ROLES OF REPLICATION PROTEIN-A SUBUNIT-2 AND SUBUNIT-3 IN DNA-REPLICATION FORK MOVEMENT IN SACCHAROMYCES-CEREVISIAE

Replication Protein-A, the eukaryotic SSB, consists of a large subunit (RPA1) with strong ssDNA binding activity and two smaller subunits (R PA2 and 3) that may cooperate with RPA1 to bind ssDNA in a higher-orde r mode. To determine the in vivo function of the two smaller subunits and the potential role of higher-order ssDNA binding, we isolated an a ssortment of heat-lethal mutations in the genes encoding RPA2 and RPA3 . At the permissive temperature, the mutants show a range of effects o n DNA replication fidelity and sensitivities to UV and MMS. Al the non permissive temperature, four out of five RPA2 mutants show a fast-stop DNA synthesis phenotype typical of a replication fork block. In contr ast, the fifth RPA2 mutant and all RPA3 mutants are able to complete a t least one round of DNA replication at the nonpermissive temperature. The effect of these mutations on the stability of the RPA complex, wa s tested using a coprecipitation assay. At the nonpermissive temperatu re, we find that RPA1 and RPA2 are dissociated in the fast-stop mutant s, but not in the slow-stop mutants. Thus, replication fork movement i n vivo requires the association of at least two subunits of RPA. This result is consistent with the hypothesis that RPA functions in vivo by binding ssDNA in a higher-order mode.