Abstract
Rad51 is the key protein in homologous recombination that plays important roles during DNA replication and repair. Auxiliary factors regulate Rad51 activity to facilitate productive recombination, and prevent inappropriate, untimely or excessive events, which could lead to genome instability. Previous genetic analyses identified a function for Rrp1 (a member of the Rad5/16-like group of SWI2/SNF2 translocases) in modulating Rad51 function, shared with the Rad51 mediator Swi5-Sfr1 and the Srs2 anti-recombinase. Here, we show that Rrp1 overproduction alleviates the toxicity associated with excessive Rad51 levels in a manner dependent on Rrp1 ATPase domain. Purified Rrp1 binds to DNA and has a DNA-dependent ATPase activity. Importantly, Rrp1 directly interacts with Rad51 and removes it from double-stranded DNA, confirming that Rrp1 is a translocase capable of modulating Rad51 function. Rrp1 affects Rad51 binding at centromeres. Additionally, we demonstrate in vivo and in vitro that Rrp1 possesses E3 ubiquitin ligase activity with Rad51 as a substrate, suggesting that Rrp1 regulates Rad51 in a multi-tiered fashion.
Highlights
Homologous recombination (HR) is a highly conserved pathway for the repair of DNA double-strand breaks (DSBs), and many key HR proteins have a critical role during DNA replication [1]
We propose that these translocase and ubiquitin ligase activities allow Rrp1 to counteract the genotoxic effects of excessive Rad51 binding to specific regions of chromatin, such as centromeric regions
Rad51 overproduction leads to its excessive accumulation on chromatin and has a negative effect on cell growth and chromosome stability that is aggravated in mutants devoid of
Summary
Homologous recombination (HR) is a highly conserved pathway for the repair of DNA double-strand breaks (DSBs), and many key HR proteins have a critical role during DNA replication [1]. Another complex, MMS22L-TONSL, has been shown in human cells to limit RAD51 binding to dsDNA and stimulate HR-mediated restart of arrested replication forks [37]. The RAD51 paralog RAD51C has been shown to prevent proteasomal degradation of RAD51 in human cells, especially after DNA damage [42], suggesting that RAD51 can be regulated by ubiquitylation. Regulation of Rad by Fbh, an F-box helicase and E3 ubiquitin ligase, is more complex and involves both activities of this protein. Ubiquitylates Rad and is able to displace it from dsDNA We propose that these translocase and ubiquitin ligase activities allow Rrp to counteract the genotoxic effects of excessive Rad binding to specific regions of chromatin, such as centromeric regions
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