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NAR Top Articles - Genome Integrity, Repair and Replication

Genome Integrity, Repair and Replication

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May 2015

PARP3 affects the relative contribution of homologous recombination and nonhomologous end-joining pathways
Beck, C; Boehler, C; Barbat, JG; Bonnet, ME; Illuzzi, G; Ronde, P; Gauthier, LR; Magroun, N; Rajendran, A; Lopez, BS; Scully, R; Boussin, FD; Schreiber, V; Dantzer, F
Nucleic Acids Res. 2014, 42, 5616-5632
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The repair of toxic double-strand breaks (DSB) is critical for the maintenance of genome integrity. The major mechanisms that cope with DSB are: homologous recombination (HR) and classical or alternative nonhomologous end joining (C-NHEJ versus A-EJ). Because these pathways compete for the repair of DSB, the choice of the appropriate repair pathway is pivotal. Among the mechanisms that influence this choice, deoxyribonucleic acid (DNA) end resection plays a critical role by driving cells to HR, while accurate C-NHEJ is suppressed. Furthermore, end resection promotes error-prone A-EJ. Increasing evidence define Poly(ADP-ribose) polymerase 3 (PARP3, also known as ARTD3) as an important player in cellular response to DSB. In this work, we reveal a specific feature of PARP3 that together with Ku80 limits DNA end resection and thereby helps in making the choice between HR and NHEJ pathways. PARP3 interacts with and PARylates Ku70/Ku80. The depletion of PARP3 impairs the recruitment of YFP-Ku80 to laser-induced DNA damage sites...

PARP-2 and PARP-3 are selectively activated by 5'' phosphorylated DNA breaks through an allosteric regulatory mechanism shared with PARP-1
Langelier, MF; Riccio, AA; Pascal, JM
Nucleic Acids Res. 2014, 42, 7762-7775
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PARP-1, PARP-2 and PARP-3 are DNA-dependent PARPs that localize to DNA damage, synthesize poly(ADP-ribose) (PAR) covalently attached to target proteins including themselves, and thereby recruit repair factors to DNA breaks to increase repair efficiency. PARP-1, PARP-2 and PARP-3 have in common two C-terminal domains-Trp-Gly-Arg (WGR) and catalytic (CAT). In contrast, the N-terminal region (NTR) of PARP-1 is over 500 residues and includes four regulatory domains, whereas PARP-2 and PARP-3 have smaller NTRs (70 and 40 residues, respectively) of unknown structural composition and function. Here, we show that PARP-2 and PARP-3 are preferentially activated by DNA breaks harboring a 5' phosphate (5'P), suggesting selective activation in response to specific DNA repair intermediates, in particular structures that are competent for DNA ligation. In contrast to PARP-1, the NTRs of PARP-2 and PARP-3 are not strictly required for DNA binding or for DNA-dependent activation. Rather, the WGR domain is the central regulatory domain of PARP-2 and PARP-3. Finally, PARP-1, PARP-2 and PARP-3 share an allosteric regulatory mechanism of DNA-dependent catalytic activation...

DNA damage triggers SAF-A and RNA biogenesis factors exclusion from chromatin coupled to R-loops removal
Britton, S; Dernoncourt, E; Delteil, C; Froment, C; Schiltz, O; Salles, B; Frit, P; Calsou, P
Nucleic Acids Res. 2014, 42, 9047-9062
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We previously identified the heterogeneous ribonucleoprotein SAF-A/hnRNP U as a substrate for DNA-PK, a protein kinase involved in DNA damage response (DDR). Using laser micro-irradiation in human cells, we report here that SAF-A exhibits a two-phase dynamics at sites of DNA damage, with a rapid and transient recruitment followed by a prolonged exclusion. SAF-A recruitment corresponds to its binding to Poly(ADP-ribose) while its exclusion is dependent on the activity of ATM, ATR and DNA-PK and reflects the dissociation from chromatin of SAF-A associated with ongoing transcription. Having established that SAF-A RNA-binding domain recapitulates SAF-A dynamics, we show that this domain is part of a complex comprising several mRNA biogenesis proteins of which at least two, FUS/TLS and TAFII68/TAF15, exhibit similar biphasic dynamics at sites of damage. Using an original reporter for live imaging of DNA: RNA hybrids (R-loops), we show a transient transcription-dependent accumulation of R-loops at sites of DNA damage that is prolonged upon inhibition of RNA biogenesis factors exclusion...

The PARP inhibitor Olaparib disrupts base excision repair of 5-aza-2''-deoxycytidine lesions
Orta, ML; Hoglund, A; Calderon-Montano, JM; Dominguez, I; Burgos-Moron, E; Visnes, T; Pastor, N; Strom, C; Lopez-Iazaro, M; Helleday, T
Nucleic Acids Res. 2014, 42, 9108-9120
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Decitabine (5-aza-2'-deoxycytidine, 5-azadC) is used in the treatment of Myelodysplatic syndrome (MDS) and Acute Myeloid Leukemia (AML). Its mechanism of action is thought to involve reactivation of genes implicated in differentiation and transformation, as well as induction of DNA damage by trapping DNA methyltranferases (DNMT) to DNA. We demonstrate for the first time that base excision repair (BER) recognizes 5-azadC-induced lesions in DNA and mediates repair. We find that BER (XRCC1) deficient cells are sensitive to 5-azadC and display an increased amount of DNA single- and double-strand breaks. The XRCC1 protein co-localizes with DNMT1 foci after 5-azadC treatment, suggesting a novel and specific role of XRCC1 in the repair of trapped DNMT1. 5-azadC-induced DNMT foci persist in XRCC1 defective cells, demonstrating a role for XRCC1 in repair of 5-azadC-induced DNA lesions. Poly (ADP-ribose) polymerase (PARP) inhibition prevents XRCC1 relocation to DNA damage sites, disrupts XRCC1-DNMT1 co-localization and thereby efficient BER....

Opposing roles for 53BP1 during homologous recombination
Kakarougkas, A; Ismail, A; Klement, K; Goodarzi, AA; Conrad, S; Freire, R; Shibata, A; Lobrich, M; Jeggo, PA
Nucleic Acids Res. 2013, 41, 9719-9731
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Although DNA non-homologous end-joining repairs most DNA double-strand breaks (DSBs) in G2 phase, late repairing DSBs undergo resection and repair by homologous recombination (HR). Based on parallels to the situation in G1 cells, previous work has suggested that DSBs that undergo repair by HR predominantly localize to regions of heterochromatin (HC). By using H3K9me3 and H4K20me3 to identify HC regions, we substantiate and extend previous evidence, suggesting that HC-DSBs undergo repair by HR. Next, we examine roles for 53BP1 and BRCA1 in this process. Previous studies have shown that 53BP1 is pro-non-homologous end-joining and anti-HR. Surprisingly, we demonstrate that in G2 phase, 53BP1 is required for HR at HC-DSBs with its role being to promote phosphorylated KAP-1 foci formation. BRCA1, in contrast, is dispensable for pKAP-1 foci formation but relieves the barrier caused by 53BP1. As 53BP1 is retained at irradiation-induced foci during HR, we propose that BRCA1 promotes displacement but retention of 53BP1 to allow resection and any necessary HC modifications to complete HR...

Genome-wide single-cell-level screen for protein abundance and localization changes in response to DNA damage in S. cerevisiae
Mazumder, A; Pesudo, LQ; McRee, S; Bathe, M; Samson, LD
Nucleic Acids Res. 2013, 41, 9310-9324
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An effective response to DNA damaging agents involves modulating numerous facets of cellular homeostasis in addition to DNA repair and cell-cycle checkpoint pathways. Fluorescence microscopy-based imaging offers the opportunity to simultaneously interrogate changes in both protein level and subcellular localization in response to DNA damaging agents at the single-cell level. We report here results from screening the yeast Green Fluorescent Protein (GFP)-fusion library to investigate global cellular protein reorganization on exposure to the alkylating agent methyl methanesulfonate (MMS). Broad groups of induced, repressed, nucleus- and cytoplasm-enriched proteins were identified. Gene Ontology and interactome analyses revealed the underlying cellular processes. Transcription factor (TF) analysis identified principal regulators of the response, and targets of all major stress-responsive TFs were enriched amongst the induced proteins. An unexpected partitioning of biological function according to the number of TFs targeting individual genes was revealed...

Swi5-Sfr1 protein stimulates Rad51-mediated DNA strand exchange reaction through organization of DNA bases in the presynaptic filament
Fornander, LH; Renodon-Corniere, A; Kuwabara, N; Ito, K; Tsutsui, Y; Shimizu, T; Iwasaki, H; Norden, B; Takahashi, M
Nucleic Acids Res. 2014, 42, 2358-2365
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The Swi5-Sfr1 heterodimer protein stimulates the Rad51-promoted DNA strand exchange reaction, a crucial step in homologous recombination. To clarify how this accessory protein acts on the strand exchange reaction, we have analyzed how the structure of the primary reaction intermediate, the Rad51/single-stranded DNA (ssDNA) complex filament formed in the presence of ATP, is affected by Swi5-Sfr1. Using flow linear dichroism spectroscopy, we observe that the nucleobases of the ssDNA are more perpendicularly aligned to the filament axis in the presence of Swi5-Sfr1, whereas the bases are more randomly oriented in the absence of Swi5-Sfr1. When using a modified version of the natural protein where the N-terminal part of Sfr1 is deleted, which has no affinity for DNA but maintained ability to stimulate the strand exchange reaction, we still observe the improved perpendicular DNA base orientation. This indicates that Swi5-Sfr1 exerts its activating effect through interaction with the Rad51 filament mainly and not with the DNA...

Oxidative DNA damage is instrumental in hyperreplication stress-induced inviability of Escherichia coli
Charbon, G; Bjorn, L; Mendoza-Chamizo, B; Frimodt-Moller, J; Lobner-Olesen, A
Nucleic Acids Res. 2014, 42, 13228-13241
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In Escherichia coli, an increase in the ATP bound form of the DnaA initiator protein results in hyperinitiation and inviability. Here, we show that such replication stress is tolerated during anaerobic growth. In hyperinitiating cells, a shift from anaerobic to aerobic growth resulted in appearance of fragmented chromosomes and a decrease in terminus concentration, leading to a dramatic increase in ori/ter ratio and cessation of cell growth. Aerobic viability was restored by reducing the level of reactive oxygen species (ROS) or by deleting mutM (Fpg glycosylase). The double-strand breaks observed in hyperinitiating cells therefore results from replication forks encountering single-stranded DNA lesions generated while removing oxidized bases, primarily 8-oxoG, from the DNA. We conclude that there is a delicate balance between chromosome replication and ROS inflicted DNA damage so the number of replication forks can only increase when ROS formation is reduced or when the pertinent repair is compromised.

Clustered DNA damage induces pan-nuclear H2AX phosphorylation mediated by ATM and DNA-PK
Meyer, B; Voss, KO; Tobias, F; Jakob, B; Durante, M; Taucher-Scholz, G
Nucleic Acids Res. 2013, 41, 6109-6118
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DNA double-strand breaks (DSB) are considered as the most deleterious DNA lesions, and their repair is further complicated by increasing damage complexity. However, the molecular effects of clustered lesions are yet not fully understood. As the locally restricted phosphorylation of H2AX to form gamma H2AX is a key step in facilitating efficient DSB repair, we investigated this process after localized induction of clustered damage by ionizing radiation. We show that in addition to foci at damaged sites, H2AX is also phosphorylated in undamaged chromatin over the whole-cell nucleus in human and rodent cells, but this is not related to apoptosis. This pan-nuclear gamma H2AX is mediated by the kinases ataxia telangiectasia mutated and DNA-dependent protein kinase (DNA-PK) that also phosphorylate H2AX at DSBs. The pan-nuclear response is dependent on the amount of DNA damage and is transient even under conditions of impaired DSB repair. Using fluorescence recovery after photobleaching (FRAP), we found that MDC1, but not 53BP1, binds to the nuclear-wide gamma H2AX. Consequently, the accumulation of MDC1 at DSBs is reduced...

Abundance of the Fanconi anaemia core complex is regulated by the RuvBL1 and RuvBL2 AAA+ ATPases
Rajendra, E; Garaycoechea, JI; Patel, KJ; Passmore, LA
Nucleic Acids Res. 2014, 42, 13736-13748
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Fanconi anaemia (FA) is a genome instability disease caused by defects in the FA DNA repair pathway that senses and repairs damage caused by DNA interstrand crosslinks. At least 8 of the 16 genes found mutated in FA encode proteins that assemble into the FA core complex, a multisubunit monoubiquitin E3 ligase. Here, we show that the RuvBL1 and RuvBL2 AAA+ ATPases co-purify with FA core complex isolated under stringent but native conditions from a vertebrate cell line. Depletion of the RuvBL1-RuvBL2 complex in human cells causes hallmark features of FA including DNA crosslinker sensitivity, chromosomal instability and defective FA pathway activation. Genetic knockout of RuvBL1 in a murine model is embryonic lethal while conditional inactivation in the haematopoietic stem cell pool confers profound aplastic anaemia. Together these findings reveal a function for RuvBL1-RuvBL2 in DNA repair through a physical and functional association with the FA core complex. Surprisingly, depletion of RuvBL1-RuvBL2 leads to co-depletion of the FA core complex in human cells...

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