STRAND EXCHANGE PROTEIN-1 (SEP1) FROM SACCHAROMYCES-CEREVISIAE DOES NOT PROMOTE BRANCH MIGRATION IN-VITRO

It has been shown in vitro that Saccharomyces cerevisiae strand exchan ge protein 1 (Sep1) promotes the transfer of one strand of a linear du plex DNA to a homologous single-stranded DNA circle, Sep1 also has an exonuclease active on DNA and RNA. By using exonuclease III-treated li near duplex DNA with various lengths of single-stranded tail as well a s Ca2+ to inhibit the exonuclease activity of Sep1, we show that the p rocessivity of exonuclease activity of Sep1 is greater than previously reported. The results in this work also demonstrate that the joint mo lecule between the linear duplex and single-stranded circle observed f rom the Sep1-promoted strand transfer reaction is just the pairing bet ween the long single-stranded tail of the linear duplex DNA (generated by the exonuclease activity of Sep1) and the single-stranded circular DNA. When a synthetic Holliday junction was used as substrate, branch migration facilitated by Sep1 could not be detected. Finally, using e lectron microscopy no alpha-structure, a joint molecule with displaced single-stranded DNA tail that indicates branch migration could be obs erved. The results imply that Sep1 cannot promote branch migration in vitro. Further investigation is needed to determine the role of Sep1 i n recombination in vivo.