PURIFICATION OF RAD1 PROTEIN FROM SACCHAROMYCES-CEREVISIAE AND FURTHER CHARACTERIZATION OF THE RAD1 RAD10 ENDONUCLEASE COMPLEX/

The yeast recombination and repair proteins Rad1 and Rad10 associate w ith a 1:1 stoichiometry to form a stable complex with a relative molec ular mass of 190 kDa. This complex, which has previously been shown to degrade single-stranded DNA endonucleolytically, also cleaves superco iled duplex DNA molecules. In this reaction, supercoiled (form I) mole cules are rapidly converted to nicked, relaxed (form II) molecules, pr esumably as a result of nicking at transient single-stranded regions i n the supercoiled DNA. At high enzyme concentrations, there is a slow conversion of the form II molecules to linear (form III) molecules. Th e Rad1/Rad10 endonuclease does not preferentially cleave UV-irradiated DNA and has no detectable exonuclease activity. The nuclease activity of the Rad1/Rad10 complex is consistent with the predicted roles of t he RAD1 and RAD10 genes of Saccharomyces cerevisiae in both the incisi on events of nucleotide excision repair and the removal of nonhomologo us 3' single strands during intrachromosomal recombination between rep eated sequences. In these pathways, the specificity and reactivity of the Rad1/Rad10 endonuclease will probably be modulated by further prot ein-protein interactions.