Bulk replicative DNA synthesis in eukaryotes is highly accurate and efficient, primarily because of two DNA polymerases (Pols): Pols δ and ε. The high fidelity of these enzymes is due to their intrinsic base selectivity and proofreading exonuclease activity which, when coupled with post-replication mismatch repair, helps to maintain human mutation rates at less than one mutation per genome duplication. Conditions that reduce polymerase fidelity result in increased mutagenesis and can lead to cancer in mice. Whereas yeast Pol ε has been well characterized, human Pol ε remains poorly understood. Here, we present the first report on the fidelity of human Pol ε. We find that human Pol ε carries out DNA synthesis with high fidelity, even in the absence of its 3′→5′ exonucleolytic proofreading and is significantly more accurate than yeast Pol ε. Though its spectrum of errors is similar to that of yeast Pol ε, there are several notable exceptions. These include a preference of the human enzyme for T→A over A→T transversions. As compared with other replicative DNA polymerases, human Pol ε is particularly accurate when copying homonucleotide runs of 4–5 bases. The base pair substitution specificity and high fidelity for frameshift errors observed for human Pol ε are distinct from the errors made by human Pol δ.

Background: Ribonucleotides in DNA are associated with genome instability.Results: Human DNA polymerase ϵ catalyzes efficient incorporation of ribonucleotides and extension from primers terminating in multiple consecutive ribonucleotides.Conclusion: Human DNA polymerase ϵ is able to extend ribonucleotide-terminal primers through a reduction in its proofreading activity.Significance: Leading strand replication may have a unique relationship to ribonucleotides, RNA, and genome stability.

Tumors defective for DNA polymerase (Pol) ε proofreading have the highest tumor mutation burden identified. A major unanswered question is whether loss of Pol ε proofreading by itself is sufficient to drive this mutagenesis, or whether additional factors are necessary. To address this, we used a combination of next generation sequencing and in vitro biochemistry on human cell lines engineered to have defects in Pol ε proofreading and mismatch repair. Absent mismatch repair, monoallelic Pol ε proofreading deficiency caused a rapid increase in a unique mutation signature, similar to that observed in tumors from patients with biallelic mismatch repair deficiency and heterozygous Pol ε mutations. Restoring mismatch repair was sufficient to suppress the explosive mutation accumulation. These results strongly suggest that concomitant suppression of mismatch repair, a hallmark of colorectal and other aggressive cancers, is a critical force for driving the explosive mutagenesis seen in tumors expressing exonuclease-deficient Pol ε.

In CHO cells, CDK1/2-dependent phosphorylation of Ubc2/ Rad6 at Ser 120 stimulates its ubiquitin conjugating activity and can be replicated by a S120D point mutant (Sarcevic, B., Mawson, A., Baker, R. T., and Sutherland, R. L. (2002) EMBO J. 21, 2009 -2018). In contrast, we find that ectopic expression of wild type Ubc2b but not Ubc2bS120D or Ubc2bS120A in T47D human breast cancer cells specifically stimulates N-end rule-dependent degradation but not the Ubc2-independent unfolded protein response pathway, indicating that the former is E2 limiting in vivo and likely down-regulated by Ser 120 phosphorylation, as modeled by the S120D point mutation. In vitro kinetic analysis shows the in vivo phenotype of Ubc2bS120D and Ubc2bS120A is not due to differences in activating enzyme-catalyzed E2 transthiolation. However, the Ser 120 mutants possess marked differences in their abilities to support in vitro conjugation by the N-end rule-specific E3␣/Ubr1 ligase that presumably accounts for their in vivo effects. Initial rate kinetics of human E3␣-catalyzed conjugation of the human ␣-lactalbumin N-end rule substrate shows Ubc2bS120D is 20-fold less active than wild type E2, resulting from an 8-fold increase in K m and a 2.5-fold decrease in V max , the latter reflecting a decreased ability to support the initial step in target protein conjugation; Ubc2bS120A is 8-fold less active than wild type E2 due almost exclusively to a decrease in V max , reflecting a defect in polyubiquitin chain elongation. These studies suggest a mechanism for the integrated regulation of diverse ubiquitin-dependent signaling pathways through E2 phosphorylation that yields differential effects on its cognate ligases.The modification of specific target proteins with ubiquitin is a fundamental regulatory strategy within eukaryotes (1, 2), the principal consequence of which is commitment to 26 S proteasome-mediated degradation by assembly on the target protein of polyubiquitin chains linked principally though Lys 48 (3). In contrast, monomeric ubiquitin or polymeric ubiquitin assembled from Lys 63 linkages serve as transposable binding elements that direct functional consequences that are independent of the 26 S proteasome (for review, see Ref. 1). The diversity of these cellular roles arising from ubiquitin conjugation reflects the intrinsically greater information content represented by the constellation of surface residues present on the polypeptide and the combinatorial contributions of polyubiquitin chain formation through each of the seven lysines contained within the polypeptide. For these reasons there is considerable interest in understanding the mechanism of ubiquitin conjugation and the processes controlling this subset of post-translational modifications.Conjugation of ubiquitin to a substrate protein requires the sequential action of three enzymes catalyzing the two halfreactions characteristic of all ligases (for review, see Ref. 4 and 5). In the first half-reaction, ubiquitin-activating enzyme (Uba1) 2 catalyzes an ATP-dependent reacti...

Mutations in human DNA Polymerase (Pol) ε, one of three eukaryotic Pols required for DNA replication, have recently been found associated with an ultramutator phenotype in tumors from somatic colorectal and endometrial cancers and in a familial colorectal cancer. Possibly, Pol ε mutations reduce the accuracy of DNA synthesis, thereby increasing the mutational burden and contributing to tumor development. To test this possibility in vivo, we characterized an active site mutant allele of human Pol ε that exhibits a strong mutator phenotype in vitro when the proofreading exonuclease activity of the enzyme is inactive. This mutant has a strong bias towards mispairs opposite template pyrimidine bases, particularly T•dTTP mispairs. Expression of mutant Pol ε in human cells lacking functional mismatch repair caused an increase in mutation rate primarily due to T•dTTP mispairs. Functional mismatch repair eliminated the increased mutagenesis. The results indicate that the mutant Pol ε causes replication errors in vivo, and is at least partially dominant over the endogenous, wild type Pol ε. Since tumors from familial and somatic colorectal patients arise with Pol ε mutations in a single allele, are microsatellite stable and have a large increase in base pair substitutions, our data are consistent with a Pol ε mutation requiring additional factors to promote tumor development.