Coordination of Rad1–Rad10 interactions with Msh2–Msh3, Saw1 and RPA is essential for functional 3′ non-homologous tail removal

Abstract

Double strand DNA break repair (DSBR) comprises multiple pathways. A subset of DSBR pathways, including single strand annealing, involve intermediates with 3′ non-homologous tails that must be removed to complete repair. In Saccharomyces cerevisiae, Rad1–Rad10 is the structure-specific endonuclease that cleaves the tails in 3′ non-homologous tail removal (3′ NHTR). Rad1–Rad10 is also an essential component of the nucleotide excision repair (NER) pathway. In both cases, Rad1–Rad10 requires protein partners for recruitment to the relevant DNA intermediate. Msh2–Msh3 and Saw1 recruit Rad1–Rad10 in 3′ NHTR; Rad14 recruits Rad1–Rad10 in NER. We created two rad1 separation-of-function alleles, rad1R203A,K205A and rad1R218A; both are defective in 3′ NHTR but functional in NER. In vitro, rad1R203A,K205A was impaired at multiple steps in 3′ NHTR. The rad1R218A in vivo phenotype resembles that of msh2- or msh3-deleted cells; recruitment of rad1R218A–Rad10 to recombination intermediates is defective. Interactions among rad1R218A–Rad10 and Msh2–Msh3 and Saw1 are altered and rad1R218A–Rad10 interactions with RPA are compromised. We propose a model in which Rad1–Rad10 is recruited and positioned at the recombination intermediate through interactions, between Saw1 and DNA, Rad1–Rad10 and Msh2–Msh3, Saw1 and Msh2–Msh3 and Rad1–Rad10 and RPA. When any of these interactions is altered, 3′ NHTR is impaired.

Distinct DNA repair pathways cause genomic instability at alternative DNA structures

“…9), demonstrating that Saw1 was not required for Z-DNA-induced mutation. Since Saw1 is essential for Rad1 recruitment and cleavage at 3′ tailed recombination intermediates and 3′ NHTR 40,45 , this result suggested that the Rad1-10 complex was involved in Z-DNA processing in a pathway unique to that of 3′ NHTR. Furthermore, in the ChIP assays the primers were designed to amplify a region containing the Z-DNA-forming sequence so that a positive PCR amplification suggested that at least some of the ERCC1-XPF and MSH2-MSH3 complexes bound on the intact DNA, and not on 3′-tails generated from nicked DNA or DSBs.…”

“…DNA polymerase and ligase fill and seal the gap only after the unannealed 3′-tails are removed. Eichmiller et al (2018) reported that the Msh2-Msh3 complex could bind to the 3′-free tail and then recruit Rad1-10 and Saw1 in yeast 45 . Saw1 can directly stimulate the endonuclease activity of Rad1-10 44 in processing the 3′-tails.…”

SAW1 is increasingly required to recruit Rad10 as SSA flap-length increases from 20 to 50 bases in single-strand annealing in S. cerevisiae

“…In triple-labeled experiments in which Saw1 was also fluorescently labeled, we observed Saw1 and Rad1-Rad10 frequently localized to the repair sites at the same time, in all flap substrates. Prior literature provided evidence for a Msh2-Msh3-Saw1-Rad1-Rad10 protein complex and other evidence suggesting that Msh2-Msh3 might stabilize the annealed repair intermediate thereby recruiting Saw1 and Rad1-Rad10 to the DSB site [ 9 , 19 ]. It is possible that Msh2-Msh3 is unable to distinguish between varying flap lengths and recruits Saw1 to all flap substrates regardless of necessity.…”

Variants of the human RAD52 gene confer defects in ionizing radiation resistance and homologous recombination repair in budding yeast

“…Consequently, the relationship between the primary structure of this region of HsRAD52 and its ability to support SSA in budding yeast will be a subject of continued investigation. Additionally, the capacity of HsRAD52-FLAG to act as a replacement for ScRad52 in SSA suggests that it works in concert with other factors important for SSA in yeast [68][69][70][71][72][73][74][75][76], which will also be investigated in the future.…”