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NAR Top Articles - Genome Integrity, Repair and Replication

Genome Integrity, Repair and Replication

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December 2013

Distinct roles for DNA-PK, ATM and ATR in RPA phosphorylation and checkpoint activation in response to replication stress
Liu, SQ; Opiyo, SO; Manthey, K; Glanzer, JG; Ashley, AK; Amerin, C; Troksa, K; Shrivastav, M; Nickoloff, JA; Oakley, GG
Nucleic Acids Res. (2012) 40 (21): 10780-10794
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DNA damage encountered by DNA replication forks poses risks of genome destabilization, a precursor to carcinogenesis. Damage checkpoint systems cause cell cycle arrest, promote repair and induce programed cell death when damage is severe. Checkpoints are critical parts of the DNA damage response network that act to suppress cancer. DNA damage and perturbation of replication machinery causes replication stress, characterized by accumulation of single-stranded DNA bound by replication protein A (RPA), which triggers activation of ataxia telangiectasia and Rad3 related (ATR) and phosphorylation of the RPA32, subunit of RPA, leading to Chk1 activation and arrest. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) [a kinase related to ataxia telangiectasia mutated (ATM) and ATR] has well characterized roles in DNA double-strand break repair, but poorly understood roles in replication stress-induced RPA phosphorylation. We show that DNA-PKcs mutant cells fail to arrest replication following stress...

Cockayne Syndrome group B protein interacts with TRF2 and regulates telomere length and stability
Batenburg, NL; Mitchell, TRH; Leach, DM; Rainbow, AJ; Zhu, XD
Nucleic Acids Res. (2012) 40 (19): 9661-9674
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The majority of Cockayne syndrome (CS) patients carry a mutation in Cockayne Syndrome group B (CSB), a large nuclear protein implicated in DNA repair, transcription and chromatin remodeling. However, whether CSB may play a role in telomere metabolism has not yet been characterized. Here, we report that CSB physically interacts with TRF2, a duplex telomeric DNA binding protein essential for telomere protection. We find that CSB localizes at a small subset of human telomeres and that it is required for preventing the formation of telomere dysfunction-induced foci (TIF) in CS cells. We find that CS cells or CSB knockdown cells accumulate telomere doublets, the suppression of which requires CSB. We find that overexpression of CSB in CS cells promotes telomerase-dependent telomere lengthening, a phenotype that is associated with a decrease in the amount of telomere-bound TRF1, a negative mediator of telomere length maintenance. Furthermore, we show that CS cells or CSB knockdown cells exhibit misregulation of TERRA, a large non-coding telomere repeat-containing RNA important for telomere maintenance...

The human RecQ helicases BLM and RECQL4 cooperate to preserve genome stability
Singh, DK; Popuri, V; Kulikowicz, T; Shevelev, I; Ghosh, AK; Ramamoorthy, M; Rossi, ML; Janscak, P; Croteau, DL; Bohr, VA
Nucleic Acids Res. (2012) 40 (14): 6632-6648
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Bacteria and yeast possess one RecQ helicase homolog whereas humans contain five RecQ helicases, all of which are important in preserving genome stability. Three of these, BLM, WRN and RECQL4, are mutated in human diseases manifesting in premature aging and cancer. We are interested in determining to which extent these RecQ helicases function cooperatively. Here, we report a novel physical and functional interaction between BLM and RECQL4. Both BLM and RECQL4 interact in vivo and in vitro. We have mapped the BLM interacting site to the N-terminus of RECQL4, comprising amino acids 361-478, and the region of BLM encompassing amino acids 1-902 interacts with RECQL4. RECQL4 specifically stimulates BLM helicase activity on DNA fork substrates in vitro. The in vivo interaction between RECQL4 and BLM is enhanced during the S-phase of the cell cycle, and after treatment with ionizing radiation. The retention of RECQL4 at DNA double-strand breaks is shortened in BLM-deficient cells. Further, depletion of RECQL4 in BLM-deficient cells leads to reduced proliferative capacity and an increased frequency of sister chromatid exchanges...

Site-directed mutagenesis of the {chi} subunit of DNA polymerase III and single-stranded DNA-binding protein of E. coli reveals key residues for their interaction
Naue, N; Fedorov, R; Pich, A; Manstein, DJ; Curth, U
Nucleic Acids Res. (2011) 39 (4): 1398-1407
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During DNA replication in Escherichia coli, single-stranded DNA-binding protein (SSB) protects single-stranded DNA from nuclease action and hairpin formation. It is known that the highly conserved C-terminus of SSB contacts the chi subunit of DNA polymerase III. However, there only exists a theoretical model in which the 11 C-terminal amino acids of SSB have been docked onto the surface of chi. In order to refine this model of SSB/chi interaction, we exchanged amino acids in chi and SSB by site-directed mutagenesis that are predicted to be of key importance. Detailed characterization of the interaction of these mutants by analytical ultracentrifugation shows that the interaction area is correctly predicted by the model; however, the SSB C-terminus binds in a different orientation to the chi surface. We show that evolutionary conserved residues of chi form a hydrophobic pocket to accommodate the ultimate two amino acids of SSB, P176 and F177. This pocket is surrounded by conserved basic residues, important for the SSB/chi interaction...

CBX4-mediated SUMO modification regulates BMI1 recruitment at sites of DNA damage
Ismail, IH; Gagne, JP; Caron, MC; McDonald, D; Xu, ZZ; Masson, JY; Poirier, GG; Hendzel, MJ
Nucleic Acids Res. (2012) 40 (12): 5497-5510
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Polycomb group (PcG) proteins are involved in epigenetic silencing where they function as major determinants of cell identity, stem cell pluripotency and the epigenetic gene silencing involved in cancer development. Recently numerous PcG proteins, including CBX4, have been shown to accumulate at sites of DNA damage. However, it remains unclear whether or not CBX4 or its E3 sumo ligase activity is directly involved in the DNA damage response (DDR). Here we define a novel role for CBX4 as an early DDR protein that mediates SUMO conjugation at sites of DNA lesions. DNA damage stimulates sumoylation of BMI1 by CBX4 at lysine 88, which is required for the accumulation of BMI1 at DNA damage sites. Moreover, we establish that CBX4 recruitment to the sites of laser micro-irradiation-induced DNA damage requires PARP activity but does not require H2AX, RNF8, BMI1 nor PI-3-related kinases. The importance of CBX4 in the DDR was confirmed by the depletion of CBX4, which resulted in decreased cellular resistance to ionizing radiation. Our results reveal a direct role for CBX4 in the DDR pathway.

Terminal deoxynucleotidyl transferase requires KU80 and XRCC4 to promote N-addition at non-V(D)J chromosomal breaks in non-lymphoid cells
Boubakour-Azzouz, I; Bertrand, P; Claes, A; Lopez, BS; Rougeon, F
Nucleic Acids Res. (2012) 40 (17): 8381-8391
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Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase that increases the repertoire of antigen receptors by adding non-templated nucleotides (N-addition) to V(D)J recombination junctions. Despite extensive in vitro studies on TdT catalytic activity, the partners of TdT that enable N-addition remain to be defined. Using an intrachromosomal substrate, we show here that, in Chinese hamter ovary (CHO) cells, ectopic expression of TdT efficiently promotes N-additions at the junction of chromosomal double-strand breaks (DSBs) generated by the meganuclease I-SceI and that the size of the N-additions is comparable with that at V(D)J junctions. Importantly, no N-addition was observed in KU80- or XRCC4-deficient cells. These data show that, in a chromosomal context of non-lymphoid cells, TdT is actually able to promote N-addition at non-V(D)J DSBs, through a process that strictly requires the components of the canonical non-homologous end-joining pathway, KU80 and XRCC4.

Role of SUMO modification of human PCNA at stalled replication fork
Gali, H; Juhasz, S; Morocz, M; Hajdu, I; Fatyol, K; Szukacsov, V; Burkovics, P; Haracska, L
Nucleic Acids Res. (2012) 40 (13): 6049-6059
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DNA double-strand breaks (DSBs) can be generated not only by reactive agents but also as a result of replication fork collapse at unrepaired DNA lesions. Whereas ubiquitylation of proliferating cell nuclear antigen (PCNA) facilitates damage bypass, modification of yeast PCNA by small ubiquitin-like modifier (SUMO) controls recombination by providing access for the Srs2 helicase to disrupt Rad51 nucleoprotein filaments. However, in human cells, the roles of PCNA SUMOylation have not been explored. Here, we characterize the modification of human PCNA by SUMO in vivo as well as in vitro. We establish that human PCNA can be SUMOylated at multiple sites including its highly conserved K164 residue and that SUMO modification is facilitated by replication factor C (RFC). We also show that expression of SUMOylation site PCNA mutants leads to increased DSB formation in the Rad18(-/-) cell line where the effect of Rad18-dependent K164 PCNA ubiquitylation can be ruled out. Moreover, expression of PCNA-SUMO1 fusion prevents DSB formation as well as inhibits recombination if replication stalls at DNA lesions...

CRL4-DDB1-VPRBP ubiquitin ligase mediates the stress triggered proteolysis of Mcm10
Kaur, M; Khan, MM; Kar, A; Sharma, A; Saxena, S
Nucleic Acids Res. (2012) 40 (15): 7332-7346
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When mammalian cells experience radiation insult, DNA replication is stalled to prevent erroneous DNA synthesis. UV-irradiation triggers proteolysis of Mcm10, an essential human replication factor, inhibiting the ongoing replication. Here, we report that Mcm10 associates with E3 ubiquitin ligase comprising DNA damage-binding protein, DDB1, cullin, Cul4 and ring finger protein, Roc1. Depletion of DDB1, Roc1 or Cul4 abrogates the UV-triggered Mcm10 proteolysis, implying that Cul4-Roc1-DDB1 ubiquitin ligase mediates Mcm10 downregulation. The purified Cul4-Roc1-DDB1 complex ubiquitinates Mcm10 in vitro, proving that Mcm10 is its substrate. By screening the known DDB1 interacting proteins, we discovered that VprBP is the substrate recognition subunit that targets Mcm10 for degradation. Hence, these results establish that Cul4-DDB1-VprBP ubiquitin ligase mediates the stress-induced proteolysis of replication factor, Mcm10.

DNA polymerase zeta is required for proliferation of normal mammalian cells
Lange, SS; Wittschieben, JP; Wood, RD
Nucleic Acids Res. (2012) 40 (10): 4473-4482
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Unique among translesion synthesis (TLS) DNA polymerases, pol zeta is essential during embryogenesis. To determine whether pol zeta is necessary for proliferation of normal cells, primary mouse fibroblasts were established in which Rev3L could be conditionally inactivated by Cre recombinase. Cells were grown in 2% O-2 to prevent oxidative stress-induced senescence. Cells rapidly became senescent or apoptotic and ceased growth within 3-4 population doublings. Within one population doubling following Rev3L deletion, DNA double-strand breaks and chromatid aberrations were found in 30-50% of cells. These breaks were replication dependent, and found in G1 and G2 phase cells. Double-strand breaks were reduced when cells were treated with the reactive oxygen species scavenger N-acetyl-cysteine, but this did not rescue the cell proliferation defect, indicating that several classes of endogenously formed DNA lesions require Rev3L for tolerance or repair. T-antigen immortalization of cells allowed cell growth. In summary, even in the absence of external challenges to DNA, pol zeta is essential for preventing replication-dependent DNA breaks in every division of normal mammalian cells...

RI-1: a chemical inhibitor of RAD51 that disrupts homologous recombination in human cells
Budke, B; Logan, HL; Kalin, JH; Zelivianskaia, AS; McGuire, WC; Miller, LL; Stark, JM; Kozikowski, AP; Bishop, DK; Connell, PP
Nucleic Acids Res. (2012) 40 (15): 7347-7357
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Homologous recombination serves multiple roles in DNA repair that are essential for maintaining genomic stability. We here describe RI-1, a small molecule that inhibits the central recombination protein RAD51. RI-1 specifically reduces gene conversion in human cells while stimulating single strand annealing. RI-1 binds covalently to the surface of RAD51 protein at cysteine 319 that likely destabilizes an interface used by RAD51 monomers to oligomerize into filaments on DNA. Correspondingly, the molecule inhibits the formation of sub-nuclear RAD51 foci in cells following DNA damage, while leaving replication protein A focus formation unaffected. Finally, it potentiates the lethal effects of a DNA cross-linking drug in human cells. Given that this inhibitory activity is seen in multiple human tumor cell lines, RI-1 holds promise as an oncologic drug. Furthermore, RI-1 represents a unique tool to dissect the network of reaction pathways that contribute to DNA repair in cells.

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