Identification of functional domains within the RAD1.RAD10 repair and recombination endonuclease of Saccharomyces cerevisiae.

Saccharomyces cerevisiae rad1 and rad10 mutants are unable to carry out nucleotide excision repair and are also defective in a mitotic intrachromosomal recombination pathway. The products of these genes are subunits of an endonuclease which recognizes DNA duplex/single-strand junctions and specifically cleaves the 3' single-strand extension at or near the junction. It has been suggested that such junctions arise as a consequence of DNA lesion processing during nucleotide excision repair and the processing of double-strand breaks during intrachromosomal recombination. In this study we show that the RAD1 RAD10 complex also cleaves a more complex junction structure consisting of a duplex with a protruding 3' single-strand branch that resembles putative recombination intermediates in the RAD1 RAD10-mediated single-strand annealing pathway of mitotic recombination. Using monoclonal antibodies, we have identified two regions of RAD1 that are required for the cleavage of duplex/single-strand junctions. These reagents also inhibit nucleotide excision repair in vitro, confirming the essential role of the RAD1 RAD10 endonuclease in this pathway.