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NAR Top Articles - Genome Integrity, Repair and Replication

Genome Integrity, Repair and Replication

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February 2015


Efficient chromosomal gene modification with CRISPR/cas9 and PCR-based homologous recombination donors in cultured Drosophila cells
Bottcher, R; Hollmann, M; Merk, K; Nitschko, V; Obermaier, C; Philippou-Massier, J; Wieland, I; Gaul, U; Forstemann, K
Nucleic Acids Res. 2014, 42, e89-e89
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The ability to edit the genome is essential for many state-of-the-art experimental paradigms. Since DNA breaks stimulate repair, they can be exploited to target site-specific integration. The clustered, regularly interspaced, short palindromic repeats (CRISPR)/cas9 system from Streptococcus pyogenes has been harnessed into an efficient and programmable nuclease for eukaryotic cells. We thus combined DNA cleavage by cas9, the generation of homologous recombination donors by polymerase chain reaction (PCR) and transient depletion of the non-homologous end joining factor lig4. Using cultured Drosophila melanogaster S2-cells and the phosphoglycerate kinase gene as a model, we reached targeted integration frequencies of up to 50% in drug-selected cell populations. Homology arms as short as 29 nt appended to the PCR primer resulted in detectable integration, slightly longer extensions are beneficial. We confirmed established rules for S. pyogenes cas9 sgRNA design and demonstrate that the complementarity region allows length variation and 5'-extensions...

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...

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...

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...

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...

Processing of double-R-loops in (CAG){middle dot}(CTG) and C9orf72 (GGGGCC){middle dot}(GGCCCC) repeats causes instability
Reddy, K; Schmidt, MHM; Geist, JM; Thakkar, NP; Panigrahi, GB; Wang, YH; Pearson, CE
Nucleic Acids Res. 2014, 42, 10473-10487
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R-loops, transcriptionally-induced RNA: DNA hybrids, occurring at repeat tracts (CTG)(n), (CAG)(n), (CGG)(n), (CCG)(n) and (GAA)(n), are associated with diseases including myotonic dystrophy, Huntington's disease, fragile X and Friedreich's ataxia. Many of these repeats are bidirectionally transcribed, allowing for single-and double-R-loop configurations, where either or both DNA strands may be RNA-bound. R-loops can trigger repeat instability at (CTG).(CAG) repeats, but the mechanism of this is unclear. We demonstrate R-loop-mediated instability through processing of R-loops by HeLa and human neuron-like cell extracts. Double-R-loops induced greater instability than single-R-loops. Pre-treatment with RNase H only partially suppressed instability, supporting a model in which R-loops directly generate instability by aberrant processing, or via slipped-DNA formation upon RNA removal and its subsequent aberrant processing. Slipped-DNAs were observed to form following removal of the RNA from R-loops...

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...

The human oncoprotein MDM2 induces replication stress eliciting early intra-S-phase checkpoint response and inhibition of DNA replication origin firing
Frum, RA; Singh, S; Vaughan, C; Mukhopadhyay, ND; Grossman, SR; Windle, B; Deb, S; Deb, SP
Nucleic Acids Res. 2014, 42, 926-940
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Conventional paradigm ascribes the cell proliferative function of the human oncoprotein mouse double minute2 (MDM2) primarily to its ability to degrade p53. Here we report that in the absence of p53, MDM2 induces replication stress eliciting an early S-phase checkpoint response to inhibit further firing of DNA replication origins. Partially synchronized lung cells cultured from p53-/-:MDM2 transgenic mice enter S phase and induce S-phase checkpoint response earlier than lung cells from p53-/- mice and inhibit firing of DNA replication origins. MDM2 activates chk1 phosphorylation, elevates mixed lineage lymphoma histone methyl transferase levels and promotes checkpoint-dependent tri-methylation of histone H3 at lysine 4, known to prevent firing of late replication origins at the early S phase. In the absence of p53, a condition that disables inhibition of cyclin A expression by MDM2, MDM2 increases expression of cyclin D2 and A and hastens S-phase entry of cells...

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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