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.

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Journal Article | Research Support, N.I.H., Extramural | Research Support, Non-U.S. Gov't

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Eichmiller R, Medina-Rivera M, DeSanto R, Minca E, Kim C, Holland C, Seol JH, Schmit M, Oramus D, Smith J, ... Show all

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