In all eukaryotes, the ligation of newly synthesized DNA, also known as Okazaki fragments, is catalyzed by DNA ligase I1. An individual with a DNA ligase I deficiency exhibited growth retardation, sunlight sensitivity and severe immunosuppression2, likely due to accumulation of DNA damage. Surprisingly, not much is known about the DNA damage response (DDR) in DNA ligase I-deficient cells. Because DNA replication and DDR pathways are highly conserved in eukaryotes, we utilized Saccharomyces cerevisiae as a model system to address this question. We uncovered a novel pathway, which facilitates ubiquitination of lysine 107 of proliferating cell nuclear antigen (PCNA). Unlike ubiquitination at lysine 164 of PCNA in response to UV irradiation, which triggers translesion synthesis3, modification of lysine 107 is not dependent on the ubiquitin conjugating enzyme (E2) Rad64 nor the ubiquitin ligase (E3) Rad185, but requires the E2 variant Mms26 in conjunction with Ubc47 and the E3 Rad58,9. Surprisingly, DNA ligase I-deficient cdc9-1 cells that carry a PCNAK107R mutation are inviable, because they cannot activate a robust DDR. Furthermore, we show that ubiquitination of PCNA in response to DNA ligase I-deficiency is conserved in humans, yet the lysine that mediates this modification remains to be determined. We propose that PCNA ubiquitination provides a “DNA damage code” that allows cells to categorize different types of defects that arise during DNA replication.
Accurate replication of the genome requires the evolutionarily conserved minichromosome maintenance protein, Mcm10. Although the details of the precise role of Mcm10 in DNA replication are still debated, it interacts with the Mcm2-7 core helicase, the lagging strand polymerase, DNA polymerase-α and the replication clamp, proliferating cell nuclear antigen. Loss of these interactions caused by the depletion of Mcm10 leads to chromosome breakage and cell cycle checkpoint activation. However, whether Mcm10 has an active role in DNA damage prevention is unknown. Here, we present data that establish a novel role of the N-terminus of Mcm10 in resisting DNA damage. We show that Mcm10 interacts with the Mec3 subunit of the 9-1-1 clamp in response to replication stress evoked by UV irradiation or nucleotide shortage. We map the interaction domain with Mec3 within the N-terminal region of Mcm10 and demonstrate that its truncation causes UV light sensitivity. This sensitivity is not further enhanced by a deletion of MEC3, arguing that MCM10 and MEC3 operate in the same pathway. Since Rad53 phosphorylation in response to UV light appears to be normal in N-terminally truncated mcm10 mutants, we propose that Mcm10 may have a role in replication fork restart or DNA repair.
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