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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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Duplication of eukaryotic chromosomes begins from multiple sites called origins of replication, with DNA synthesis proceeding bidirectionally away from the origin. There is little detailed information available pertaining to whether replication initiates at specific sites or anywhere within a given origin. The development of replication initiation point (RIP) mapping has made it possible to map start sites for DNA synthesis at the nucleotide level. The key step in RIP mapping is the purification of nascent DNA, which is initiated by small RNA primers. For the removal of broken DNA fragments, we utilize lambda-exonuclease, which digests DNA, but leaves nascent strands intact as long as they have the RNA primer still attached. RIP mapping is a sensitive technique and has been successfully applied to single copy loci in both budding and fission yeast, archaebacteria, and human cells. Studies in yeast have shown that the binding site for the initiator, the origin recognition complex (ORC), lies immediately adjacent to the replication start point, which suggests that ORC directs the initiation machinery to a distinct site. Here, we present a detailed step-by-step protocol for RIP mapping of replication origins in budding yeast.

Minichromosome maintenance protein (Mcm) 10 is a part of the eukaryotic replication machinery and highly conserved throughout evolution. As a multivalent DNA scaffold, Mcm10 coordinates the action of proteins that are indispensable for lagging strand synthesis, such as the replication clamp, proliferating cell nuclear antigen (PCNA). The binding between Mcm10 and PCNA serves an essential function during DNA elongation and is mediated by the ubiquitination of Mcm10. Here we map lysine 372 as the primary attachment site for ubiquitin on S. cerevisiae Mcm10. Moreover, we identify five additional lysines that can be ubiquitinated. Mutation of lysine 372 to arginine ablates ubiquitination of overexpressed protein and causes sensitivity to the replication inhibitor hydroxyurea in cells that are S-phase checkpoint compromised. Together, these findings reveal the high selectivity of the ubiquitination machinery that targets Mcm10 and that ubiquitination has a role in suppressing replication stress.

The anti‐parallel nature of DNA permits continuous synthesis of DNA on the leading strand and discontinuous DNA synthesis on the lagging strand. On the lagging strand, Okazaki fragments are joined by the catalytic action of DNA ligase I. An individual with a DNA ligase I deficiency exhibited growth retardation, sunlight sensitivity and severe immunosuppression, likely due to accumulation of DNA damage. Surprisingly, little is known about the DNA damage response (DDR) in DNA ligase I‐deficient cells. We utilized Saccharomyces cerevisiae as a model system to address this question. We uncovered a novel pathway, which facilitates ubiquitination of proliferating cell nuclear antigen (PCNA) on lysine 107. Unlike PCNA ubiquitination at lysine 164 in response to UV irradiation, which triggers translesion synthesis and is dependent on Rad6 and Rad18, modification at lysine 107 requires the E2 variant Mms2 in conjunction with Ubc4 and Rad5. Surprisingly, DNA ligase I‐deficient cdc9‐1 cells that carry a PCNAK107R mutation are inviable, because they cannot activate a robust DDR. Our data show that depending on the type of damage that a cell encounters, PCNA is ubiquitinated at different lysine residues. We propose a “DNA Damage code” that allows cells to recognize different types of DNA damage through differential PCNA ubiquitination, allowing the cells to activate the appropriate repair response.