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NAR Top Articles - Genome Integrity, Repair and Replication

Genome Integrity, Repair and Replication

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January 2015


Efficient chromosomal gene modification with CRISPR/cas9 and PCR-based homologous recombination donors in cultured Drosophila cells
R. Bottcher, M. Hollmann, K. Merk, V. Nitschko, C. Obermaier, J. Philippou-Massier, I. Wieland, U. Gaul and K. Forstemann
Nucleic Acids Res. (2014) 42 (11): 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...

A non-catalytic role of DNA polymerase {eta} in recruiting Rad18 and promoting PCNA monoubiquitination at stalled replication forks
M. Durando, S. Tateishi and C. Vaziri
Nucleic Acids Res. (2013) 41 (5): 3079-3093
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Trans-lesion DNA synthesis (TLS) is a DNA damage-tolerance mechanism that uses low-fidelity DNA polymerases to replicate damaged DNA. The inherited cancer-propensity syndrome xeroderma pigmentosum variant (XPV) results from error-prone TLS of UV-damaged DNA. TLS is initiated when the Rad6/Rad18 complex monoubiquitinates proliferating cell nuclear antigen (PCNA), but the basis for recruitment of Rad18 to PCNA is not completely understood. Here, we show that Rad18 is targeted to PCNA by DNA polymerase eta (Poleta), the XPV gene product that is mutated in XPV patients. The C-terminal domain of Poleta binds to both Rad18 and PCNA and promotes PCNA monoubiquitination, a function unique to Poleta among Y-family TLS polymerases and dissociable from its catalytic activity. Importantly, XPV cells expressing full-length catalytically-inactive Poleta exhibit increased recruitment of other error-prone TLS polymerases (Polkappa and Poliota) after UV irradiation. These results define a novel non-catalytic role for Poleta in promoting PCNA monoubiquitination and provide a new potential mechanism for mutagenesis and genome instability in XPV individuals.

Replication of alpha-satellite DNA arrays in endogenous human centromeric regions and in human artificial chromosome
I. Erliandri, H. Fu, M. Nakano, J. H. Kim, K. H. Miga, M. Liskovykh, W. C. Earnshaw, H. Masumoto, N. Kouprina, M. I. Aladjem and V. Larionov
Nucleic Acids Res. (2014) 42 (18): 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 Human Artificial Chromosome (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...

Spatial organization of transcription machinery and its segregation from the replisome in fast-growing bacterial cells
C. Cagliero, Y. N. Zhou and D. J. Jin
Nucleic Acids Res. (2014) 42 (22): 13696-13705
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In a fast-growing Escherichia coli cell, most RNA polymerase (RNAP) is allocated to rRNA synthesis forming transcription foci at clusters of rrn operons or bacterial nucleolus, and each of the several nascent nucleoids contains multiple pairs of replication forks. The composition of transcription foci has not been determined. In addition, how the transcription machinery is three-dimensionally organized to promote cell growth in concord with replication machinery in the nucleoid remains essentially unknown. Here, we determine the spatial and functional landscapes of transcription and replication machineries in fast-growing E. coli cells using super-resolution-structured illumination microscopy. Co-images of RNAP and DNA reveal spatial compartmentation and duplication of the transcription foci at the surface of the bacterial chromosome, encompassing multiple nascent nucleoids. Transcription foci cluster with NusA and NusB, which are the rrn anti-termination system and are associated with nascent rRNAs. However, transcription foci tend to separate from SeqA and SSB foci, which track DNA replication forks and/or the replisomes...

Abundance of the Fanconi anaemia core complex is regulated by the RuvBL1 and RuvBL2 AAA+ ATPases
E. Rajendra, J. I. Garaycoechea, K. J. Patel and L. A. Passmore
Nucleic Acids Res. (2014) 42 (22): 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. This suggests that a potential mechanism for the role of RuvBL1-RuvBL2 in maintaining genome integrity...

Rad51/Dmc1 paralogs and mediators oppose DNA helicases to limit hybrid DNA formation and promote crossovers during meiotic recombination
A. Lorenz, A. Mehats, F. Osman and M. C. Whitby
Nucleic Acids Res. (2014) 42 (22): 13723-13735
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During meiosis programmed DNA double-strand breaks (DSBs) are repaired by homologous recombination using the sister chromatid or the homologous chromosome (homolog) as a template. This repair results in crossover (CO) and non-crossover (NCO) recombinants. Only CO formation between homologs provides the physical linkages guiding correct chromosome segregation, which are essential to produce healthy gametes. The factors that determine the CO/NCO decision are still poorly understood. Using Schizosaccharomyces pombe as a model we show that the Rad51/Dmc1-paralog complexes Rad55-Rad57 and Rdl1-Rlp1-Sws1 together with Swi5-Sfr1 play a major role in antagonizing both the FANCM-family DNA helicase/translocase Fml1 and the RecQ-type DNA helicase Rqh1 to limit hybrid DNA formation and promote Mus81-Eme1-dependent COs. A common attribute of these protein complexes is an ability to stabilize the Rad51/Dmc1 nucleoprotein filament, and we propose that it is this property that imposes constraints on which enzymes gain access to the recombination intermediate, thereby controlling the manner in which it is processed and resolved.

The intrinsically disordered amino-terminal region of human RecQL4: multiple DNA-binding domains confer annealing, strand exchange and G4 DNA binding
H. Keller, K. Kiosze, J. Sachsenweger, S. Haumann, O. Ohlenschlager, T. Nuutinen, J. E. Syvaoja, M. Gorlach, F. Grosse and H. Pospiech
Nucleic Acids Res. (2014) 42 (20): 12614-12627
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Human RecQL4 belongs to the ubiquitous RecQ helicase family. Its N-terminal region represents the only homologue of the essential DNA replication initiation factor Sld2 of Saccharomyces cerevisiae, and also participates in the vertebrate initiation of DNA replication. Here, we utilized a random screen to identify N-terminal fragments of human RecQL4 that could be stably expressed in and purified from Escherichia coli. Biophysical characterization of these fragments revealed that the Sld2 homologous RecQL4 N-terminal domain carries large intrinsically disordered regions. The N-terminal fragments were sufficient for the strong annealing activity of RecQL4. Moreover, this activity appeared to be the basis for an ATP-independent strand exchange activity. Both activities relied on multiple DNA-binding sites with affinities to single-stranded, double-stranded and Y-structured DNA. Finally, we found a remarkable affinity of the N-terminus for guanine quadruplex (G4) DNA, exceeding the affinities for other DNA structures by at least 60-fold...

The PARP inhibitor Olaparib disrupts base excision repair of 5-aza-2''-deoxycytidine lesions
M. L. Orta, A. Hoglund, J. M. Calderon-Montano, I. Dominguez, E. Burgos-Moron, T. Visnes, N. Pastor, C. Strom, M. Lopez-lazaro and T. Helleday
Nucleic Acids Res. (2014) 42 (14): 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...

BRCA1 modulates the autophosphorylation status of DNA-PKcs in S phase of the cell cycle
A. J. Davis, L. Chi, S. So, K. J. Lee, E. Mori, K. Fattah, J. Yang and D. J. Chen
Nucleic Acids Res. (2014) 42 (18): 11487-11501
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Non-homologous end-joining (NHEJ) and homologous recombination (HR) are the two prominent pathways responsible for the repair of DNA double-strand breaks (DSBs). NHEJ is not restricted to a cell-cycle stage, whereas HR is active primarily in the S/G2 phases suggesting there are cell cycle-specific mechanisms that play a role in the choice between NHEJ and HR. Here we show NHEJ is attenuated in S phase via modulation of the autophosphorylation status of the NHEJ factor DNA-PKcs at serine 2056 by the pro-HR factor BRCA1. BRCA1 interacts with DNA-PKcs in a cell cycle-regulated manner and this interaction is mediated by the tandem BRCT domain of BRCA1, but surprisingly in a phospho-independent manner. BRCA1 attenuates DNA-PKcs autophosphorylation via directly blocking the ability of DNA-PKcs to autophosphorylate. Subsequently, blocking autophosphorylation of DNA-PKcs at the serine 2056 phosphorylation cluster promotes HR-required DNA end processing and loading of HR factors to DSBs and is a possible mechanism by which BRCA1 promotes HR.

DNA damage triggers SAF-A and RNA biogenesis factors exclusion from chromatin coupled to R-loops removal
S. Britton, E. Dernoncourt, C. Delteil, C. Froment, O. Schiltz, B. Salles, P. Frit and P. Calsou
Nucleic Acids Res. (2014) 42 (14): 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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