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

Abstract

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 ...

Cyclin Kinase-independent role of p21CDKN1A in the promotion of nascent DNA elongation in unstressed cells

“…Primer sequences were as follows: USP1 (Forward): 5’- GGACGCGTTGCTTGGAATGT-3’ (Reverse) 5’-TGCCCATCTCAGGGTCTTCA-3’. GADPH: (Forward) 5’-AGCCTCCCGCTTCGCTCTCT-3’ (Reverse) 5’-GAGCGATGTGGCTCGGCTGG-3’ (Vallerga et al, 2015). …”

The MRE11–RAD50–NBS1 Complex Conducts the Orchestration of Damage Signaling and Outcomes to Stress in DNA Replication and Repair

“…Although the MRN nuclease function can help resolve stalled RFs, unregulated resection can be catastrophic and lead to fork degradation through MRE11 exonuclease activity (145, 146). Thus, other proteins help regulate MRN-mediated nascent DNA degradation (147–150).…”

Common Chemical Inductors of Replication Stress: Focus on Cell‐Based Studies

“…Although the mechanism of DNA damage induction may look similar to that for APH, HU induces a different spectrum of fragile sites, called early replicating fragile sites (ERFs) [233]. ERFs are also induced by c-Myc expression [11,12]. It was also reported that 10 μg/mL APH [234] (concentration that stalls the replication fork progression) leads to the generation of several kilobases long unwound DNA; however, HU treatment can generate only up to 100–200 nt long ssDNA [235].…”

“…The most common extrinsic sources of RS are all wavelengths of ultraviolet radiation (UV) [11], ionising radiation (IR) [12] and special genotoxic chemical compounds [13] which are the main focus of this review. RS-inducing chemicals can cause a broad spectrum of DNA lesions.…”