Maintaining the chemical identity of DNA depends on ribonucleotide exclusion by DNA polymerases. However, ribonucleotide exclusion during DNA synthesis in vitro is imperfect. To determine if ribonucleotides are incorporated during DNA replication in vivo, we substituted leucine or glycine for an active site methionine in yeast DNA polymerase ε (Pol ε). Compared to wild type Pol ε, ribonucleotide incorporation in vitro was 3-fold lower for M644L and 11-fold higher for M644G Pol ε. This hierarchy was re-capitulated in vivo in yeast strains lacking RNase H2. Moreover, the pol2-M644G rnh201Δ strain progressed more slowly through S-phase, had elevated dNTP pools and generated 2–5 base pair deletions in repetitive sequences at a high rate and gene orientation-dependent manner. The data indicate that ribonucleotides are incorporated during replication in vivo, that they are removed by RNase H2-dependent repair, and that defective repair results in replicative stress and genome instability via DNA strand misalignment.

Measurements of nucleoside triphosphate levels in Saccharomyces cerevisiae reveal that the four rNTPs are in 36-to 190-fold molar excess over their corresponding dNTPs. During DNA synthesis in vitro using the physiological nucleoside triphosphate concentrations, yeast DNA polymerase ε, which is implicated in leading strand replication, incorporates one rNMP for every 1,250 dNMPs. Pol δ and Pol α, which conduct lagging strand replication, incorporate one rNMP for every 5,000 or 625 dNMPs, respectively. Discrimination against rNMP incorporation varies widely, in some cases by more than 100-fold, depending on the identity of the base and the template sequence context in which it is located. Given estimates of the amount of replication catalyzed by Pols α, δ, and ε, the results are consistent with the possibility that more than 10,000 rNMPs may be incorporated into the nuclear genome during each round of replication in yeast. Thus, rNMPs may be the most common noncanonical nucleotides introduced into the eukaryotic genome. Potential beneficial and negative consequences of abundant ribonucleotide incorporation into DNA are discussed, including the possibility that unrepaired rNMPs in DNA could be problematic because yeast DNA polymerase ε has difficulty bypassing a single rNMP present within a DNA template.DNA replication | nucleotide precursors | nucleotide selectivity T he integrity of the eukaryotic genome is ensured in part by the chemical nature of the storage medium-DNA. Compared to RNA, DNA is inherently more resistant to strand cleavage due to the absence of a reactive 2′ hydroxyl on the ribose ring. The active sites of most DNA polymerases are evolved to efficiently exclude ribonucleoside triphosphates (rNTPs) from being incorporated during DNA synthesis (reviewed in (1)). However, rNTP exclusion is not absolute. Early studies (reviewed in (1, 2)) revealed that DNA polymerases do incorporate rNMPs during DNA synthesis. Kinetic studies (3-13) have further demonstrated that selectivity for insertion of dNMPs into DNA rather than rNMPs varies from 10-fold to >10 6 -fold, depending on the DNA polymerase and the dNTP/rNTP pair examined. rNMP incorporation during DNA synthesis is potentially made more probable by the fact that the concentrations of rNTPs in vivo are higher than are the concentrations of dNTPs (e.g., see refs. 2, 14 and results of this study). Thus some rNMPs are likely to be stably incorporated into DNA during replication, and possibly during DNA repair, e.g., nonhomologous end joining (NHEJ) of double strand breaks in DNA (9, 15). This possibility is supported by biochemical studies implicating RNase H2 and FEN1 in the repair of single ribonucleotides in DNA (16,17). It is therefore of interest to know just how frequently rNMPs are incorporated into DNA by the DNA polymerases that synthesize the most DNA in a eukaryotic cell, namely DNA polymerases α, δ, and ε. Here we investigate this by first measuring the rNTP and dNTP concentrations in budding yeast. We then use these concentrations in DNA sy...

DNA polymerase delta (Pol delta) and DNA polymerase epsilon (Pol epsilon) are both required for efficient replication of the nuclear genome, yet the division of labor between these enzymes has remained unclear for many years. Here we investigate the contribution of Pol delta to replication of the leading and lagging strand templates in Saccharomyces cerevisiae using a mutant Pol delta allele (pol3-L612M) whose error rate is higher for one mismatch (e.g., T x dGTP) than for its complement (A x dCTP). We find that strand-specific mutation rates strongly depend on the orientation of a reporter gene relative to an adjacent replication origin, in a manner implying that >90% of Pol delta replication is performed using the lagging strand template. When combined with recent evidence implicating Pol epsilon in leading strand replication, these data support a model of the replication fork wherein the leading and lagging strand templates are primarily copied by Pol epsilon and Pol delta, respectively.

Three Pol X family members have been linked to nonhomologous end joining (NHEJ) in mammals. Template-independent TdT promotes diversity during NHEJ-dependent repair of V(D)J recombination intermediates, but the roles of the template-dependent polymerases mu and lambda in NHEJ remain unclear. We show here that pol mu and pol lambda are similarly recruited by NHEJ factors to fill gaps when ends have partially complementary overhangs, suggesting equivalent roles promoting accuracy in NHEJ. However, only pol mu promotes accuracy during immunoglobulin kappa recombination. This distinctive in vivo role correlates with the TdT-like ability of pol mu, but not pol lambda, to act when primer termini lack complementary bases in the template strand. However, unlike TdT, synthesis by pol mu in this context is primarily instructed by a template from another DNA molecule. This apparent gradient of template dependence is largely attributable to a small structural element that is present but different in all three polymerases.

Genetic experiments have determined that Ku, XRCC4, and ligase IV are required for repair of double-strand breaks by the end-joining pathway. The last two factors form a tight complex in cells. However, ligase IV is only one of three known mammalian ligases and is intrinsically the least active in intermolecular ligation; thus, the biochemical basis for requiring this ligase has been unclear. We demonstrate here a direct physical interaction between the XRCC4-ligase IV complex and Ku. This interaction is stimulated once Ku binds to DNA ends. Since XRCC4-ligase IV alone has very low DNA binding activity, Ku is required for effective recruitment of this ligase to DNA ends. We further show that this recruitment is critical for efficient end-joining activity in vitro. Preformation of a complex containing Ku and XRCC4-ligase IV increases the initial ligation rate 20-fold, indicating that recruitment of the ligase is an important limiting step in intermolecular ligation. Recruitment by Ku also allows XRCC4-ligase IV to use Ku's high affinity for DNA ends to rapidly locate and ligate ends in an excess of unbroken DNA, a necessity for end joining in cells. These properties are conferred only on ligase IV, because Ku does not similarly interact with the other mammalian ligases. We have therefore defined cell-free conditions that reflect the genetic requirement for ligase IV in cellular end joining and consequently can explain in molecular terms why this factor is required.

Mammalian DNA polymerase (pol ) is related to terminal deoxynucleotidyl transferase, but its biological role is not yet clear. We show here that after exposure of cells to ionizing radiation (IR), levels of pol protein increase. pol also forms discrete nuclear foci after IR, and these foci are largely coincident with IR-induced foci of ␥H2AX, a previously characterized marker of sites of DNA double-strand breaks. pol is thus part of the cellular response to DNA double-strand breaks. pol also associates in cell extracts with the nonhomologous end-joining repair factor Ku and requires both Ku and another end-joining factor, XRCC4-ligase IV, to form a stable complex on DNA in vitro. pol in turn facilitates both stable recruitment of XRCC4-ligase IV to Ku-bound DNA and ligase IV-dependent end joining. In contrast, the related mammalian DNA polymerase ␤ does not form a complex with Ku and XRCC4-ligase IV and is less effective than pol in facilitating joining mediated by these factors. Our data thus support an important role for pol in the end-joining pathway for repair of double-strand breaks.

DNA polymerases are defined as such because they use deoxynucleotides instead of ribonucleotides with high specificity. We show here that polymerase mu (pol ), implicated in the nonhomologous end-joining pathway for repair of DNA double-strand breaks, incorporates both ribonucleotides and deoxynucleotides in a templatedirected manner. pol has an approximately 1,000-fold-reduced ability to discriminate against ribonucleotides compared to that of the related pol ␤, although pol 's substrate specificity is similar to that of pol ␤ in most other respects. Moreover, pol more frequently incorporates ribonucleotides when presented with nucleotide concentrations that approximate cellular pools. We therefore addressed the impact of ribonucleotide incorporation on the activities of factors required for double-strand break repair by nonhomologous end joining. We determined that the ligase required for this pathway readily joined strand breaks with terminal ribonucleotides. Most significantly, pol frequently introduced ribonucleotides into the repair junctions of an in vitro nonhomologous end-joining reaction, an activity that would be expected to have important consequences in the context of cellular double-strand break repair.A large number of mammalian DNA polymerases have been identified within the last 3 years, and initial characterization suggests that they possess a surprisingly diverse array of substrate specificities. We investigate here the substrate specificity of one of these recently described polymerases, polymerase mu (pol ).pol , in addition to pol ␤, pol , pol , and terminal deoxynucleotidyltransferase (TdT), is a member of the Pol X family (5). pol is most similar to TdT; the two polymerases have similar domain organizations and are ϳ40% identical in sequence, but both are less than 25% identical to remaining Pol X family members (e.g., pol ␤) (7). Moreover, pol and TdT are likely both involved in the nonhomologous end-joining (or end-joining) pathway for repair of double-strand breaks (DSBs) in vertebrates. We have recently demonstrated that pol and TdT form essentially identical complexes with the endjoining factors Ku and the XRCC4-ligase IV complex (X4-LIV) (14). End joining is a major pathway for repair of DSBs introduced by exogenous sources (e.g., ionizing radiation) in all cell types and is required for resolution of DSB intermediates in V(D)J recombination, the lymphocyte-specific process required for assembly of mature antigen receptor genes (reviewed in reference 9). TdT is expressed only in cells active in V(D)J recombination (reviewed in reference 10) and thus participates in end-joining reactions only in this context. In contrast, the more widely expressed pol seems likely to play an important role in general end-joining DSB repair.TdT's substrate specificity is uniquely suited for its role in promoting diversity in V(D)J recombination. TdT is template independent, adding random nucleotides only to 3Ј singlestranded or blunt DNA ends (reviewed in reference 6). Indeed, TdT shows generally redu...

Eukaryotic replication begins at origins and on the lagging strand with RNA-primed DNA synthesis of a few nucleotides by polymerase alpha, which lacks proofreading activity. A polymerase switch then allows chain elongation by proofreading-proficient pol delta and pol epsilon. Pol delta and pol epsilon are essential, but their roles in replication are not yet completely defined . Here, we investigate their roles by using yeast pol alpha with a Leu868Met substitution . L868M pol alpha copies DNA in vitro with normal activity and processivity but with reduced fidelity. In vivo, the pol1-L868M allele confers a mutator phenotype. This mutator phenotype is strongly increased upon inactivation of the 3' exonuclease of pol delta but not that of pol epsilon. Several nonexclusive explanations are considered, including the hypothesis that the 3' exonuclease of pol delta proofreads errors generated by pol alpha during initiation of Okazaki fragments. Given that eukaryotes encode specialized, proofreading-deficient polymerases with even lower fidelity than pol alpha, such intermolecular proofreading could be relevant to several DNA transactions that control genome stability.