Genetic and functional interactions between Dna2/Fen1 and DNA ligase I

Abstract

The culminating events in eukaryotic lagging strand DNA synthesis include the removal of flaps by processing enzymes such as Dna2 and Fen1 and the subsequent sealing of resulting nicks by DNA ligase. In this study, we identified CDC9 (encoding DNA ligase I) as a multi-copy suppressor of two mutant alleles of Saccharomyces cerevisiae DNA2, the gene encoding a single-strand specific endonuclease that plays a critical role in Okazaki fragment maturation. We found that the catalytic activity of Cdc9 is not essential for suppressing the growth defect of the two dna2 mutant alleles while the elevated levels of Cdc9 could alleviate the requirements of Dna2. We also found that purified recombinant Cdc9 was able to stimulate in vitro endonuclease activities of both Rad27 (yeast Fen1) and Dna2, suggesting a certain mechanism by which Cdc9 could suppress the dna2 growth defects. Our data in vivo and in vitro suggest that, while Cdc9 participates directly in the maturation of lagging strand synthesis using its catalytic activity, it also contributes to Okazaki fragment maturation via its specific protein-protein interactions with Rad27 and Dna2, the two main endonucleases of the Okazaki fragment maturation. In vitro experiments with the various mutant versions of Cdc9 reveal the crucial role of N-terminal part of Cdc9, which has been known to contain the interacting site with PCNA and nuclear location signal, and they confirm again that the ligase activity of Cdc9 is not necessary for the interaction with Rad27 and Dna2.