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1.
《Cell cycle (Georgetown, Tex.)》2013,12(11):1765-1774
The Rad9/Rad1/Hus1 complex functions to facilitate the ATR-mediated phosphorylation of several substrates that control the checkpoint arrest induced by DNA damage. Here we show that in response to genotoxic stress induced by different types of damaging agents, Rad9 rapidly relocalized to sites of single stranded DNA, as visualized by discrete nuclear foci that co-localize with RPA. UV light-induced Rad9 foci also colocalized with TopBP1 and γ-H2AX. Interestingly, Rad9 foci were predominately formed in G1 and S phase after UV light, while treatment of cells with ionizing radiation (IR) resulted in accumulation of Rad9 into foci in S and G2. Photobleaching experiments in living cells revealed that the Rad9 protein is highly mobile in undamaged cells. However, genotoxic stress induced the immobilization of a large proportion of the protein. The proportion of Rad9 immobilization was larger in S phase and the accumulation to sites of locally damaged areas induced by UV-laser irradiation was faster during DNA replication. Inactivation of nucleotide excision repair by knock down of XPA and XPC resulted in a decrease of G1 phase cells that displayed Rad9 foci in response to UV light, whereas IR-induced Rad9 foci were not affected. In contrast, downregulation of CtIP, which promotes DSB resection, abrogated the IR-induced Rad9 foci. These findings show that due to processing of DNA lesions into a common intermediate, which occurs in a cell cycle-dependent manner, Rad9 is able to respond to different types of genotoxic stress. 相似文献
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Tan TL Essers J Citterio E Swagemakers SM de Wit J Benson FE Hoeijmakers JH Kanaar R 《Current biology : CB》1999,9(6):325-328
Error-free repair by homologous recombination of DNA double-strand breaks induced by ionizing radiation (IR) requires the Rad52 group proteins, including Rad51 and Rad54, in the yeast Saccharomyces cerevisiae [1]. The formation of a 'joint' molecule between the damaged DNA and the homologous repair template is a key step in recombination mediated by Rad51 and stimulated by Rad54 [2] [3] [4] [5]. Mammalian homologs of Rad51 and Rad54 have been identified [2] [3] [6]. Here, we demonstrate that mouse Rad54 (mRad54) formed IR-induced nuclear foci that colocalized with mRad51. Interaction between mRad51 and mRad54 was induced by genotoxic stress, but only when lesions that required mRad54 for their repair were formed. Interestingly, mRad54 was essential for the formation of IR-induced mRad51 foci. Rad54 belongs to the SWI2/SNF2 protein family, members of which modulate protein-DNA interactions in an ATP-driven manner [7]. Results of a topological assay suggested that purified human Rad54 (hRad54) protein can unwind double-stranded (ds) DNA at the expense of ATP hydrolysis. Unwinding of the homologous repair template could promote the formation or stabilization of hRad51-mediated joint molecules. Rad54 appears to be required downstream of other Rad52 group proteins, such as Rad52 and the Rad55-Rad57 heterodimer, that assist Rad51 in interacting with the broken DNA [2] [3] [4]. 相似文献
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A DNA Damage-Regulated BRCT-Containing Protein, TopBP1, Is Required for Cell Survival 总被引:15,自引:0,他引:15
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BRCA1 carboxyl-terminal (BRCT) motifs are present in a number of proteins involved in DNA repair and/or DNA damage-signaling pathways. Human DNA topoisomerase II binding protein 1 (TopBP1) contains eight BRCT motifs and shares sequence similarity with the fission yeast Rad4/Cut5 protein and the budding yeast DPB11 protein, both of which are required for DNA damage and/or replication checkpoint controls. We report here that TopBP1 is phosphorylated in response to DNA double-strand breaks and replication blocks. TopBP1 forms nuclear foci and localizes to the sites of DNA damage or the arrested replication forks. In response to DNA strand breaks, TopBP1 phosphorylation depends on the ataxia telangiectasia mutated protein (ATM) in vivo. However, ATM-dependent phosphorylation of TopBP1 does not appear to be required for focus formation following DNA damage. Instead, focus formation relies on one of the BRCT motifs, BRCT5, in TopBP1. Antisense Morpholino oligomers against TopBP1 greatly reduced TopBP1 expression in vivo. Similar to that of ataxia telangiectasia-related protein (ATR), Chk1, or Hus1, downregulation of TopBP1 leads to reduced cell survival, probably due to increased apoptosis. Taken together, the data presented here suggest that, like its putative counterparts in yeast species, TopBP1 may be involved in DNA damage and replication checkpoint controls. 相似文献
6.
Role for caspase-mediated cleavage of Rad51 in induction of apoptosis by DNA damage 总被引:4,自引:0,他引:4
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Huang Y Nakada S Ishiko T Utsugisawa T Datta R Kharbanda S Yoshida K Talanian RV Weichselbaum R Kufe D Yuan ZM 《Molecular and cellular biology》1999,19(4):2986-2997
We report here that the Rad51 recombinase is cleaved in mammalian cells during the induction of apoptosis by ionizing radiation (IR) exposure. The results demonstrate that IR induces Rad51 cleavage by a caspase-dependent mechanism. Further support for involvement of caspases is provided by the finding that IR-induced proteolysis of Rad51 is inhibited by Ac-DEVD-CHO. In vitro studies show that Rad51 is cleaved by caspase 3 at a DVLD/N site. Stable expression of a Rad51 mutant in which the aspartic acid residues were mutated to alanines (AVLA/N) confirmed that the DVLD/N site is responsible for the cleavage of Rad51 in IR-induced apoptosis. The functional significance of Rad51 proteolysis is supported by the finding that, unlike intact Rad51, the N- and C-terminal cleavage products fail to exhibit recombinase activity. In cells, overexpression of the Rad51(D-A) mutant had no effect on activation of caspase 3 but did abrogate in part the apoptotic response to IR exposure. We conclude that proteolytic inactivation of Rad51 by a caspase-mediated mechanism contributes to the cell death response induced by DNA damage. 相似文献
7.
Megan McKerlie John R. Walker Taylor R. H. Mitchell Florence R. Wilson Xu-Dong Zhu 《Nucleic acids research》2013,41(22):10268-10282
TRF1, a duplex telomeric DNA-binding protein, plays an important role in telomere metabolism. We have previously reported that a fraction of endogenous TRF1 can stably exist free of telomere chromatin when it is phosphorylated at T371 by Cdk1; however, the role of this telomere-free (pT371)TRF1 has yet to be fully characterized. Here we show that phosphorylated (pT371)TRF1 is recruited to sites of DNA damage, forming damage-induced foci in response to ionizing radiation (IR), etoposide and camptothecin. We find that IR-induced (pT371)TRF1 foci formation is dependent on the ATM- and Mre11/Rad50/Nbs1-mediated DNA damage response. While loss of functional BRCA1 impairs the formation of IR-induced (pT371)TRF1 foci, depletion of either 53BP1 or Rif1 stimulates IR-induced (pT371)TRF1 foci formation. In addition, we show that TRF1 depletion or the lack of its phosphorylation at T371 impairs DNA end resection and repair of nontelomeric DNA double-strand breaks by homologous recombination. The lack of TRF1 phosphorylation at T371 also hampers the activation of the G2/M checkpoint and sensitizes cells to PARP inhibition, IR and camptothecin. Collectively, these results reveal a novel but important function of phosphorylated (pT371)TRF1 in facilitating DNA double-strand break repair and the maintenance of genome integrity. 相似文献
8.
《Cell cycle (Georgetown, Tex.)》2013,12(2):308-322
The promyelocytic leukemia (PML) protein is the main structural component of subnuclear domains termed PML nuclear bodies (PML NBs), which are implicated in tumor suppression by regulating apoptosis, cell senescence, and DNA repair. Previously, we demonstrated that ATM kinase can regulate changes in PML NB number in response to DNA double-strand breaks (DSBs). PML NBs make extensive contacts with chromatin and ATM mediates DNA damage-dependent changes in chromatin structure in part by the phosphorylation of the KRAB-associated protein 1 (KAP1) at S824. We now demonstrate that in the absence of DNA damage, reduced KAP1 expression results in a constitutive increase in PML NB number in both human U2-OS cells and normal human diploid fibroblasts. This increase in PML NB number correlated with decreased nuclear lamina-associated heterochromatin and a 30% reduction in chromatin density as observed by electron microscopy, which is reminiscent of DNA damaged chromatin. These changes in chromatin ultrastructure also correlated with increased histone H4 acetylation, and treatment with the HDAC inhibitor TSA failed to further increase PML NB number. Although PML NB number could be restored by complementation with wild-type KAP1, both the loss of KAP1 or complementation with phospho-mutants of KAP1 inhibited the early increase in PML NB number and reduced the fold induction of PML NBs by 25-30% in response to etoposide-induced DNA DSBs. Together these data implicate KAP1-dependent changes in chromatin structure as one possible mechanism by which ATM may regulate PML NB number in response to DNA damage. 相似文献
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The RAD52 epistasis group of proteins, including Rad51, Rad52, and Rad54, plays an important role in the homologous recombination repair of double strand breaks. A well characterized feature associated with the ability of these proteins to repair double strand breaks is inducible nuclear foci formation at the sites of damage. How the process is functionally regulated in response to DNA damage, however, remains elusive. We show here that c-Abl tyrosine kinase associates with and phosphorylates Rad52 on tyrosine 104. Importantly, the very same site of Rad52 is phosphorylated on exposure of cells to ionizing radiation (IR). The functional significance of c-Abl-dependent phosphorylation of Rad52 is underscored by our findings that cells that express the phosphorylation-resistant Rad52 mutant, in which tyrosine 104 is replaced by phenylalanine, exhibit compromised nuclear foci formation in response to IR. Furthermore, IR-induced Rad52 nuclear foci formation is markedly suppressed by the expression of dominant-negative c-Abl. Together our data support a mode of post-translational regulation of Rad52 mediated by the c-Abl tyrosine kinase. 相似文献
10.
Furuya K Miyabe I Tsutsui Y Paderi F Kakusho N Masai H Niki H Carr AM 《Molecular cell》2010,40(4):606-618
When inappropriate DNA structures arise, they are sensed by DNA structure-dependent checkpoint pathways and subsequently repaired. Recruitment of checkpoint proteins to such structures precedes recruitment of proteins involved in DNA metabolism. Thus, checkpoints can regulate DNA metabolism. We show that fission yeast Rad9, a 9-1-1 heterotrimeric checkpoint-clamp component, is phosphorylated by Hsk1(Cdc7), the Schizosaccharomyces pombe?Dbf4-dependent kinase (DDK) homolog, in response to replication-induced DNA damage. Phosphorylation of Rad9 disrupts its interaction with replication protein A (RPA) and is dependent on 9-1-1 chromatin loading, the Rad9-associated protein Rad4/Cut5(TopBP1), and prior phosphorylation by Rad3(ATR). rad9 mutants defective in DDK phosphorylation show wild-type checkpoint responses but abnormal DNA repair protein foci and decreased viability after replication stress. We propose that Rad9 phosphorylation by DDK releases Rad9 from DNA damage sites to facilitate DNA repair. 相似文献
11.
《DNA Repair》2019
The bulk of DNA damage caused by ionizing radiation (IR) is generally repaired within hours, yet a subset of DNA lesions may persist even for long periods of time. Such persisting IR-induced foci (pIRIF) co-associate with PML nuclear bodies (PML-NBs) and are among the characteristics of cellular senescence. Here we addressed some fundamental questions concerning the nature and determinants of this co-association, the role of PML-NBs at such sites, and the reason for the persistence of DNA damage in human primary cells. We show that the persistent DNA lesions are devoid of homologous recombination (HR) proteins BRCA1 and Rad51. Our super-resolution microscopy-based analysis showed that PML-NBs are juxtaposed to and partially overlap with the pIRIFs. Notably, depletion of 53BP1 resulted in decreased intersection between PML-NBs and pIRIFs implicating the RNF168-53BP1 pathway in their interaction. To test whether the formation and persistence of IRIFs is PML-dependent and to investigate the role of PML in the context of DNA repair and senescence, we genetically deleted PML in human hTERT-RPE-1 cells. Unexpectedly, upon high-dose IR treatment, cells displayed similar DNA damage signalling, repair dynamics and kinetics of cellular senescence regardless of the presence or absence of PML. In contrast, the PML knock-out cells showed increased sensitivity to low doses of IR and DNA-damaging agents mitomycin C, cisplatin and camptothecin that all cause DNA lesions requiring repair by HR. These results, along with enhanced sensitivity of the PML knock-out cells to DNA-PK and PARP inhibitors implicate PML as a factor contributing to HR-mediated DNA repair. 相似文献
12.
Phosphorylation of BLM, dissociation from topoisomerase IIIalpha, and colocalization with gamma-H2AX after topoisomerase I-induced replication damage 总被引:1,自引:0,他引:1
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Rao VA Fan AM Meng L Doe CF North PS Hickson ID Pommier Y 《Molecular and cellular biology》2005,25(20):8925-8937
Topoisomerase I-associated DNA single-strand breaks selectively trapped by camptothecins are lethal after being converted to double-strand breaks by replication fork collisions. BLM (Bloom's syndrome protein), a RecQ DNA helicase, and topoisomerase IIIalpha (Top3alpha) appear essential for the resolution of stalled replication forks (Holliday junctions). We investigated the involvement of BLM in the signaling response to Top1-mediated replication DNA damage. In BLM-complemented cells, BLM colocalized with promyelocytic leukemia protein (PML) nuclear bodies and Top3alpha. Fibroblasts without BLM showed an increased sensitivity to camptothecin, enhanced formation of Top1-DNA complexes, and delayed histone H2AX phosphorylation (gamma-H2AX). Camptothecin also induced nuclear relocalization of BLM, Top3alpha, and PML protein and replication-dependent phosphorylation of BLM on threonine 99 (T99p-BLM). T99p-BLM was also observed following replication stress induced by hydroxyurea. Ataxia telangiectasia mutated (ATM) protein and AT- and Rad9-related protein kinases, but not DNA-dependent protein kinase, appeared to play a redundant role in phosphorylating BLM. Following camptothecin treatment, T99p-BLM colocalized with gamma-H2AX but not with Top3alpha or PML. Thus, BLM appears to dissociate from Top3alpha and PML following its phosphorylation and facilitates H2AX phosphorylation in response to replication double-strand breaks induced by Top1. A defect in gamma-H2AX signaling in response to unrepaired replication-mediated double-strand breaks might, at least in part, explain the camptothecin-sensitivity of BLM-deficient cells. 相似文献
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Rad4TopBP1, a scaffold protein, plays separate roles in DNA damage and replication checkpoints and DNA replication 总被引:1,自引:0,他引:1
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Rad4TopBP1, a BRCT domain protein, is required for both DNA replication and checkpoint responses. Little is known about how the multiple roles of Rad4TopBP1 are coordinated in maintaining genome integrity. We show here that Rad4TopBP1 of fission yeast physically interacts with the checkpoint sensor proteins, the replicative DNA polymerases, and a WD-repeat protein, Crb3. We identified four novel mutants to investigate how Rad4TopBP1 could have multiple roles in maintaining genomic integrity. A novel mutation in the third BRCT domain of rad4+TopBP1 abolishes DNA damage checkpoint response, but not DNA replication, replication checkpoint, and cell cycle progression. This mutant protein is able to associate with all three replicative polymerases and checkpoint proteins Rad3ATR-Rad26ATRIP, Hus1, Rad9, and Rad17 but has a compromised association with Crb3. Furthermore, the damaged-induced Rad9 phosphorylation is significantly reduced in this rad4TopBP1 mutant. Genetic and biochemical analyses suggest that Crb3 has a role in the maintenance of DNA damage checkpoint and influences the Rad4TopBP1 damage checkpoint function. Taken together, our data suggest that Rad4TopBP1 provides a scaffold to a large complex containing checkpoint and replication proteins thereby separately enforcing checkpoint responses to DNA damage and replication perturbations during the cell cycle. 相似文献
15.
Yoon Jeon Mi Kyung Park Sun Mi Kim June Sung Bae Chang Woo Lee Ho Lee 《Biochemical and biophysical research communications》2019,508(3):722-728
Topoisomerase IIβ-binding protein 1 (TopBP1) is BRCT domain-containing protein that is required for DNA double-strand break (DSB) repair and DNA damage responses; however, its function during the early stage of spermatogenesis is still unclear. To investigate the physiological role of TopBP1, we have generated germ cell-specific TopBP1-depleted mouse model. TopBP1-deleted mice were infertile, showed a loss of germ cells and had meiotic defects. Conditional TopBP1 deletion resulted in reduced testis size, reduced number of epididymal sperm, increased apoptosis, and severely compromised fertility. TopBP1 deficiency caused defects in DMC1 and Rad51 foci formation, abnormal synaptonemal complexes and meiotic chromosome defects. Collectively, these results suggest that TopBP1 deficiency during spermatogenesis impairs the localization of proteins involved in early recombination at DSBs, results in meiotic chromosome defects and leads to infertility. 相似文献
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The Mre11 complex has been implicated in diverse aspects of the cellular response to DNA damage. We used in situ fractionation of human fibroblasts to carry out cytologic analysis of Mre11 complex proteins in the double-strand break (DSB) response. In situ fractionation removes most nucleoplasmic protein, permitting immunofluorescent localization of proteins that become more avidly bound to nuclear structures after induction of DNA damage. We found that a fraction of the Mre11 complex was bound to promyelocyte leukemia protein bodies in undamaged cells. Within 10 min after gamma irradiation, nuclear retention of the Mre11 complex in small granular foci was observed and persisted until 2 h postirradiation. In light of the previous demonstration that the Mre11 complex associated with ionizing radiation (IR)-induced DSBs, we infer that the protein retained under these conditions was associated with DNA damage. We also observed increased retention of Rad51 following IR treatment, although IR induced Rad51 foci were distinct from Mre11 foci. The ATM kinase, which phosphorylates Nbs1 during activation of the S-phase checkpoint, was not required for the Mre11 complex to associate with DNA damage. These data suggest that the functions of the Mre11 complex in the DSB response are implicitly dependent upon its ability to detect DNA damage. 相似文献
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DPB11/TopBP1 is an essential evolutionarily conserved gene involved in initiation of DNA replication and checkpoint signaling. Here, we show that Saccharomyces cerevisiae Dpb11 forms nuclear foci that localize to sites of DNA damage in G1, S and G2 phase, a recruitment that is conserved for its homologue TopBP1 in Gallus gallus. Damage-induced Dpb11 foci are distinct from Sld3 replication initiation foci. Further, Dpb11 foci are dependent on the checkpoint proteins Mec3 (9-1-1 complex) and Rad24, and require the C-terminal domain of Dpb11. Dpb11 foci are independent of the checkpoint kinases Mec1 and Tel1, and of the checkpoint mediator Rad9. In a site-directed mutagenesis screen, we identify a separation-of-function mutant, dpb11-PF, that is sensitive to DSB-inducing agents yet remains proficient for DNA replication and the S-phase checkpoint at the permissive temperature. The dpb11-PF mutant displays altered rates of heteroallelic and direct-repeat recombination, sensitivity to DSB-inducing drugs as well as delayed kinetics of mating-type switching with a defect in the DNA synthesis step thus implicating Dpb11 in homologous recombination. We conclude that Dpb11/TopBP1 plays distinct roles in replication, checkpoint response and recombination processes, thereby contributing to chromosomal stability. 相似文献
18.
ATM-dependent phosphorylation of human Rad9 is required for ionizing radiation-induced checkpoint activation 总被引:9,自引:0,他引:9
Chen MJ Lin YT Lieberman HB Chen G Lee EY 《The Journal of biological chemistry》2001,276(19):16580-16586
ATM (ataxia-telangiectasia-mutated) is a Ser/Thr kinase involved in cell cycle checkpoints and DNA repair. Human Rad9 (hRad9) is the homologue of Schizosaccharomyces pombe Rad9 protein that plays a critical role in cell cycle checkpoint control. To examine the potential signaling pathway linking ATM and hRad9, we investigated the modification of hRad9 in response to DNA damage. Here we show that hRad9 protein is constitutively phosphorylated in undamaged cells and undergoes hyperphosphorylation upon treatment with ionizing radiation (IR), ultraviolet light (UV), and hydroxyurea (HU). Interestingly, hyperphosphorylation of hRad9 induced by IR is dependent on ATM. Ser(272) of hRad9 is phosphorylated directly by ATM in vitro. Furthermore, hRad9 is phosphorylated on Ser(272) in response to IR in vivo, and this modification is delayed in ATM-deficient cells. Expression of hRad9 S272A mutant protein in human lung fibroblast VA13 cells disturbs IR-induced G(1)/S checkpoint activation and increased cellular sensitivity to IR. Together, our results suggest that the ATM-mediated phosphorylation of hRad9 is required for IR-induced checkpoint activation. 相似文献
19.
Jean-Marc Brondello Michael N. Boddy Beth Furnari Paul Russell 《Molecular and cellular biology》1999,19(6):4262-4269
Six checkpoint Rad proteins (Rad1, Rad3, Rad9, Rad17, Rad26, and Hus1) are needed to regulate checkpoint protein kinases Chk1 and Cds1 in fission yeast. Chk1 is required to prevent mitosis when DNA is damaged by ionizing radiation (IR), whereas either kinase is sufficient to prevent mitosis when DNA replication is inhibited by hydroxyurea (HU). Checkpoint Rad proteins are required for IR-induced phosphorylation of Chk1 and HU-induced activation of Cds1. IR activates Cds1 only during the DNA synthesis (S) phase, whereas HU induces Chk1 phosphorylation only in cds1 mutants. Here, we investigate the basis of the checkpoint signal specificity of Chk1 phosphorylation and Cds1 activation. We show that IR fails to induce Chk1 phosphorylation in HU-arrested cells. Release from the HU arrest following IR causes substantial Chk1 phosphorylation. These and other data indicate that Cds1 prevents Chk1 phosphorylation in HU-arrested cells, which suggests that Cds1 actively suppresses a repair process that leads to Chk1 phosphorylation. Cds1 becomes more highly concentrated in the nucleus only during the S phase of the cell cycle. This finding correlates with S-phase specificity of IR-induced activation of Cds1. However, constitutive nuclear localization of Cds1 does not enhance IR-induced activation of Cds1. This result suggests that Cds1 activation requires DNA structures or protein activities that are present only during S phase. These findings help to explain how Chk1 and Cds1 respond to different checkpoint signals. 相似文献
20.
Kerstin Herzer Anna Carbow Svenja Sydor Jan-Peter Sowa Stefan Biesterfeld Thomas-Georg Hofmann Peter-Robert Galle Guido Gerken Ali Canbay 《PloS one》2012,7(9)
Overwhelming lines of epidemiological evidence have indicated that persistent infection with hepatitis C virus (HCV) is a major risk for the development of hepatocellular carcinoma (HCC). We have recently shown that HCV core protein mediates functional inactivation of the promyelocytic leukemia (PML) tumor suppressor pathway. However, the role of PML in HCC development yet remains unclear. To clarify the function of PML in liver carcinogenesis and HCV-associated pathogenesis we crossed PML-deficient mice with HCV transgene (HCV-Tg) expressing mice and treated the resulting animals with DEN/Phenobarbital, an established protocol for liver carcinogenesis. Seven months after treatment, livers were examined macroscopically and histologically. Genetic depletion of the tumor suppressor PML coincided with an increase in hepatocyte proliferation, resulting in development of multiple dysplastic nodules in 100% of the PML-deficient livers and of HCCs in 53%, establishing a tumor suppressive function of PML in the liver. In animals expressing the HCV-transgene in PML-deficient background, HCC development occurred even in 73%, while only 7% of their wildtype littermates developed HCC. The neoplastic nature of the tumors was confirmed by histology and expression of the HCC marker glutamine synthetase. Several pro- and antiapoptotic factors were tested for differential expression and liver carcinogenesis was associated with impaired expression of the proapoptotic molecule TRAIL in PML-deficient mice. In conclusion, this study provides first in vivo evidence that the tumor suppressor PML acts as an important barrier in liver carcinogenesis and HCV-dependent liver pathology. 相似文献