Phosphorylation of Histone H2AX and Activation of Mre11, Rad50, and Nbs1 in Response to Replication-dependent DNA Double-strand Breaks Induced by Mammalian DNA Topoisomerase I Cleavage Complexes (original) (raw)
Related papers
Journal of Biological …, 2003
DNA double-strand breaks originating from diverse causes in eukaryotic cells are accompanied by the formation of phosphorylated H2AX (␥H2AX) foci. Here we show that ␥H2AX formation is also a cellular response to topoisomerase I cleavage complexes known to induce DNA double-strand breaks during replication. In HCT116 human carcinoma cells exposed to the topoisomerase I inhibitor camptothecin, the resulting ␥H2AX formation can be prevented with the phosphatidylinositol 3-OH kinase-related kinase inhibitor wortmannin; however, in contrast to ionizing radiation, only camptothecin-induced ␥H2AX formation can be prevented with the DNA replication inhibitor aphidicolin and enhanced with the checkpoint abrogator 7-hydroxystaurosporine. This ␥H2AX formation is suppressed in ATR (ataxia telangiectasia and Rad3-related) deficient cells and markedly decreased in DNA-dependent protein kinase-deficient cells but is not abrogated in ataxia telangiectasia cells, indicating that ATR and DNA-dependent protein kinase are the kinases primarily involved in ␥H2AX formation at the sites of replication-mediated DNA doublestrand breaks. Mre11-and Nbs1-deficient cells are still able to form ␥H2AX. However, H2AX؊/؊ mouse embryonic fibroblasts exposed to camptothecin fail to form Mre11, Rad50, and Nbs1 foci and are hypersensitive to camptothecin. These results demonstrate a conserved ␥H2AX response for double-strand breaks induced by replication fork collision. ␥H2AX foci are required for recruiting repair and checkpoint protein complexes to the replication break sites.
The EMBO Journal, 1999
Replication protein A (RPA) is a DNA single-strand binding protein essential for DNA replication, recombination and repair. In human cells treated with the topoisomerase inhibitors camptothecin or etoposide (VP-16), we find that RPA2, the middle-sized subunit of RPA, becomes rapidly phosphorylated. This response appears to be due to DNA-dependent protein kinase (DNA-PK) and to be independent of p53 or the ataxia telangiectasia mutated (ATM) protein. RPA2 phosphorylation in response to camptothecin required ongoing DNA replication. Camptothecin itself partially inhibited DNA synthesis, and this inhibition followed the same kinetics as DNA-PK activation and RPA2 phosphorylation. DNA-PK activation and RPA2 phosphorylation were prevented by the cell-cycle checkpoint abrogator 7-hydroxystaurosporine (UCN-01), which markedly potentiates camptothecin cytotoxicity. The DNA-PK catalytic subunit (DNA-PKcs) was found to bind RPA which was replaced by the Ku autoantigen upon camptothecin treatment. DNA-PKcs interacted directly with RPA1 in vitro. We propose that the encounter of a replication fork with a topoisomerase-DNA cleavage complex could lead to a juxtaposition of replication fork-associated RPA and DNA double-strand end-associated DNA-PK, leading to RPA2 phosphorylation which may signal the presence of DNA damage to an S-phase checkpoint mechanism.
Journal of Biological Chemistry, 2006
The Mre11⅐Rad50⅐Nbs1 (MRN) complex binds DNA double strand breaks to repair DNA and activate checkpoints. We report MRN deficiency in three of seven colon carcinoma cell lines of the NCI Anticancer Drug Screen. To study the involvement of MRN in replication-mediated DNA double strand breaks, we examined checkpoint responses to camptothecin, which induces replication-mediated DNA double strand breaks after replication forks collide with topoisomerase I cleavage complexes. MRN-deficient cells were deficient for Chk2 activation, whereas Chk1 activation was independent of MRN. Chk2 activation was ataxia telangiectasia mutated (ATM)-dependent and associated with phosphorylation of Mre11 and Nbs1. Mre11 complementation in MRNdeficient HCT116 cells restored Chk2 activation as well as Rad50 and Nbs1 levels. Conversely, Mre11 down-regulation by small interference RNA (siRNA) in HT29 cells inhibited Chk2 activation and down-regulated Nbs1 and Rad50. Proteasome inhibition also restored Rad50 and Nbs1 levels in HCT116 cells suggesting that Mre11 stabilizes Rad50 and Nbs1. Chk2 activation was also defective in three of four MRN-proficient colorectal cell lines because of low Chk2 levels. Thus, six of seven colon carcinoma cell lines from the NCI Anticancer Drug Screen are functionally Chk2-deficient in response to replication-mediated DNA double strand breaks. We propose that Mre11 stabilizes Nbs1 and Rad50 and that MRN activates Chk2 downstream from ATM in response to replication-mediated DNA double strand breaks. Chk2 deficiency in HCT116 is associated with defective S-phase checkpoint, prolonged G 2 arrest, and hypersensitivity to camptothecin. The high frequency of MRN and Chk2 deficiencies may contribute to genomic instability and therapeutic response to camptothecins in colorectal cancers.
1997
We previously identified a conserved multiprotein complex that includes hMre11 and hRad50. In this study, we used immunofluorescence to investigate the role of this complex in DNA double-strand break (DSB) repair. hMre11 and hRad50 form discrete nuclear foci in response to treatment with DSB-inducing agents but not in response to UV irradiation. hMre11 and hRad50 foci colocalize after treatment with ionizing radiation and are distinct from those of the DSB repair protein, hRad51. Our data indicate that an irradiated cell is competent to form either hMre11-hRad50 foci or hRad51 foci, but not both. The multiplicity of hMre11 and hRad50 foci is much higher in the DSB repair-deficient cell line 180BR than in repair-proficient cells. hMre11-hRad50 focus formation is markedly reduced in cells derived from ataxia-telangiectasia patients, whereas hRad51 focus formation is markedly increased. These experiments support genetic evidence from Saccharomyces cerevisiae indicating that Mre11-Rad50 have roles distinct from that of Rad51 in DSB repair. Further, these data indicate that hMre11-hRad50 foci form in response to DNA DSBs and are dependent upon a DNA damage-induced signaling pathway.
Pathways of DNA Double-Strand Break Repair during the Mammalian Cell Cycle
Molecular and Cellular Biology, 2003
Little is known about the quantitative contributions of nonhomologous end joining (NHEJ) and homologous recombination (HR) to DNA double-strand break (DSB) repair in different cell cycle phases after physiologically relevant doses of ionizing radiation. Using immunofluorescence detection of ␥-H2AX nuclear foci as a novel approach for monitoring the repair of DSBs, we show here that NHEJ-defective hamster cells (CHO mutant V3 cells) have strongly reduced repair in all cell cycle phases after 1 Gy of irradiation. In contrast, HR-defective CHO irs1SF cells have a minor repair defect in G 1 , greater impairment in S, and a substantial defect in late S/G 2 . Furthermore, the radiosensitivity of irs1SF cells is slight in G 1 but dramatically higher in late S/G 2 , while V3 cells show high sensitivity throughout the cell cycle. These findings show that NHEJ is important in all cell cycle phases, while HR is particularly important in late S/G 2 , where both pathways contribute to repair and radioresistance. In contrast to DSBs produced by ionizing radiation, DSBs produced by the replication inhibitor aphidicolin are repaired entirely by HR. irs1SF, but not V3, cells show hypersensitivity to aphidicolin treatment. These data provide the first evaluation of the cell cycle-specific contributions of NHEJ and HR to the repair of radiation-induced versus replication-associated DSBs.
Molecular Biology of the Cell, 2008
The interaction of ataxia-telangiectasia mutated (ATM) and the Mre11/Rad50/Nbs1 (MRN) complex is critical for the response of cells to DNA double-strand breaks; however, little is known of the role of these proteins in response to DNA replication stress. Here, we report a mutant allele of MRE11 found in a colon cancer cell line that sensitizes cells to agents causing replication fork stress. The mutant Mre11 weakly interacts with Rad50 relative to wild type and shows little affinity for Nbs1. The mutant protein lacks 3-5 exonuclease activity as a result of loss of part of the conserved nuclease domain; however, it retains binding affinity for single-stranded DNA (ssDNA), double-stranded DNA with a 3 singlestrand overhang, and fork-like structures containing ssDNA regions. In cells, the mutant protein shows a time-and dose-dependent accumulation in chromatin after thymidine treatment that corresponds with increased recruitment and hyperphosphorylation of replication protein A. ATM autophosphorylation, Mre11 foci, and thymidine-induced homologous recombination are suppressed in cells expressing the mutant allele. Together, our results suggest that the mutant Mre11 suppresses the cellular response to replication stress by binding to ssDNA regions at disrupted forks and impeding replication restart in a dominant negative manner.
Inviability of a DNA2 deletion mutant is due to the DNA damage checkpoint
Cell Cycle, 2011
Chromosomal DNA is subjected to constant damage not only from exogenous agents, such as ionizing radiation, ultraviolet radiation and base-damaging drugs, but also from damage arising from faulty DNA replication and spontaneous base loss. Unrepaired or misrepaired damage can lead to cell lethality or genome instability, such as translocations or aneuploidy. The DNA damage checkpoint involves a cascade of protein kinases that arrests or slows down the cell cycle, allowing time to repair the damage. Additional functions of the checkpoint include stabilizing the DNA replication fork and stimulating DNA repair. Yeast (Saccharomyces cerevisiae) Mec1 and Tel1 are two phosphoinositol kinase-related protein kinases that initiate the DNA damage response in response to single-strand and double-strand DNA breaks. 1,2 Two downstream kinases, Rad53 and Chk1, are activated by Mec1/Tel1 kinases after DNA damage, although Mec1 is the primary upstream kinase. 3 Activated Rad53 then positively regulates an additional kinase, Dun1. 4 Dun1 regulates levels of ribonucleotide reductase and arrests cells having DNA damage in late anaphase. 4 Additional activities of Rad53 after DNA damage include stimulation of DNA repair by phosphorylation of Rad55, inhibition of firing of late origins, induction of transcription of DNA repair genes and phosphorylation of the nuclease Exo1 and its apparent inhibition. 5-7 Exo1 is a 5' to 3' nuclease involved in mismatch base repair, double-strand break (DSB) repair and 5' to 3' degradation of uncapped telomeres. Dna2 is a dual polarity exo/endonuclease, and 5' to 3' DNA helicase involved in Okazaki Fragment Processing (OFP) and Double-Strand Break (DSB) Repair. In yeast, DNA2 is an essential gene, as expected for a DNA replication protein.
2020
ABSTRACTUnrepaired O6-methylguanine lesions induced by the alkylating chemotherapy agent temozolomide lead to replication-associated single-ended DNA double-strand breaks (seDSBs) that are repaired predominantly through RAD51-mediated homologous recombination (HR). Here, we show that loss of the pre-mRNA splicing and DNA repair protein XAB2 leads to increased temozolomide sensitivity in glioblastoma cells, which reflects abortive HR due to Ku retention on resected seDSBs. XAB2-dependent Ku eviction also occurred at seDSBs generated by the topoisomerase I poison campthotecin and operated in parallel to an ATM-dependent pathway previously described. Although Ku retention elicited by loss of XAB2 did not prevent RAD51 focus formation, the resulting RAD51-ssDNA associations were unproductive, leading to increased engagement of non-homologous-end-joining in S/G2 and genetic instability. Overexpression of RAD51 or the single-stranded DNA annealing factor RAD52 rescued the XAB2 defects. RA...
2007
b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 7 6 0 -7 7 2 telangiectasia-mutated; ATR, ATM and Rad3-related; DSBs, DNA doublestranded breaks; IR, ionizing radiation; chk1, checkpoint kinase 1; g-H2Ax, phosphorylated-histone 2Ax; sh/siRNA, short hairpin/short interference RNA; PARP, poly(ADP-ribose) polymerase; PCNA, proliferating cell nuclear antigen; MDC1, mediator of DNA damage checkpoint protein 1; 53BP, p53 binding protein 1; BRCA1, breast cancer 1 early onset; cdk2, cyclin dependent kinase 2; cdc25, cell division cycle 25; NBS1, Nijmegen breakage syndrome 1 a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b i o c h e m p h a r m 0006-2952/$ -see front matter #