USE OF MONOCLONAL-ANTIBODIES IN THE FUNCTIONAL-CHARACTERIZATION OF THE SACCHAROMYCES-CEREVISIAE SEP1 PROTEIN

The Saccharomyces cerevisiae strand-exchange protein 1 (Sep1 also know n as Xm1, Kem1, Rar5, Stp beta/DST2) has been demonstrated to mediate the formation of hybrid DNA from model substrates of linear double-str anded and circular single-stranded DNA in vitro. To delineate the mech anism by which Sep1 acts in the strand-exchange reaction, we analyzed mouse anti-Sep1 monoclonal antibodies for inhibition of the Sep1 in vi tro activity. Of 12 class-G immunoglobulins tested, four were found to consistently inhibit the Sep1-mediated strand-exchange reaction. The inhibiting antibodies were tested for inhibition of a variety of Sep1- catalyzed DNA reactions including exonuclease activity on double-stran ded and single-stranded DNA, renaturation of complementary single-stra nded DNA and condensation of DNA into large aggregates. All four inhib iting antibodies had no effect on the exonuclease activity of Sep1. Th ree antibodies specifically blocked DNA aggregation. In addition, one antibody inhibited renaturation of complementary single-stranded DNA. This inhibition pattern underlines the importance of condensation of D NA into large aggregates in conjunction with double-stranded DNA exonu clease activity for the in vitro homologous pairing activity of Sep1. The implications of these data for the interpretation of proteins whic h promote homologous pairing of DNA are discussed, in particular in li ght of the reannealing activity of the p53 human tumor-suppressor prot ein.