The Intra-S Phase Checkpoint Targets Dna2 to Prevent Stalled Replication Forks from Reversing

Hu, Jiazhi; Sun, Lei; Shen, Fenfen; Chen, Yufei; Yu, Hua-Zhong; Liu, Yang; Zhang, Mian; Hu, Yiren; Wang, Qingsong; Xu, Wei; Sun, Fei; Ji, Jianguo; Murray, Johanne M.; Carr, Antony; Kong, Delong

doi:10.1016/j.cell.2012.04.030

Cited by 147 publications

(173 citation statements)

References 44 publications

(62 reference statements)

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“…7). These restart events may entail nucleolytic degradation rather than branch migration of the reversed forks (12), in agreement with the nucleolytic processing of reversed forks previously reported in yeast (38,39). Regardless of their source, postreplicative ssDNA gaps can certainly contribute to explain the observed accumulation of RAD51 in PARG-depleted cells.…”

Section: Discussionsupporting

confidence: 70%

Poly(ADP-Ribosyl) Glycohydrolase Prevents the Accumulation of Unusual Replication Structures during Unperturbed S Phase

Chaudhuri

Ahuja

Herrador

et al. 2015

Molecular and Cellular Biology

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Poly(ADP-ribosyl)ation (PAR) has been implicated in various aspects of the cellular response to DNA damage and genome stability. Although 17 human poly(ADP-ribose) polymerase (PARP) genes have been identified, a single poly(ADP-ribosyl) glycohydrolase (PARG) mediates PAR degradation. Here we investigated the role of PARG in the replication of human chromosomes. We show that PARG depletion affects cell proliferation and DNA synthesis, leading to replication-coupled H2AX phosphorylation. Furthermore, PARG depletion or inhibition per se slows down individual replication forks similarly to mild chemotherapeutic treatment. Electron microscopic analysis of replication intermediates reveals marked accumulation of reversed forks and single-stranded DNA (ssDNA) gaps in unperturbed PARG-defective cells. Intriguingly, while we found no physical evidence for chromosomal breakage, PARG-defective cells displayed both ataxia-telangiectasia-mutated (ATM) and ataxia-Rad3-related (ATR) activation, as well as chromatin recruitment of standard double-strand-break-repair factors, such as 53BP1 and RAD51. Overall, these data prove PAR degradation to be essential to promote resumption of replication at endogenous and exogenous lesions, preventing idle recruitment of repair factors to remodeled replication forks. Furthermore, they suggest that fork remodeling and restarting are surprisingly frequent in unperturbed cells and provide a molecular rationale to explore PARG inhibition in cancer chemotherapy.C ellular responses are crucial for the adaptability and survival of a cell exposed to different types of endogenous and exogenous stress. The DNA damage response (DDR) consists of one such defense mechanism in response to different types of insults to the DNA. Poly(ADP)ribosylation of proteins is one of the quickest cellular responses to DNA damage and is brought about by proteins of the poly(ADP) ribose polymerase family (PARP), mostly PARP1 (1). Upon being recruited to sites of the DNA damage, NAD ϩ is used as a substrate by PARP to synthesize negatively charged poly(ADP-ribosyl)ation (PAR) polymers onto itself and also its target proteins (1). Through this posttranslational modification, PARP targets a variety of nuclear proteins to facilitate the recruitment of DNA repair factors to sites of damage (2, 3). Accordingly, PARP-1 or PARP-2-deficient mice and mouse embryonic fibroblasts show chromosomal aberrations and various DNA repair defects (4-6).Inhibition of PARP has become a promising therapeutic approach for the treatment of certain types of cancer (7). It was shown that PARP inhibitors could selectively kill homologous recombination (HR)-deficient cancer cells (8, 9). The reason behind the sensitivity of HR-deficient cells to PARP inhibition is thought to be the accumulation of single-stranded DNA (ssDNA) breaks in the absence of PAR synthesis, leading to replication fork collapse and double-stranded breaks (DSBs), which would then require HR factors for repair (8,10). Recently, PARP activity has also been reported to play a ro...

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Section: Discussionsupporting

confidence: 70%

Poly(ADP-Ribosyl) Glycohydrolase Prevents the Accumulation of Unusual Replication Structures during Unperturbed S Phase

Chaudhuri

Ahuja

Herrador

et al. 2015

Molecular and Cellular Biology

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“…In general, the cells require DNA2 for repression of replicative stress. DNA2 can trigger proper intra-S-phase checkpoints, 49 prevent replication fork reversal, 50 and restart replication when forks have already undergone reversal. 51 However, in response to ICL-inducing agents, DNA2 contributes to over-resection of DNA due to an imbalanced hyperactivation of its nuclease activity in response to loss of FancD2 and/or to impairment of the replication fork stabilizer BOD1L.…”

Section: Ganetespib Carboplatin Combinationmentioning

confidence: 99%

Strong antitumor synergy between DNA crosslinking and HSP90 inhibition causes massive premitotic DNA fragmentation in ovarian cancer cells

et al. 2016

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All current regimens for treating ovarian cancer center around carboplatin as standard first line. The HSP90 inhibitor ganetespib is currently being assessed in advanced clinical oncology trials. Thus, we tested the combined effects of ganetespib and carboplatin on a panel of 15 human ovarian cancer lines. Strikingly, the two drugs strongly synergized in cytotoxicity in tumor cells lacking wild-type p53. Mechanistically, ganetespib and carboplatin in combination, but not individually, induced persistent DNA damage causing massive global chromosome fragmentation. Live-cell microscopy revealed chromosome fragmentation occurring to a dramatic degree when cells condensed their chromatin in preparation for mitosis, followed by cell death in mitosis or upon aberrant exit from mitosis. HSP90 inhibition caused the rapid decay of key components of the Fanconi anemia pathway required for repair of carboplatin-induced interstrand crosslinks (ICLs), as well as of cell cycle checkpoint mediators. Overexpressing FancA rescued the DNA damage induced by the drug combination, indicating that FancA is indeed a key client of Hsp90 that enables cancer cell survival in the presence of ICLs. Conversely, depletion of nuclease DNA2 prevented chromosomal fragmentation, pointing to an imbalance of defective repair in the face of uncontrolled nuclease activity as mechanistic basis for the observed premitotic DNA fragmentation. Importantly, the drug combination induced robust antitumor activity in xenograft models, again accompanied with depletion of FancA. In sum, our findings indicate that ganetespib strongly potentiates the antitumor efficacy of carboplatin by causing combined inhibition of DNA repair and cell cycle control mechanisms, thus triggering global chromosome disruption, aberrant mitosis and cell death. 6 This provides a strong rationale for HSP90 inhibitors in cancer therapy 7 and raises the possibility that HSP90 inhibitors can be used in combination with DNAdamaging chemotherapeutics. 8 Ovarian cancer is among the most common gynecological tumor types and carries the worst prognosis. HSP90 is highly expressed in advanced ovarian carcinoma 9 and thus constitutes a potential therapeutic drug target. 10 In support, HSP90 inhibitors were found effective against ovarian cancer in preclinical models, 11-14 alone or in combination with conventional chemotherapy. 15,16 Carboplatin is the key component of all current first-line therapies for ovarian carcinoma. However, while initially often responding with regression, most tumors relapse and develop resistance within less than a year. 17,18 Like other platinum compounds, carboplatin acts by forming crosslinks within DNA. 17 Platinum compounds form intrastrand DNA lesions but also interstrand crosslinks (ICLs) by covalently linking opposite DNA strands. Such ICLs represent major obstacles to the DNA replication fork 19 and are therefore considered the principal toxic lesion of carboplatin's mode of action. The ICLs can only be removed by a sophisticated DNA repair systemthe Fanconi ...

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“…As described under "Materials and Methods," G 1 -arrested cells were released into media containing 20 M CPT or 0.033% MMS. MMS is known to activate the intra-S-phase checkpoint by interfering with progression of replication fork progression (14,47,48), whereas CTP fails to do so (42). DNA was purified from the samples taken at regular (20 or 30 min) intervals after release.…”

Section: Unimpeded S-phase Progression Is Observed In Dna2⌬405n Follomentioning

confidence: 99%

“…In Saccharomyces cerevisiae, the endonuclease activity of Dna2 is essential for viability (3,4), whereas its helicase activity is dispensable under some growth conditions (12,13). Recently, it was shown that Dna2 contributes to the protection of stalled replication forks by inhibiting reversion of replication forks in a manner dependent on the intra-S-phase checkpoint in Schizosaccharomyces pombe (14). The enzymatic properties of Dna2 endonuclease are suited to remove flaps generated from 5Ј-ends of Okazaki fragments by DNA displacement activity of DNA polymerase (pol) ␦ (13,15).…”

mentioning

confidence: 99%

The N-terminal 45-kDa Domain of Dna2 Endonuclease/Helicase Targets the Enzyme to Secondary Structure DNA

Lee

Kang

et al. 2013

Journal of Biological Chemistry

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Background:The biochemical function of variable N-terminal regions of eukaryotic Dna2 remains unclear. Results: The N-terminal 45-kDa domain of yeast Dna2 targets the enzyme specifically to a secondary structure flap. Conclusion:The hairpin binding activity of Dna2 contributes to efficient removal of hairpin flaps together with endonuclease and helicase activities during Okazaki fragment processing. Significance: The hairpin binding activity of Dna2 is critical for genome stability.

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The Intra-S Phase Checkpoint Targets Dna2 to Prevent Stalled Replication Forks from Reversing

Cited by 147 publications

References 44 publications

Poly(ADP-Ribosyl) Glycohydrolase Prevents the Accumulation of Unusual Replication Structures during Unperturbed S Phase

Poly(ADP-Ribosyl) Glycohydrolase Prevents the Accumulation of Unusual Replication Structures during Unperturbed S Phase

Strong antitumor synergy between DNA crosslinking and HSP90 inhibition causes massive premitotic DNA fragmentation in ovarian cancer cells

The N-terminal 45-kDa Domain of Dna2 Endonuclease/Helicase Targets the Enzyme to Secondary Structure DNA

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