Alterations in the exonuclease domain of DNA polymerase ε (Polε) cause ultramutated tumors. Severe mutator effects of the most common variant, Polε-P286R, modeled in yeast suggested that its pathogenicity involves yet unknown mechanisms beyond simple proofreading deficiency. We show that, despite producing a catastrophic amount of replication errors in vivo, the yeast Polε-P286R analog retains partial exonuclease activity and is more accurate than exonuclease-dead Polε. The major consequence of the arginine substitution is a dramatically increased DNA polymerase activity. This is manifested as a superior ability to copy synthetic and natural templates, extend mismatched primer termini, and bypass secondary DNA structures. We discuss a model wherein the cancer-associated substitution limits access of the 3’-terminus to the exonuclease site and promotes binding at the polymerase site, thus stimulating polymerization. We propose that the ultramutator effect results from increased polymerase activity amplifying the contribution of Polε errors to the genomic mutation rate.
During eukaryotic replication, DNA polymerases e (Pole) and δ (Polδ) synthesize the leading and lagging strands, respectively. In a long-known contradiction to this model, defects in the fidelity of Pole have a much weaker impact on mutagenesis than analogous Polδ defects. It has been previously proposed that Polδ contributes more to mutation avoidance because it proofreads mismatches created by Pole in addition to its own errors. However, direct evidence for this model was missing. We show that, in yeast, the mutation rate increases synergistically when a Pole nucleotide selectivity defect is combined with a Polδ proofreading defect, demonstrating extrinsic proofreading of Pole errors by Polδ. In contrast, combining Polδ nucleotide selectivity and Pole proofreading defects produces no synergy, indicating that Pole cannot correct errors made by Polδ. We further show that Polδ can remove errors made by exonuclease-deficient Pole in vitro. These findings illustrate the complexity of the one-strand-one-polymerase model where synthesis appears to be largely divided, but Polδ proofreading operates on both strands.DNA replication | extrinsic proofreading | DNA polymerase δ |
Substitutions in the exonuclease domain of DNA polymerase ϵ cause ultramutated human tumors. Yeast and mouse mimics of the most common variant, P286R, produce mutator effects far exceeding the effect of Polϵ exonuclease deficiency. Yeast Polϵ-P301R has increased DNA polymerase activity, which could underlie its high mutagenicity. We aimed to understand the impact of this increased activity on the strand-specific role of Polϵ in DNA replication and the action of extrinsic correction systems that remove Polϵ errors. Using mutagenesis reporters spanning a well-defined replicon, we show that both exonuclease-deficient Polϵ (Polϵ-exo−) and Polϵ-P301R generate mutations in a strictly strand-specific manner, yet Polϵ-P301R is at least ten times more mutagenic than Polϵ-exo− at each location analyzed. Thus, the cancer variant remains a dedicated leading-strand polymerase with markedly low accuracy. We further show that P301R substitution is lethal in strains lacking Polδ proofreading or mismatch repair (MMR). Heterozygosity for pol2-P301R is compatible with either defect but causes strong synergistic increases in the mutation rate, indicating that Polϵ-P301R errors are corrected by Polδ proofreading and MMR. These data reveal the unexpected ease with which polymerase exchange occurs in vivo, allowing Polδ exonuclease to prevent catastrophic accumulation of Polϵ-P301R-generated errors on the leading strand.
With advances in next generation sequencing (NGS) technologies, efforts have been made to develop personalized medicine, targeting the specific genetic makeup of an individual. Somatic or germline DNA Polymerase epsilon (PolE) mutations cause ultramutated (>100 mutations/Mb) cancer. In contrast to mismatch repair-deficient hypermutated (>10 mutations/Mb) cancer, PolE-associated cancer is primarily microsatellite stable (MSS) In this article, we provide a comprehensive review of this PolE-associated ultramutated tumor. We describe its molecular characteristics, including the mutation sites and mutation signature of this type of tumor and the mechanism of its ultramutagenesis. We discuss its good clinical prognosis and elucidate the mechanism for enhanced immunogenicity with a high tumor mutation burden, increased neoantigen load, and enriched tumor-infiltrating lymphocytes. We also provide the rationale for immune checkpoint inhibitors in PolE-mutated tumors.
bReplication factor C (RFC) is known to function in loading proliferating cell nuclear antigen (PCNA) onto primed DNA, allowing PCNA to tether DNA polymerase for highly processive DNA synthesis in eukaryotic and archaeal replication. In this report, we show that an RFC complex from the hyperthermophilic archaea of the genus Sulfolobus physically interacts with DNA polymerase B1 (PolB1) and enhances both the polymerase and 3=-5= exonuclease activities of PolB1 in an ATP-independent manner. Stimulation of the PolB1 activity by RFC is independent of the ability of RFC to bind DNA but is consistent with the ability of RFC to facilitate DNA binding by PolB1 through protein-protein interaction. These results suggest that Sulfolobus RFC may play a role in recruiting DNA polymerase for efficient primer extension, in addition to clamp loading, during DNA replication.A ll forms of cellular life replicate their chromosomal DNA in a strikingly similar fashion, employing clamp loader proteins to assemble ring-shaped sliding clamps in ATP-dependent reactions at new RNA-primed sites, where the clamp tethers DNA polymerase for highly processive DNA synthesis (1). Although clamp loader proteins of different origins differ at the amino acid sequence level and in subunit composition, they share both overall structures and molecular mechanisms in clamp-loading processes (2). In Escherichia coli, the clamp loader, known as the ␥ complex, consists of five subunits, i.e., ␦, ␦=, and three copies of /␥ (3). In eukarya, the clamp loader, referred to as replication factor C (RFC), has four different small subunits (RFC S ) and one large subunit (RFC L ) (2). Archaea, the third domain of life, replicate DNA in a eukaryotic-like fashion. While most of the archaeal clamp loaders are a pentameric complex of one large subunit and four identical small subunits (4-6), RFCs from some archaea exhibit variations in subunit composition. For example, RFC from Methanosarcina acetivorans shows a stoichiometry of one large subunit to three small subunits to one even smaller subunit (7).PCNA loading by the RFC heteropentamer (1 RFC L /4 RFC S ) from the thermoacidophilic crenarchaeon Sulfolobus solfataricus has been extensively studied (8,9). Unlike eukaryotes and euryarchaea, which possess a homotrimeric PCNA, crenarchaea have an unusual heterotrimeric PCNA comprising PCNA1, PCNA2, and PCNA3 (8). The S. solfataricus RFC has been shown to interact with the PCNA1 and PCNA2 subunits through RFC S and with PCNA3 through RFC L (8). Opening of the PCNA ring by RFC at the PCNA3-PCNA1 interface allows the passage of DNA into the PCNA ring (9). In addition to its role in PCNA loading, RFC also interacts with the eukaryotic-type primase (10). As the result of this interaction, primer synthesis by the primase is inhibited while the ATPase activity of RFC is stimulated, suggesting that RFC may serve roles in regulating primer synthesis and transfer of primers to DNA polymerase.In the present study, we show that Sulfolobus RFC interacts physically with DNA polymera...
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