Rad51 replication fork recruitment is required for DNA damage tolerance

González-Prieto, Román; Muñoz-Cabello, Ana M.; Cabello-Lobato, María J.; Prado, Félix

doi:10.1038/emboj.2013.73

Cited by 80 publications

(122 citation statements)

References 62 publications

(151 reference statements)

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“…Specifically, the effect of Rad52 has been found to be more pronounced than Rad51 at stalled forks [26], [28], indicating that Rad52 binding at stalled forks may have additional repair independent roles that are nevertheless important for maintaining the stability and integrity of these stall sites. Emerging views on the role of repair proteins at the centromeres suggest that they are primarily involved in stabilization/protection of stalled replication forks while deleterious end results of HR such as cross-over recombination or gross chromosomal rearrangements are carefully prevented [58].…”

Section: Discussionmentioning

confidence: 99%

Rad51–Rad52 Mediated Maintenance of Centromeric Chromatin in Candida albicans

et al. 2014

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Specification of the centromere location in most eukaryotes is not solely dependent on the DNA sequence. However, the non-genetic determinants of centromere identity are not clearly defined. While multiple mechanisms, individually or in concert, may specify centromeres epigenetically, most studies in this area are focused on a universal factor, a centromere-specific histone H3 variant CENP-A, often considered as the epigenetic determinant of centromere identity. In spite of variable timing of its loading at centromeres across species, a replication coupled early S phase deposition of CENP-A is found in most yeast centromeres. Centromeres are the earliest replicating chromosomal regions in a pathogenic budding yeast Candida albicans. Using a 2-dimensional agarose gel electrophoresis assay, we identify replication origins (ORI7-LI and ORI7-RI) proximal to an early replicating centromere (CEN7) in C. albicans. We show that the replication forks stall at CEN7 in a kinetochore dependent manner and fork stalling is reduced in the absence of the homologous recombination (HR) proteins Rad51 and Rad52. Deletion of ORI7-RI causes a significant reduction in the stalled fork signal and an increased loss rate of the altered chromosome 7. The HR proteins, Rad51 and Rad52, have been shown to play a role in fork restart. Confocal microscopy shows declustered kinetochores in rad51 and rad52 mutants, which are evidence of kinetochore disintegrity. CENP-ACaCse4 levels at centromeres, as determined by chromatin immunoprecipitation (ChIP) experiments, are reduced in absence of Rad51/Rad52 resulting in disruption of the kinetochore structure. Moreover, western blot analysis reveals that delocalized CENP-A molecules in HR mutants degrade in a similar fashion as in other kinetochore mutants described before. Finally, co-immunoprecipitation assays indicate that Rad51 and Rad52 physically interact with CENP-ACaCse4 in vivo. Thus, the HR proteins Rad51 and Rad52 epigenetically maintain centromere functioning by regulating CENP-ACaCse4 levels at the programmed stall sites of early replicating centromeres.

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Section: Discussionmentioning

confidence: 99%

Rad51–Rad52 Mediated Maintenance of Centromeric Chromatin in Candida albicans

et al. 2014

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“…Our data indicate that Rad51 indirectly promotes polη loading onto DNA and accelerates TLS onset, probably by avoiding nascent DNA shortening. The functional link between Rad51 and TLS might extend to other scenarios, such as TLS-mediated gap filling (36,37).…”

Section: Resultsmentioning

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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“…Alternative, recombination-based, (legitimate) template switching can also rescue stalled forks. However, template switching in yeast depends on RAD51 (Gonzalez-Prieto et al, 2013; Urulangodi et al, 2015) which was not recruited to the stalled forks on the DMs. We also observed the recruitment of BRCA2 to the stalled forks.…”

Section: Discussionmentioning

confidence: 99%

Impediment of Replication Forks by Long Non-coding RNA Provokes Chromosomal Rearrangements by Error-Prone Restart

et al. 2017

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Summary Naturally stalled replication forks are considered to cause structurally abnormal chromosomes in tumor cells. However, underlying mechanisms remain speculative, as capturing naturally stalled forks has been a challenge. Here, we captured naturally stalled forks in tumor cells and delineated molecular processes underlying the structural evolution of circular mini-chromosomes (double minute chromosomes, DMs). Replication forks stalled on the DM by the co-directional collision with the transcription machinery for long non-coding RNA. RPA, BRCA2, and DNA polymerase eta (Polη), were recruited to the stalled forks. The recruitment of Polη was critical for replication to continue, as Polη knockdown resulted in DM loss. Rescued stalled forks were error-prone and switched replication templates repeatedly to create complex fusions of multiple short genomic segments. In mice, such complex fusions circularized the genomic region surrounding MYC to create a DM during tumorigenesis. Our results define a molecular path that guides stalled replication forks to complex chromosomal rearrangements.

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Rad51 replication fork recruitment is required for DNA damage tolerance

Cited by 80 publications

References 62 publications

Rad51–Rad52 Mediated Maintenance of Centromeric Chromatin in Candida albicans

Rad51–Rad52 Mediated Maintenance of Centromeric Chromatin in Candida albicans

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

Impediment of Replication Forks by Long Non-coding RNA Provokes Chromosomal Rearrangements by Error-Prone Restart

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