Pol II at a DNA lesion triggers replacement of the elongation factor DSIF by CSB, which binds to PAF and moves upstream DNA to SPT6. The resulting elongation complex, ECTCR, uses the CSA-stimulated translocase activity of CSB to pull on upstream DNA and push Pol II forward. If the lesion cannot be bypassed, CRL4CSA spans over the Pol II clamp and ubiquitylates the RPB1 residue K1268, enabling recruitment of TFIIH to UVSSA and DNA repair. Conformational changes in CRL4CSA lead to ubiquitylation of CSB and to release of transcription-coupled DNA repair factors before transcription may continue over repaired DNA.

DOI: https://doi.org/10.1038/s41586-021-03906-4
Blood Adv. 2021 Sep 16. pii: bloodadvances.2021004638. [Epub ahead of print]

Poly (ADP-ribose) polymerase-1 (PARP1) as a therapeutic target in acute myeloid leukemia and myelodysplastic syndrome.

Christina-Nefeli Kontandreopoulou, Panagiotis T Diamantopoulos, Despina Tiblalexi, Nefeli Giannakopoulou, Nora-Athina Viniou.

Poly (ADP-ribose) polymerase-1 (PARP1) is a key mediator of various forms of DNA damage repair and plays an important role in the progression of several cancer types. The enzyme is activated by binding to DNA single-strand and double-strand breaks. Its contribution to chromatin remodeling makes PARP1 crucial for gene expression regulation. Inhibition of its activity with small molecules, leads to the synthetic lethal effect by impeding DNA repair in the treatment of cancer cells. At first PARP1 inhibitors (PARPi) were developed to target BRCA mutated cancer cells. Currently, PARPi are being studied to be used in a broader variety of patients either as single agents or in combination with chemotherapy, antiangiogenic agents, ionizing radiation, and immune checkpoint inhibitors. Ongoing clinical trials on olaparib, rucaparib, niraparib, veliparib and the most recent talazoparib show the advantage of these agents in overcoming PARPi resistance and underline their efficacy in targeted treatment of several hematologic malignancies. In this review, focusing on the crucial role of PARP1 in physiological and pathological effects in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), we give an outline of the enzyme's mechanisms of action and its role in the pathophysiology and prognosis of MDS/AML and we analyze the available data on the use of PARPi, highlighting their promising advances in clinical application.

DOI: https://doi.org/10.1182/bloodadvances.2021004638
Cancer Discov. 2021 Sep 16. pii: candisc.1849.2020. [Epub ahead of print]

SLC5A3-dependent myo-inositol auxotrophy in acute myeloid leukemia.

Yiliang Wei, Yu-Han Huang, Damianos S Skopelitis, Shruti V Iyer, Ana S H Costa, Zhaolin Yang, Melissa Kramer, Emmalee R Adelman, Olaf Klingbeil, Osama E Demerdash, Sofya A Polyanskaya, Kenneth Chang, Sara Goodwin, Emily Hodges, W Richard McCombie, Maria E Figueroa, Christopher R Vakoc.

An enhanced requirement for nutrients is a hallmark property of cancer cells. Here, we optimized an in vivo genetic screening strategy in acute myeloid leukemia (AML), which led to the identification of the myo-inositol transporter SLC5A3 as a dependency in this disease. We demonstrate that SLC5A3 is essential to support a myo-inositol auxotrophy in AML. The commonality among SLC5A3-dependent AML lines is the transcriptional silencing of ISYNA1, which encodes the rate limiting enzyme for myo-inositol biosynthesis, inositol-3-phosphate synthase 1. We use gain- and loss-of-function experiments to reveal a synthetic lethal genetic interaction between ISYNA1 and SLC5A3 in AML, which function redundantly to sustain intracellular myo-inositol. Transcriptional silencing and DNA hyper-methylation of ISYNA1 occur in a recurrent manner in human AML patient samples, in association with IDH1/IDH2 and CEBPA mutations. Our findings reveal myo-inositol as a nutrient dependency in AML caused by the aberrant silencing of a biosynthetic enzyme.

DOI: https://doi.org/10.1158/2159-8290.CD-20-1849
Front Mol Biosci. 2021 ;8 741718

Single-Molecule Insights Into the Dynamics of Replicative Helicases.

Richard R Spinks, Lisanne M Spenkelink, Nicholas E Dixon, Antoine M van Oijen.

  Helicases are molecular motors that translocate along single-stranded DNA and unwind duplex DNA. They rely on the consumption of chemical energy from nucleotide hydrolysis to drive their translocation. Specialized helicases play a critically important role in DNA replication by unwinding DNA at the front of the replication fork. The replicative helicases of the model systems bacteriophages T4 and T7, Escherichia coli and Saccharomyces cerevisiae have been extensively studied and characterized using biochemical methods. While powerful, their averaging over ensembles of molecules and reactions makes it challenging to uncover information related to intermediate states in the unwinding process and the dynamic helicase interactions within the replisome. Here, we describe single-molecule methods that have been developed in the last few decades and discuss the new details that these methods have revealed about replicative helicases. Applying methods such as FRET and optical and magnetic tweezers to individual helicases have made it possible to access the mechanistic aspects of unwinding. It is from these methods that we understand that the replicative helicases studied so far actively translocate and then passively unwind DNA, and that these hexameric enzymes must efficiently coordinate the stepping action of their subunits to achieve unwinding, where the size of each step is prone to variation. Single-molecule fluorescence microscopy methods have made it possible to visualize replicative helicases acting at replication forks and quantify their dynamics using multi-color colocalization, FRAP and FLIP. These fluorescence methods have made it possible to visualize helicases in replication initiation and dissect this intricate protein-assembly process. In a similar manner, single-molecule visualization of fluorescent replicative helicases acting in replication identified that, in contrast to the replicative polymerases, the helicase does not exchange. Instead, the replicative helicase acts as the stable component that serves to anchor the other replication factors to the replisome.

Keywords:  DNA replication; dynamics; fluorescence; helicases; multi-protein complexes; replisome; single-molecule

DOI:  https://doi.org/10.3389/fmolb.2021.741718
Cell Biol Toxicol. 2021 Sep 14.

Exploring the ATR-CHK1 pathway in the response of doxorubicin-induced DNA damages in acute lymphoblastic leukemia cells.

Andrea Ghelli Luserna Di Rorà, Martina Ghetti, Lorenzo Ledda, Anna Ferrari, Matteo Bocconcelli, Antonella Padella, Roberta Napolitano, Maria Chiara Fontana, Chiara Liverani, Enrica Imbrogno, Maria Teresa Bochicchio, Matteo Paganelli, Valentina Robustelli, Seydou Sanogo, Claudio Cerchione, Monica Fumagalli, Michela Rondoni, Annalisa Imovilli, Gerardo Musuraca, Giovanni Martinelli, Giorgia Simonetti.

  Doxorubicin (Dox) is one of the most commonly used anthracyclines for the treatment of solid and hematological tumors such as B-/T cell acute lymphoblastic leukemia (ALL). Dox compromises topoisomerase II enzyme functionality, thus inducing structural damages during DNA replication and causes direct damages intercalating into DNA double helix. Eukaryotic cells respond to DNA damages by activating the ATM-CHK2 and/or ATR-CHK1 pathway, whose function is to regulate cell cycle progression, to promote damage repair, and to control apoptosis. We evaluated the efficacy of a new drug schedule combining Dox and specific ATR (VE-821) or CHK1 (prexasertib, PX) inhibitors in the treatment of human B-/T cell precursor ALL cell lines and primary ALL leukemic cells. We found that ALL cell lines respond to Dox activating the G2/M cell cycle checkpoint. Exposure of Dox-pretreated ALL cell lines to VE-821 or PX enhanced Dox cytotoxic effect. This phenomenon was associated with the abrogation of the G2/M cell cycle checkpoint with changes in the expression pCDK1 and cyclin B1, and cell entry in mitosis, followed by the induction of apoptosis. Indeed, the inhibition of the G2/M checkpoint led to a significant increment of normal and aberrant mitotic cells, including those showing tripolar spindles, metaphases with lagging chromosomes, and massive chromosomes fragmentation. In conclusion, we found that the ATR-CHK1 pathway is involved in the response to Dox-induced DNA damages and we demonstrated that our new in vitro drug schedule that combines Dox followed by ATR/CHK1 inhibitors can increase Dox cytotoxicity against ALL cells, while using lower drug doses. • Doxorubicin activates the G2/M cell cycle checkpoint in acute lymphoblastic leukemia (ALL) cells. • ALL cells respond to doxorubicin-induced DNA damages by activating the ATR-CHK1 pathway. • The inhibition of the ATR-CHK1 pathway synergizes with doxorubicin in the induction of cytotoxicity in ALL cells. • The inhibition of ATR-CHK1 pathway induces aberrant chromosome segregation and mitotic spindle defects in doxorubicin-pretreated ALL cells.

Keywords:  ATR; Acute lymphoblastic leukemia; CHK1; Cell cycle; Doxorubicin

DOI:  https://doi.org/10.1007/s10565-021-09640-x
Front Mol Biosci. 2021 ;8 724870

SAMHD1 Phosphorylation at T592 Regulates Cellular Localization and S-phase Progression.

Stephanie Batalis, LeAnn C Rogers, Wayne O Hemphill, Christopher H Mauney, David A Ornelles, Thomas Hollis.

  SAMHD1 activity is regulated by a network of mechanisms including phosphorylation, oxidation, oligomerization, and others. Significant questions remain about the effects of phosphorylation on SAMHD1 function and activity. We investigated the effects of a SAMHD1 T592E phosphorylation mimic on its cellular localization, catalytic activity, and cell cycle progression. We found that the SAMHD1 T592E is a catalytically active enzyme that is inhibited by protein oxidation. SAMHD1 T592E is retained in the nucleus at higher levels than the wild-type protein during growth factor-mediated signaling. This nuclear localization protects SAMHD1 from oxidation by cytoplasmic reactive oxygen species. The SAMHD1 T592E phosphomimetic further inhibits the cell cycle S/G2 transition. This has significant implications for SAMHD1 function in regulating innate immunity, antiviral response and DNA replication.

Keywords:  SAMHD1; cell cycle; dNTP; phosphorylation; protein localization; protein oxidation

DOI:  https://doi.org/10.3389/fmolb.2021.724870
Front Endocrinol (Lausanne). 2021 ;12 731648

Nucleotide Metabolism Behind Epigenetics.

Tamaki Suganuma, Jerry L Workman.

  The mechanisms of epigenetic gene regulation-histone modifications, chromatin remodeling, DNA methylation, and noncoding RNA-use metabolites as enzymatic cofactors and substrates in reactions that allow chromatin formation, nucleotide biogenesis, transcription, RNA processing, and translation. Gene expression responds to demands from cellular processes that use specific metabolites and alters or maintains cellular metabolic status. However, the roles of metabolites-particularly nucleotides-as regulatory molecules in epigenetic regulation and biological processes remain largely unknown. Here we review the crosstalk between gene expression, nucleotide metabolism, and cellular processes, and explore the role of metabolism in epigenetics as a critical regulator of biological events.

Keywords:  ADP-ribosylation; DNA damage; NAD; RNA editing; chromatin modifiers; histone modifications; metabolism; nucleotide metabolism

DOI:  https://doi.org/10.3389/fendo.2021.731648
Cell Death Dis. 2021 Sep 12. 12(9): 844

EMP3 negatively modulates breast cancer cell DNA replication, DNA damage repair, and stem-like properties.

Kailing Zhou, Yu Sun, Dan Dong, Chenghai Zhao, Wei Wang.

Enhanced DNA damage repair capacity attenuates cell killing of DNA-damaging chemotherapeutic agents. In silico analysis showed that epithelial membrane protein 3 (EMP3) is associated with favorable survival, and negatively regulates cell cycle S-phase. Consistently, loss and gain of function studies demonstrated that EMP3 inhibits breast cancer cell S-phage entry, DNA replication, DNA damage repair, and stem-like properties. Moreover, EMP3 blocks Akt-mTOR signaling activation and induces autophagy. EMP3 negatively modulates BRCA1 and RAD51 expression, indicating EMP3 suppresses homologous recombination repair of DNA double-strand breaks. Accordingly, EMP3 sensitizes breast cancer cells to the DNA-damaging drug Adriamycin. EMP3 downregulates YTHDC1, a RNA-binding protein involved in m6a modification, which at least in part mediates the effects of EMP3 on breast cancer cells. Taken together, these data indicate that EMP3 is a putative tumor suppressor in breast cancer, and EMP3 downregulation may be responsible for breast cancer chemoresistance.

DOI: https://doi.org/10.1038/s41419-021-04140-6
Nat Commun. 2021 Sep 13. 12(1): 5404

SMARCA4/2 loss inhibits chemotherapy-induced apoptosis by restricting IP3R3-mediated Ca2+ flux to mitochondria.

Yibo Xue, Jordan L Morris, Kangning Yang, Zheng Fu, Xianbing Zhu, Fraser Johnson, Brian Meehan, Leora Witkowski, Amber Yasmeen, Tunde Golenar, Mackenzie Coatham, Geneviève Morin, Anie Monast, Virginie Pilon, Pierre Olivier Fiset, Sungmi Jung, Anne V Gonzalez, Sophie Camilleri-Broet, Lili Fu, Lynne-Marie Postovit, Jonathan Spicer, Walter H Gotlieb, Marie-Christine Guiot, Janusz Rak, Morag Park, William Lockwood, William D Foulkes, Julien Prudent, Sidong Huang.

Inactivating mutations in SMARCA4 and concurrent epigenetic silencing of SMARCA2 characterize subsets of ovarian and lung cancers. Concomitant loss of these key subunits of SWI/SNF chromatin remodeling complexes in both cancers is associated with chemotherapy resistance and poor prognosis. Here, we discover that SMARCA4/2 loss inhibits chemotherapy-induced apoptosis through disrupting intracellular organelle calcium ion (Ca2+) release in these cancers. By restricting chromatin accessibility to ITPR3, encoding Ca2+ channel IP3R3, SMARCA4/2 deficiency causes reduced IP3R3 expression leading to impaired Ca2+ transfer from the endoplasmic reticulum to mitochondria required for apoptosis induction. Reactivation of SMARCA2 by a histone deacetylase inhibitor rescues IP3R3 expression and enhances cisplatin response in SMARCA4/2-deficient cancer cells both in vitro and in vivo. Our findings elucidate the contribution of SMARCA4/2 to Ca2+-dependent apoptosis induction, which may be exploited to enhance chemotherapy response in SMARCA4/2-deficient cancers.

DOI: https://doi.org/10.1038/s41467-021-25260-9
Cancer Drug Resist. 2021 ;4 837-841

Targeting DNA repair pathways to overcome cancer drug resistance.

Robert C A M van Waardenburg, Eddy S Yang.

DOI: https://doi.org/10.20517/cdr.2021.80