Accumulation of unligated Okazaki fragments induces PCNA ubiquitination at lysine 107 and Rad59-dependent replication fork progression

Abstract

DNA ligase I, encoded by the <italic>CDC9</italic> gene in <italic>Saccharomyces cerevisiae</italic>, is an essential enzyme that catalyzes the ligation of newly synthesized DNA on the lagging strand called Okazaki fragments. In humans, approximately 30 million Okazaki fragments are synthesized during every S phase and require further processing prior to DNA ligation. An individual harboring DNA ligase I mutations exhibited growth retardation, sunlight sensitivity, severe immunosuppression and developed lymphoma, indicating a link between defects in Okazaki fragment maturation and cancer predisposition. How cells monitor and suppress such accumulation of DNA damage that arises due to defective Okazaki fragment processing is unclear. Using <italic>S. cerevisiae</italic> as a model system, we uncovered a novel and conserved ubiquitination pathway that targets proliferating cell nuclear antigen (PCNA) at lysine 107 when DNA ligase I activity is inhibited. The modification at K107 is catalyzed by the E2 variant Mms2 together with Ubc4 and the E3 ubiquitin ligase Rad5. Most importantly, this signal is crucial to activate the S phase checkpoint, which promotes cell cycle arrest. In support of this notion, a <italic>pol30K107</italic> mutation alleviated cell cycle arrest in <italic>cdc9</italic> mutants. To determine whether PCNA ubiquitination occurred in response to nicks or the lack of PCNA-DNA ligase interaction, we complemented <italic>cdc9</italic> cells either with wild-type DNA ligase I or <italic>Chlorella</italic> virus ligase, the latter of which fails to interact with PCNA. Both enzymes reversed PCNA ubiquitination, arguing that the modification is likely triggered directly by nicks. To further understand how cells cope with nicks during replication, we utilized <italic>cdc9-1</italic> in a genome-wide synthetic lethality screen and identified <italic>RAD59</italic> as a strong negative interactor. <italic>cdc9 rad59</italic> double mutants did not alter PCNA ubiquitination but enhanced phosphorylation of the mediator of the replication checkpoint, Mrc1, indicative of increased replication fork stalling. Thus, Rad59 promotes fork progression when Okazaki fragment processing is compromised and counteracts PCNA-K107 mediated cell cycle arrest.