Mammalian DNA polymerase ␦ (pol ␦), a key enzyme of chromosomal DNA replication, consists of four subunits as follows: the catalytic subunit; p125, which is tightly associated with the p50 subunit; p68, a proliferating cell nuclear antigen (PCNA)-binding protein; and a fourth subunit, p12. In this study, the functional roles of the p12 subunit of pol ␦ were studied. The inter-subunit interactions of the p12 subunit were determined by yeast two-hybrid assays and by pulldown assays. These assays revealed that p12 interacts with p125 as well as p50. This dual interaction of p12 suggests that it may serve to stabilize the p125-p50 interaction. p12 was shown to be a novel PCNA-binding protein. This was confirmed by identification of a PCNA-binding motif at its N terminus by binding assays and by site-directed mutagenesis. The activities and reaction products of recombinant pol ␦ containing a p12 mutant defective in PCNA binding, as well as purified recombinant pol ␦ and its subassemblies, were analyzed. Our results indicate that p12 contributes to PCNA-dependent pol ␦ activity, i.e. the p12-PCNA interaction is functional. Our data indicate that both p12 and p68 are required for optimal pol ␦ activity. This supports the hypothesis that the interaction between pol ␦ and PCNA is a divalent one that involves p12 and p68. We propose a model in which pol ␦ interacts with PCNA via at least two of its subunits, and one in which p12 could play a role in stabilizing the overall pol ␦-PCNA complex as well as pol ␦ itself.Chromosomal DNA replication in eukaryotic cells requires the following three distinct DNA polymerases: polymerase ␣, polymerase ␦ (pol ␦), 3 and polymerase ⑀. DNA pol ␦ is the key enzyme that is thought to play a central role in the elongation of both the leading and the lagging strands of DNA and the maturation of Okazaki fragments (1-3). DNA pol ␦ was originally identified as a new type of DNA polymerase possessing an intrinsic 3Ј-5Ј-exonuclease activity (4). Mammalian pol ␦ holoenzyme consists of the p125 catalytic subunit (which harbors both 5Ј-3Ј DNA polymerase and 3Ј-5Ј-exonuclease activities) and a tightly associated second subunit p50; this core is associated with two other subunits, p68 and p12, that are also referred to as the third and fourth subunits (5-9). The function of pol ␦ as a chromosomal DNA polymerase is dependent on its association with PCNA, which functions as a molecular sliding clamp (10, 11). The third subunits of pol ␦ in both mammalian (p68/p66) and in yeast cells (Cdc27 in Schizosaccharomyces pombe and pol 32 in Saccharomyces cerevisiae) harbor a PCNA-binding motif, and it has been shown that this provides a PCNA interaction site for pol ␦ (12-16). However, the exact nature of the subunit contacts of mammalian pol ␦ with PCNA has yet to be clarified; we (17-20) and others (8) have reported that human pol ␦ p125 binds to PCNA, although other reports have come to the opposite conclusion (14, 21). There is also a report that the p50 subunit of mammalian pol ␦ binds to PCNA (21).The fou...

In both budding and fission yeast, a large number of ribonucleotides are incorporated into DNA during replication by the major replicative polymerases (Pols α, δ, and ε). They are subsequently removed by RNase H2-dependent repair, which if defective leads to replication stress and genome instability. To extend these studies to humans, where an RNase H2 defect results in an autoimmune disease, here we compare the ability of human and yeast Pol δ to incorporate, proofread, and bypass ribonucleotides during DNA synthesis. In reactions containing nucleotide concentrations estimated to be present in mammalian cells, human Pol δ stably incorporates one rNTP for approximately 2,000 dNTPs, a ratio similar to that for yeast Pol δ. This result predicts that human Pol δ may introduce more than a million ribonucleotides into the nuclear genome per replication cycle, an amount recently reported to be present in the genome of RNase H2-defective mouse cells. Consistent with such abundant stable incorporation, we show that the 3´-exonuclease activity of yeast and human Pol δ largely fails to edit ribonucleotides during polymerization. We also show that, like yeast Pol δ, human Pol δ pauses as it bypasses ribonucleotides in DNA templates, with four consecutive ribonucleotides in a DNA template being more problematic than single ribonucleotides. In conjunction with recent studies in yeast and mice, this ribonucleotide incorporation may be relevant to impaired development and disease when RNase H2 is defective in mammals. As one tool to investigate ribonucleotide incorporation by Pol δ in human cells, we show that human Pol δ containing a Leu606Met substitution in the polymerase active site incorporates 7-fold more ribonucleotides into DNA than does wild type Pol δ.

Human DNA polymerase δ (Pol δ4), a key enzyme in chromosomal replication, is a heterotetramer composed of the p125, p50, p68 and p12 subunits. Genotoxic agents such as UV and alkylating chemicals trigger a DNA damage response in which Pol δ4 is converted to a trimer (Pol δ3) by degradation of p12. We show that Pol δ3 has altered enzymatic properties: it is less able to perform translesion synthesis on templates containing base lesions (O6-MeG, 8-oxoG, an abasic site or a thymine-thymine dimer); a greater proofreading activity; an increased exonuclease/polymerase activity ratio; a decreased tendency for the insertion of wrong nucleotides, and for the extension of mismatched primers. Overall, our findings indicate that Pol δ3 exhibits an enhanced ability for the detection of errors in both primers and templates over its parent enzyme. These alterations in Pol δ3 show that p12 plays a major role in Pol δ4 catalytic functions, and provides significant insights into the rationale for the conversion of Pol δ4 to Pol δ3 in the cellular response to DNA damage.

SUMMARY Cancer cells rely on the activation of telomerase or the alternative lengthening of telomeres (ALT) pathways for telomere maintenance and survival. ALT involves homologous recombination (HR)-dependent exchange and/or HR-associated synthesis of telomeric DNA. Utilizing proximity-dependent biotinylation (BioID), we sought to determine the proteome of telomeres in cancer cells that employ these distinct telomere elongation mechanisms. Our analysis reveals that multiple DNA repair networks converge at ALT telomeres. These include the specialized translesion DNA synthesis (TLS) proteins FANCJ-RAD18-PCNA and, most notably, DNA polymerase eta (Polη). We observe that the depletion of Polη leads to increased ALT activity and late DNA polymerase δ (Polδ)-dependent synthesis of telomeric DNA in mitosis. We propose that Polη fulfills an important role in managing replicative stress at ALT telomeres, maintaining telomere recombination at tolerable levels and stimulating DNA synthesis by Polδ.

Mammalian DNA polymerase δ (Pol δ), a four-subunit enzyme, plays a crucial and versatile role in DNA replication and DNA repair processes. We have reconstituted human Pol δ complexes in insect cells infected with a single baculovirus into which one or more subunits were assembled. This system allowed for the efficient expression of the tetrameric Pol δ holoenzyme, the p125/p50 core dimer, the core+p68 trimer and the core+p12 trimer, as well as the p125 catalytic subunit. These were isolated in milligram amounts with reproducible purity and specific activities by a highly standardized protocol. We have systematically compared their activities in order to gain insights into the roles of the p12 and p68 subunits, as well as their responses to PCNA. The relative specific activities (apparent k cat) of the Pol δ holoenzyme, core+p68, core+p12 and p125/p50 core were 100, 109, 40, and 29. The corresponding apparent K d's for PCNA were 7.1, 8.7, 9.3 and 73 nM. Our results support the hypothesis that Pol δ interacts with PCNA through multiple interactions, and that there may be a redundancy in binding interactions that may permit Pol δ to adopt flexible configurations with PCNA. The abilities of the Pol δ complexes to fully extend singly primed M13 DNA were examined. All the subassemblies except the core+p68 were defective in their abilities to completely extend the primer, showing that the p68 subunit has an important function in synthesis of long stretches of DNA in this assay. The core+p68 trimer could be reconstituted by addition of p12.