DNA2 drives processing and restart of reversed replication forks in human cells

Thangavel, Saravanabhavan; Berti, Matteo; Levikova, Maryna; Pinto, Cosimo; Gomathinayagam, Shivasankari; Vujanovic, Marko; Zellweger, Ralph; Moore, Hayley R.; Lee, Eu Han; Hendrickson, Eric A.; Ćejka, Petr; Stewart, Sheila A.; Lopes, Massimo; Vindigni, Alessandro

doi:10.1083/jcb.201406100

Cited by 307 publications

(465 citation statements)

References 60 publications

(116 reference statements)

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“…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. 33,52 These considerations are in line with our observation of phospho-RPA32 accumulation (Figure 2c), a marker for ssDNA intermediates and DNA resection, as phospho-RPA recruits DNA2 to chromatin.…”

Section: Ganetespib Carboplatin Combinationsupporting

confidence: 79%

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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Section: Ganetespib Carboplatin Combinationsupporting

confidence: 79%

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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“…Mre11-mediated nucleolytic degradation of nascent DNA in BCRA2-and FANCD2-depleted, HU-treated cells was suggested to take place at the unprotected ends of reversed forks, which may mimic DSBs (6-8). Conversely, two recent reports suggest that Rad51 prevents pathological Mre11-dependent nucleolytic degradation of nonreversed stalled forks and promotes controlled DNA2-dependent exonucleolytic processing of reversed forks (15,21).DSB-independent Rad51 functions were revealed by the use of agents that cause a significant degree (more than 40%) of replication fork stalling, such as HU, camptothecin (CPT), and mitomycin C (MMC) (15). Much less is known about the participation of Rad51 in the replication across DNA lesions that do not persistently halt replication forks and only cause a moderate reduction in the replication speed (e.g., UV-induced DNA lesions) (22).…”

mentioning

confidence: 99%

Rad51 recombinase prevents Mre11 nuclease-dependent degradation and excessive PrimPol-mediated elongation of nascent DNA after UV irradiation

Vallerga

Mansilla

Federico

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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After UV irradiation, DNA polymerases specialized in translesion DNA synthesis (TLS) aid DNA replication. However, it is unclear whether other mechanisms also facilitate the elongation of UVdamaged DNA. We wondered if Rad51 recombinase (Rad51), a factor that escorts replication forks, aids replication across UV lesions. We found that depletion of Rad51 impairs S-phase progression and increases cell death after UV irradiation. Interestingly, Rad51 and the TLS polymerase polη modulate the elongation of nascent DNA in different ways, suggesting that DNA elongation after UV irradiation does not exclusively rely on TLS events. In particular, Rad51 protects the DNA synthesized immediately before UV irradiation from degradation and avoids excessive elongation of nascent DNA after UV irradiation. In Rad51-depleted samples, the degradation of DNA was limited to the first minutes after UV irradiation and required the exonuclease activity of the double strand break repair nuclease (Mre11). The persistent dysregulation of nascent DNA elongation after Rad51 knockdown required Mre11, but not its exonuclease activity, and PrimPol, a DNA polymerase with primase activity. By showing a crucial contribution of Rad51 to the synthesis of nascent DNA, our results reveal an unanticipated complexity in the regulation of DNA elongation across UV-damaged templates.PrimPol | polκ | polη | DNA damage tolerance | DNA replication T he DNA-binding protein Rad51 is a central component of homologous recombination repair (HRR). HRR repairs doublestrand breaks (DSBs) in an error-free way and processes one-ended DSBs to reactivate collapsed replication forks (1). During HRR, DSBs are processed by the 3′-to-5′ exonuclease activity of the double strand break repair nuclease (Mre11) to generate protruding 3′ ssDNA at DSBs. The ssDNA is then coated with Rad51, a factor that catalyzes homology search and strand invasion. The loading and stabilization of Rad51/ssDNA complexes are supported by multiple mediators, such as the tumor suppressor BRCA2 (breast cancer 2) (1). Moreover, Rad51 promotes XPF1-and Exo1-mediated DSB formation after gemcitabine-induced irreversible ribonucleotide reductase inhibition, thus promoting cell death (2). The signals that redirect Rad51 into a DSB formation pathway rather than DSB repair are not yet known.The functions of Rad51 are not limited to the processing/ generation of DSBs. Over the past few years, it has become evident that Rad51 escorts ongoing replication forks regardless of the presence of DSBs (3-5). Specifically, Rad51 protects persistently stalled replication forks from Mre11-mediated nucleolytic degradation and facilitates replication fork restart when the replication-halting agent hydroxyurea (HU) or aphidicolin (APH) is removed (6-19). Such novel functions of Rad51 require many HRR factors, including BCRA2, FANCD2 (Fanconi Anemia Complementation group protein D2), CtIP, BRCA1, and the WRN helicase, but are independent of HRR effectors, such as Rad54 (6, 7). Rad51-dependent fork-restart and fork-protection ...

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“…On the other hand, DNA2 acts together with the Werner syndrome ATP-dependent helicase (WRN) at reversed replication forks (Thangavel et al 2015). The current model proposes that DNA2 and MRE11 accumulate on different types of stalled forks depending on their structures and the moment at which they appear (Karanja et al 2014, Higgs et al 2015, Thangavel et al 2015. Nevertheless, the recent finding that the Fanconi-associated nuclease 1 (FAN1) is also implicated in replication fork recovery (Lachaud et al 2016, Chaudhury et al 2014 suggests that this model is much more complex than initially anticipated.…”

Section: Brca2 Protects Stalled Replication Forks From Nucleolytic Dementioning

confidence: 60%

“…While the exact mechanism by which resection contributes to the processing of stalled replication forks is currently unknown, both its inhibition and its overactivation are detrimental for replication fork restart (Buis et al 2008, Hashimoto et al 2010. Two key nucleases, the meiotic recombination 11 (MRE11) and DNA2, are thought to drive this step (Costanzo et al 2001, Trenz et al 2006, Hashimoto et al 2010, Thangavel et al 2015; however, the mechanism(s) by which these nucleases recognize and process stalled forks remain unclear. Recent studies suggest that poly (ADP-ribose) polymerase 1 (PARP1) and the histone methyltransferase complex PTIP/mixedlineage leukemia protein 3 et 4 (MLL3/4) promote the recruitment of MRE11 to stalled forks (Bryant et al 2009, Ying et al 2012, Chaudhuri et al 2016.…”

Section: Brca2 Protects Stalled Replication Forks From Nucleolytic Dementioning

confidence: 99%

“…Recent studies suggest that poly (ADP-ribose) polymerase 1 (PARP1) and the histone methyltransferase complex PTIP/mixedlineage leukemia protein 3 et 4 (MLL3/4) promote the recruitment of MRE11 to stalled forks (Bryant et al 2009, Ying et al 2012, Chaudhuri et al 2016. On the other hand, DNA2 acts together with the Werner syndrome ATP-dependent helicase (WRN) at reversed replication forks (Thangavel et al 2015). The current model proposes that DNA2 and MRE11 accumulate on different types of stalled forks depending on their structures and the moment at which they appear (Karanja et al 2014, Higgs et al 2015, Thangavel et al 2015.…”

Section: Brca2 Protects Stalled Replication Forks From Nucleolytic Dementioning

confidence: 99%

See 1 more Smart Citation

BRCA2 functions: from DNA repair to replication fork stabilization

Fradet-Turcotte¹,

Sitz²,

Grapton³

et al. 2016

Endocrine-Related Cancer

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Maintaining genomic integrity is essential to preserve normal cellular physiology and to prevent the emergence of several human pathologies including cancer. The breast cancer susceptibility gene 2 (BRCA2, also known as the Fanconi anemia (FA) complementation group D1 (FANCD1)) is a potent tumor suppressor that has been extensively studied in DNA double-stranded break (DSB) repair by homologous recombination (HR). However, BRCA2 participates in numerous other processes central to maintaining genome stability, including DNA replication, telomere homeostasis and cell cycle progression. Consequently, inherited mutations in BRCA2 are associated with an increased risk of breast, ovarian and pancreatic cancers. Furthermore, bi-allelic mutations in BRCA2 are linked to FA, a rare chromosome instability syndrome characterized by aplastic anemia in children as well as susceptibility to leukemia and cancer. Here, we discuss the recent developments underlying the functions of BRCA2 in the maintenance of genomic integrity. The current model places BRCA2 as a central regulator of genome stability by repairing DSBs and limiting replication stress. These findings have direct implications for the development of novel anticancer therapeutic approaches.

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DNA2 drives processing and restart of reversed replication forks in human cells

Abstract: Following prolonged genotoxic stress, DNA2 and WRN functionally interact to degrade reversed replication forks and promote replication restart, thereby preventing aberrant processing of unresolved replication intermediates

Cited by 307 publications

References 60 publications

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

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

Rad51 recombinase prevents Mre11 nuclease-dependent degradation and excessive PrimPol-mediated elongation of nascent DNA after UV irradiation

BRCA2 functions: from DNA repair to replication fork stabilization

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