Abstract
Deficiency in DNA ligase I, encoded by CDC9 in budding yeast, leads to the accumulation of unligated Okazaki fragments and triggers PCNA ubiquitination at a non-canonical lysine residue. This signal is crucial to activate the S phase checkpoint, which promotes cell cycle delay. We report here that a pol30-K107 mutation alleviated cell cycle delay in cdc9 mutants, consistent with the idea that the modification of PCNA at K107 affects the rate of DNA synthesis at replication forks. To determine whether PCNA ubiquitination occurred in response to nicks or was triggered by the lack of PCNA-DNA ligase interaction, we complemented cdc9 cells with either wild-type DNA ligase I or a mutant form, which fails to interact with PCNA. Both enzymes reversed PCNA ubiquitination, arguing that the modification is likely an integral part of a novel nick-sensory mechanism and not due to non-specific secondary mutations that could have occurred spontaneously in cdc9 mutants. To further understand how cells cope with the accumulation of nicks during DNA replication, we utilized cdc9-1 in a genome-wide synthetic lethality screen, which identified RAD59 as a strong negative interactor. In comparison to cdc9 single mutants, cdc9 rad59Δ double mutants did not alter PCNA ubiquitination but enhanced phosphorylation of the mediator of the replication checkpoint, Mrc1. Since Mrc1 resides at the replication fork and is phosphorylated in response to fork stalling, these results indicate that Rad59 alleviates nick-induced replication fork slowdown. Thus, we propose that Rad59 promotes fork progression when Okazaki fragment processing is compromised and counteracts PCNA-K107 mediated cell cycle arrest.
Highlights
Replication fork arrest in response to DNA lesions, such as UVinduced thymine dimers that physically block DNA synthesis and lead to exposure of unreplicated, single-stranded DNA has been studied extensively in multiple different model organisms [1]
Cells could directly sense the accumulation of nicks; second, cells might recognize the absence of the PCNACdc9 interaction or third, because cdc9 mutants are known to be highly mutagenic [5], secondary defects unrelated to the generation of nicks could cause the ubiquitination of PCNA
Cdc9-1 mutants expressing PCNA-K164R, which renders cells sensitive to UVirradiation and methyl methanesulfonate (MMS) [18], did not exhibit any additional temperature sensitivity as compared to cdc9-1 single mutants (Figure 1A). These results indicated that the survival of cdc9-1 mutants did not depend on K164 of PCNA, but rather on the reconstitution of DNA ligase I activity
Summary
Replication fork arrest in response to DNA lesions, such as UVinduced thymine dimers that physically block DNA synthesis and lead to exposure of unreplicated, single-stranded (ss) DNA has been studied extensively in multiple different model organisms [1]. Given that human cells produce on the order of 30 million Okazaki fragments that need to be processed and ligated during a single round of replication, a tracking system should be in place to account for possible errors that could lead to the accumulation of nicked DNA. The importance of such a surveillance system is underscored by mutations impinging on proper Okazaki fragment processing that have been identified in human cancer patients and whose cancer-causing effect has been recapitulated in animal studies [2,3]. DNA ligase catalyzes the nucleophilic attack of the 39-OH to the DNA-adenylate to covalently join the two ends of the DNA strands and release AMP
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