STIMULATION OF RTH1 NUCLEASE OF THE YEAST SACCHAROMYCES-CEREVISIAE BYREPLICATION PROTEIN-A

The RTH1 nuclease is involved in the replication of chromosomal DNA as well as in the repair of DNA damage. Replication protein A (RPA) is a lso an integral part of the DNA replication and repair processes. We h ave investigated the roles(s) of RPA in the function of RTH1 nuclease, including its structure specific endonuclease activity. Initial in vi tro studies, which employed a ''flap'' or a ''pseudo Y'' substrate con taining a short 14 bp duplex region, showed the effect of RPA to be mi nimal or inhibitory. As RPA inhibition is unwarranted for a protein pa rticipating in the DNA replication process, we have further investigat ed the mechanism of such inhibition. Alternate flap and pseudo Y subst rates with a long duplex region (50 bp) were prepared using M13mp19 ss DNA and synthetic oligonucleotides. Yeast RPA stimulated the endonucle ase activity of RTH1 endonuclease with these substrates in a dose-depe ndent manner. Kinetic analysis suggested that yRPA exerted a bipartite effect on the nuclease reaction: (i) the ''load time'' of RTH1 nuclea se onto the DNA substrate decreased from similar to 5 to 2 min in the presence of RPA, and (ii) following initiation of the nuclease reactio n, the initial rate of the reaction increased 10-fold in the presence of yRPA. Further analysis of the interaction of RPA with various endon uclease substrates indicated that RPA has a weak helix destabilizing e ffect and could melt small, 14 bp, regions of duplex DNA. RTH1 endonuc lease cleaves the DNA strand at the junction of single- and double-str anded DNA; consequently, the observed inhibition with small duplex sub strates was likely due to duplex melting. Our studies also demonstrate d that RPA stimulated the RNase H activity of RTH1 nuclease significan tly. In both instances (RTH1 endonuclease and RNase Fl), the stimulati on may involve a specific interaction of RPA with the RTH1 nuclease ra ther than a structural positioning of the DNA substrate by RPA.