The Rad51 paralogs facilitate a novel DNA strand specific damage tolerance pathway

Rosenbaum, Joel C.; Bonilla, Braulio; Hengel, Sarah R; Mertz, Tony M.; Herken, Benjamin W.; Kazemier, Hinke G.; Pressimone, Catherine A.; Ratterman, Timothy C.; MacNary, Ellen; Magis, Alessio De; Kwon, Youngho; Godin, Stephen K.; Houten, Bennett Van; Normolle, Daniel P.; Sung, Patrick; Das, Sauvik; Paeschke, Katrin; Roberts, Steven A.; VanDemark, Andrew P.; Bernstein, Kara A.

doi:10.1038/s41467-019-11374-8

Cited by 30 publications

(45 citation statements)

References 53 publications

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“…We can only speculate that either Pol α maybe somehow inhibited at bulky lesions in mammalian cells, or that PrimPol might have additional roles in promoting HR at gaps, for example by interacting with DNA repair factors or due to its preference to synthesize DNA primers 12 . Interestingly, recent work also suggests that different DNA damage tolerance pathways may be active at the leading versus the lagging strand in budding yeast 44 . It will be important, if challenging, to further investigate how re-priming via PrimPol specifically promotes HR-dependent gap repair.…”

Section: Discussionmentioning

confidence: 99%

PrimPol-dependent single-stranded gap formation mediates homologous recombination at bulky DNA adducts

et al. 2020

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Stalled replication forks can be restarted and repaired by RAD51-mediated homologous recombination (HR), but HR can also perform post-replicative repair after bypass of the obstacle. Bulky DNA adducts are important replication-blocking lesions, but it is unknown whether they activate HR at stalled forks or behind ongoing forks. Using mainly BPDE-DNA adducts as model lesions, we show that HR induced by bulky adducts in mammalian cells predominantly occurs at post-replicative gaps formed by the DNA/RNA primase PrimPol. RAD51 recruitment under these conditions does not result from fork stalling, but rather occurs at gaps formed by PrimPol re-priming and resection by MRE11 and EXO1. In contrast, RAD51 loading at double-strand breaks does not require PrimPol. At bulky adducts, PrimPol promotes sister chromatid exchange and genetic recombination. Our data support that HR at bulky adducts in mammalian cells involves post-replicative gap repair and define a role for PrimPol in HR-mediated DNA damage tolerance.

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Section: Discussionmentioning

confidence: 99%

PrimPol-dependent single-stranded gap formation mediates homologous recombination at bulky DNA adducts

et al. 2020

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“…During evolution, duplications of the Rad51 gene have resulted in so-called Rad51 paralogues [ 3 ], a set of factors that are found in variable numbers in different organisms and whose spectrum of roles remain somewhat undefined, at least in part because they can belong to a number of protein complexes. Nonetheless, Rad51 paralogues have been implicated in directly modulating HR, acting on Rad51 HR intermediates [ 4 – 6 ], and in wider repair activities for cell cycle progression [ 7 , 8 ]. HR reactions mediated by Rad51 [ 9 – 12 ] and modulated by the Rad51 paralogues [ 13 ] are also required for resolving DNA replication impediments, by promoting protection and restart of stalled replication forks during replication stress.…”

Section: Introductionmentioning

confidence: 99%

Conditional knockout of RAD51-related genes in Leishmania major reveals a critical role for homologous recombination during genome replication

et al. 2020

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Homologous recombination (HR) has an intimate relationship with genome replication, both during repair of DNA lesions that might prevent DNA synthesis and in tackling stalls to the replication fork. Recent studies led us to ask if HR might have a more central role in replicating the genome of Leishmania, a eukaryotic parasite. Conflicting evidence has emerged regarding whether or not HR genes are essential, and genome-wide mapping has provided evidence for an unorthodox organisation of DNA replication initiation sites, termed origins. To answer this question, we have employed a combined CRISPR/Cas9 and DiCre approach to rapidly generate and assess the effect of conditional ablation of RAD51 and three RAD51-related proteins in Leishmania major. Using this approach, we demonstrate that loss of any of these HR factors is not immediately lethal but in each case growth slows with time and leads to DNA damage and accumulation of cells with aberrant DNA content. Despite these similarities, we show that only loss of RAD51 or RAD51-3 impairs DNA synthesis and causes elevated levels of genome-wide mutation. Furthermore, we show that these two HR factors act in distinct ways, since ablation of RAD51, but not RAD51-3, has a profound effect on DNA replication, causing loss of initiation at the major origins and increased DNA synthesis at subtelomeres. Our work clarifies questions regarding the importance of HR to survival of Leishmania and reveals an unanticipated, central role for RAD51 in the programme of genome replication in a microbial eukaryote.

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“…In mammalian cells, with Pol α constantly repriming on the lagging strand and possibly also the leading strand, one would expect at most half of gap- and RAD51 foci formation to be PrimPol-dependent. Recent work has suggested that in budding yeast, different pathways act on the leading and lagging strands 40 it will be interesting to investigate whether only re-priming via PrimPol promotes HR-dependent gap repair, possibly due to PrimPol’s preference to synthesise DNA primers.…”

Section: Discussionmentioning

confidence: 99%

PrimPol-dependent single-stranded gap formation mediates homologous recombination at bulky DNA adducts

Piberger

Bowry

Kelly

et al. 2019

Preprint

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Obstacles on the DNA template can lead to DNA replication fork stalling and genomic rearrangements. RAD51-mediated homologous recombination (HR) can promote restart and repair of stalled forks, but also post-replicative repair once the obstacle has been bypassed. Bulky DNA adducts are important replication-blocking lesions induced by environmental carcinogens, but it is not known whether they activate HR directly at stalled forks, or at gaps left behind ongoing forks. Here we show that in mammalian cells, bulky adducts predominantly induce HR at post-replicative gaps formed by the DNA/RNA primase PrimPol. Using BPDE and UV model lesions, we report that RAD51 is not recruited to stalled or collapsed forks, but instead to long gaps formed by PrimPol re-priming activity and resection by MRE11 and EXO1. In contrast, RAD51 loading at DSBs does not require PrimPol. At bulky adducts, PrimPol is required for the induction of sister chromatid exchanges and genetic recombination. Our data support that HR at bulky adducts in mammalian cells is primarily associated with post-replicative gap repair and define a role for PrimPol in DNA damage tolerance by homologous recombination.

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The Rad51 paralogs facilitate a novel DNA strand specific damage tolerance pathway

Cited by 30 publications

References 53 publications

PrimPol-dependent single-stranded gap formation mediates homologous recombination at bulky DNA adducts

PrimPol-dependent single-stranded gap formation mediates homologous recombination at bulky DNA adducts

Conditional knockout of RAD51-related genes in Leishmania major reveals a critical role for homologous recombination during genome replication

PrimPol-dependent single-stranded gap formation mediates homologous recombination at bulky DNA adducts

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