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NAR Top Articles - Genome Integrity, Repair and Replication

Genome Integrity, Repair and Replication

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April 2015

Mutant DnaAs of Escherichia coli that are refractory to negative control
Chodavarapu, S; Felczak, MM; Simmons, LA; Murillo, A; Kaguni, JM
Nucleic Acids Res. 2013, 41, 10254-10267
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DnaA is the initiator of DNA replication in bacteria. A mutant DnaA named DnaAcos is unusual because it is refractory to negative regulation. We developed a genetic method to isolate other mutant DnaAs that circumvent regulation to extend our understanding of mechanisms that control replication initiation. Like DnaAcos, one mutant bearing a tyrosine substitution for histidine 202 (H202Y) withstands the regulation exerted by datA, hda and dnaN (beta clamp), and both DnaAcos and H202Y resist inhibition by the Hda-beta clamp complex in vitro. Other mutant DnaAs carrying G79D, E244K, V303M or E445K substitutions are either only partially sensitive or refractory to inhibition by the Hda-b clamp complex in vitro but are responsive to hda expression in vivo. All mutant DnaAs remain able to interact directly with Hda. Of interest, both DnaAcos and DnaAE244K bind more avidly to Hda. These mutants, by sequestrating Hda, may limit its availability to regulate other DnaA molecules, which remain active to induce extra rounds of DNA replication. Other evidence suggests that a mutant bearing a V292M substitution hyperinitiates by escaping the effect of an unknown regulatory factor...

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...

The Werner syndrome protein limits the error-prone 8-oxo-dG lesion bypass activity of human DNA polymerase kappa
Maddukuri, L; Ketkar, A; Eddy, S; Zafar, MK; Eoff, RL
Nucleic Acids Res. 2014, 42, 12027-12040
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Human DNA polymerase kappa (hpol kappa) is the only Y-family member to preferentially insert dAMP opposite 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) during translesion DNA synthesis. We have studied the mechanism of action by which hpol kappa activity is modulated by the Werner syndrome protein (WRN), a RecQ helicase known to influence repair of 8-oxo-dG. Here we show that WRN stimulates the 8-oxodG bypass activity of hpol kappa in vitro by enhancing the correct base insertion opposite the lesion, as well as extension from dC: 8-oxo-dG base pairs. Steady-state kinetic analysis reveals that WRN improves hpol kappa-catalyzed dCMP insertion opposite 8-oxo-dG similar to 10-fold and extension from dC: 8-oxo-dG by 2.4-fold. Stimulation is primarily due to an increase in the rate constant for polymerization (k(pol)), as assessed by pre-steady-state kinetics, and it requires the RecQ C-terminal (RQC) domain. In support of the functional data, recombinant WRN and hpol kappa were found to physically interact through the exo and RQC domains of WRN, and co-localization of WRN and hpol kappa was observed in human cells treated with hydrogen peroxide...

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...

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...

Replication of alpha-satellite DNA arrays in endogenous human centromeric regions and in human artificial chromosome
Erliandri, I; Fu, HQ; Nakano, M; Kim, JH; Miga, KH; Liskovykh, M; Earnshaw, WC; Masumoto, H; Kouprina, N; Aladjem, MI; Larionov, V
Nucleic Acids Res. 2014, 42, 11502-11516
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In human chromosomes, centromeric regions comprise megabase-size arrays of 171 bp alpha-satellite DNA monomers. The large distances spanned by these arrays preclude their replication from external sites and imply that the repetitive monomers contain replication origins. However, replication within these arrays has not previously been profiled and the role of alpha-satellite DNA in initiation of DNA replication has not yet been demonstrated. Here, replication of alpha-satellite DNA in endogenous human centromeric regions and in de novo formed (H) under bar uman (A) under bar rtificial (C) under bar hromosome (HAC) was analyzed. We showed that alpha-satellite monomers could function as origins of DNA replication and that replication of alphoid arrays organized into centrochromatin occurred earlier than those organized into heterochromatin. The distribution of inter-origin distances within centromeric alphoid arrays was comparable to the distribution of inter-origin distances on randomly selected non-centromeric chromosomal regions...

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...

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...

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