Summary Mcm10 is an essential eukaryotic DNA replication protein required for assembly and progression of the replication fork. The highly conserved internal domain (Mcm10-ID) has been shown to physically interact with single-stranded (ss) DNA, DNA polymerase α, and PCNA. The crystal structure of Xenopus laevis Mcm10-ID presented here reveals a novel DNA binding architecture composed of an OB-fold followed in tandem by a variant and highly basic zinc finger. NMR chemical shift perturbation and mutational studies of DNA binding activity in vitro reveal how Mcm10 uses this unique surface to engage ssDNA. Corresponding mutations in Saccharomyces cerevisiae result in increased sensitivity to replication stress, demonstrating the functional importance of DNA binding by this region of Mcm10 to replication. In addition, mapping Mcm10 mutations known to disrupt PCNA, pol α, and DNA interactions onto the crystal structure provides insight into how Mcm10 may coordinate protein and DNA binding within the replisome.

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.

The S-phase checkpoint kinases Mec1 and Rad53 in the budding yeast, Saccharomyces cerevisiae, are activated in response to replication stress that induces replication fork arrest. In the absence of a functional S-phase checkpoint, stalled replication forks collapse and give rise to chromosome breakage. In an attempt to better understand replication dynamics in S-phase checkpoint mutants, we developed a replication origin array for budding yeast that contains 424 of 432 previously identified potential origin regions. As expected, mec1-1 and rad53-1 mutants failed to inhibit late origin activation. Surprisingly however, 17 early-firing regions were not replicated efficiently in these mutants. This was not due to a lack of initiation, but rather to problems during elongation, as replication forks arrested in close proximity to these origins, resulting in the accumulation of small replication intermediates and eventual replication fork collapse. Importantly, these regions were not only prone to chromosome breakage in the presence of exogenous stress but also in its absence, similar to fragile sites in the human genome.

The accurate duplication of chromosomal DNA is required to maintain genomic integrity. However, from an evolutionary point of view, a low mutation rate during DNA replication is desirable. One way to strike the right balance between accuracy and limited mutagenesis is to use a DNA polymerase that lacks proofreading activity but contributes to DNA replication in a very restricted manner. DNA polymerase-␣ fits this purpose exactly, but little is known about its regulation at the replication fork. Minichromosome maintenance protein (Mcm) 10 regulates the stability of the catalytic subunit of pol-␣ in budding yeast and human cells. Cdc17, the catalytic subunit of pol-␣ in yeast, is rapidly degraded after depletion of Mcm10. Here we show that Ubc4 and Not4 are required for Cdc17 destabilization. Disruption of Cdc17 turnover resulted in sensitivity to hydroxyurea, suggesting that this pathway is important for DNA replication. Furthermore, overexpression of Cdc17 in ubc4 and not4 mutants caused slow growth and synthetic dosage lethality, respectively. Our data suggest that Cdc17 levels are very tightly regulated through the opposing forces of Ubc4 and Not4 (destabilization) and Mcm10 (stabilization). We conclude that regular turnover of Cdc17 via Ubc4 and Not4 is required for proper cell proliferation. INTRODUCTIONThe accurate duplication of the genome is crucial for the prolonged health of eukaryotic organisms. Inaccurate DNA replication and/or replication of any portion of the genome more than once can result in genomic instability, which is a consistently observed hallmark of cancer cells (Vaziri et al., 2003;Venkatesan et al., 2007). It is crucial, therefore, to understand the entire process of DNA replication. The initiation of DNA replication requires the coordinated recruitment of several proteins. Prereplicative complexes (Diffley et al., 1994), including the core helicase Mcm2-7 (Bochman and Schwacha, 2008), form at origins of replication, are converted to pre-initiation complexes (Zou and Stillman, 1998), and DNA is subsequently unwound (reviewed in Bell and Dutta, 2002). Once the DNA is unwound, the accuracy of DNA replication depends in large part upon DNA polymerases (pol)-␣, -␦, and -, all of which coordinate to synthesize the nascent copy of DNA during replication in eukaryotes (Burgers, 2009). Highlighting the importance of accurate DNA replication, 43% of mice homozygous for a proofreading-deficient allele of pol-␦ develop cancer, primarily lymphoma, but also squamous-cell carcinoma (Goldsby et al., 2001). Interestingly, pol-␣, the only enzyme that can synthesize DNA de novo, naturally lacks proofreading activity (Morrison et al., 1991). Unlike pol-␦, pol-␣ is foremost a replicative -and not a repair -polymerase (Wu et al., 2001;Wang et al., 2004). The lack of proofreading activity likely reflects one mechanism by which nature enforces evolution. However, this raises the question of how humans use a potentially mutagenic polymerase responsible for the initiation of more than 30 million Okazaki fragmen...

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.

In lagging strand synthesis, Okazaki fragments are initiated by DNA polymerase‐alpha/primase, which produces a short RNA/DNA primer. Subsequently, a switch occurs from pol‐alpha to pol‐delta. We believe that minichromosome maintenance protein (Mcm) 10 coordinates this switch as it interacts with pol‐alpha and proliferating cell nuclear antigen (PCNA), the accessory factor for pol‐delta. This polymerase switch may be triggered by the non‐proteolytic ubiquitination of Mcm10, which is a pre‐requisite for Mcm10 to bind PCNA. Importantly, ubiquitinated Mcm10 can no longer interact with pol‐alpha. Because we have shown that the catalytic subunit of pol‐alpha is rapidly degraded in the absence of Mcm10, this modification might provide a mechanism to release pol‐alpha from the replication fork. We are currently trying to understand how Mcm10 is ubiquitinated and whether the Mcm10‐regulated degradation of pol‐alpha plays a role during replication.