Mutation at the Polymerase Active Site of Mouse DNA Polymerase δ Increases Genomic Instability and Accelerates Tumorigenesis
| Autor: | Lawrence A. Loeb, Piper M. Treuting, Ted Gooley, Bradley D. Preston, Ranga N. Venkatesan, Warren C. Ladiges, Evan D. Fuller, Thomas H. Norwood, Robert E. Goldsby |
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| Rok vydání: | 2007 |
| Předmět: |
Heterozygote
Base pair DNA repair DNA polymerase Longevity Molecular Sequence Data Loss of Heterozygosity Eukaryotic DNA replication Genomic Instability Protein Structure Secondary Histones Mice Neoplasms Animals Amino Acid Sequence Phosphorylation Molecular Biology Cells Cultured Polymerase DNA Polymerase III Genetics Binding Sites Base Sequence biology POLD1 DNA replication Articles Cell Biology Fibroblasts Embryo Mammalian Chromosomes Mammalian Survival Analysis Molecular biology Cell Transformation Neoplastic Phenotype Mutation biology.protein DNA mismatch repair DNA Damage |
| Zdroj: | Molecular and Cellular Biology. 27:7669-7682 |
| ISSN: | 1098-5549 |
| Popis: | Eukaryotic DNA polymerase δ (Pol δ) is an essential, highly conserved enzyme that participates in DNA replication, DNA repair, and genetic recombination. Pol δ is believed to replicate a large portion of the genome, synthesizing most of the lagging strand and perhaps contributing to leading-strand synthesis as well (14, 22, 43) The 125-kDa catalytic subunit, encoded at the mouse Pold1 locus, contains a polymerase domain near the carboxyl terminus that catalyzes DNA synthesis and an exonuclease domain near the amino terminus that catalyzes 3′→5′ exonucleolytic proofreading (5, 18). Pol δ is highly processive in association with PCNA and synthesizes DNA with great accuracy, catalyzing about one error in 10−5 to 10−6 nucleotides polymerized (11, 46, 61). Discrimination between correct and incorrect base pairs at the polymerase active site confers most of the fidelity (error rate, ca. 1 × 10−5), while proofreading increases accuracy approximately 10- to 60-fold (11). Partitioning of the newly formed primer terminus between the polymerase and exonuclease active sites is an important determinant of accuracy as well (11, 47). In cells, mismatch repair adds an additional correction mechanism for errors that escape proofreading, reducing the overall error rate to about 1 × 10−9 bp (19, 35, 36, 55). Accurate DNA replication is essential for the maintenance of genomic stability and suppression of carcinogenesis. Mutants of prokaryotic and eukaryotic DNA polymerases that harbor amino acid substitutions in the polymerase domain or that lack proofreading exhibit increased mutation frequencies in vitro and in vivo (2, 8, 34, 42, 44, 47, 60). In the case of Pol δ, inactivation of the 3′→5′ exonuclease confers a mutator phenotype, increasing mutation rates in haploid yeast and in homozygous mouse embryo fibroblasts (MEFs) (7, 16, 17, 58). The homozygous mutant mice display elevated cancer incidence, suggesting that unrepaired Pol δ errors may contribute to tumorigenesis (16, 17). Given the essentiality of Pol δ and its central role in eukaryotic DNA replication, it is important to assess the nature and severity of the consequences of polymerase active site mutations in mammals. For this study, we created mice harboring mutations at a conserved Leu residue, L604, in motif A of the Pol δ polymerase domain. We elected to study L604 mutants because the effects on accuracy of mutation at homologous residues in other DNA polymerases have been studied extensively. For example, our laboratory has shown that substitution at the homologous Ile in both Escherichia coli Pol I and Taq Pol I creates mutator mutants (40, 41, 57). In phage T4 DNA polymerase, substitution for the homologous Leu can generate either mutator or antimutator mutants (47). In yeast and human DNA polymerase α (Pol α), substitution for the orthologous Leu generates mutators of various strengths (38). Most recently, we and others have shown that replacement of L612 in Saccharomyces cerevisiae Pol δ with each of eight different amino acids increases spontaneous mutation rates up to 37-fold (28, 62). The L612G and L612K yeast Pol δ alleles, which are homologous to the L604G and L604K mutations we studied here, elevated the mutation rate 17- and 13-fold, respectively (62). The present work represents the first description of mammals harboring a mutation in the polymerase domain of a major replicative DNA polymerase. Given the significant phenotypic defects we observed in haploid yeast (62) and the central role of Pol δ in replication, it was not certain that the murine L604G and L604K alleles would yield viable animals. However, when heterozygous, these mutator mutations are compatible with mammalian development and reproduction and do not produce a significant overall increase in end-of-life pathology, indicative of extensive compensation for the defective mutant polymerases. Interestingly, both alleles result in a mutator phenotype, and a single Pold1L604K allele, but not a Pold1L604G allele, accelerates tumorigenesis and reduces life span. We discuss the implications of our results for the ongoing search for Pol δ mutations in human cancers. |
| Databáze: | OpenAIRE |
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