Saccharomyces cerevisiae RNase H(35) functions in RNA primer removal during lagging-strand DNA synthesis, most efficiently in cooperation with Rad27 nuclease

Correct removal of RNA primers of Okazaki fragments during lagging-strand D NA synthesis is a critical process for the maintenance of genome integrity. Disturbance of this process has severe mutagenic consequences and could co ntribute to the development of cancer. The role of the mammalian nucleases RNase HI and FEN-1 in RNA primer removal has been substantiated by several studies. Recently, RNase H(35), the Saccharomyces cerevisiae homologue of m ammalian RNase HI, was identified and its possible role in DNA replication was proposed (P. Frank, C. Braunshofer-Reiter, and U. Wintersberger, FEBS L ett. 421:23-26, 1998). This led to the possibility of moving to the genetic ally powerful yeast system for studying the homologues of RNase HI and FEN- 1, i.e., RNase H(35) and Rad27p, respectively. In this study, we have bioch emically defined the substrate specificities and the cooperative as well as independent cleavage mechanisms of S. cerevisiae RNase H(35) and Rad27 nuc lease by using Okazaki fragment model substrates. We have also determined t he additive and compensatory pathological effects of gene deletion and over expression of these two enzymes. Furthermore, the mutagenic consequences of the nuclease deficiencies have been analyzed. Based on our findings, we su ggest that three alternative RNA primer removal pathways of different effic iencies involve RNase H(35) and Rad27 nucleases in yeast.