Mammalian DNA polymerase ␦ (Pol ␦) is believed to replicate a large portion of the genome and to synthesize DNA in DNA repair and genetic recombination pathways. The effects of mutation in the polymerase domain of this essential enzyme are unknown. Here, we generated mice harboring an L604G or L604K substitution in highly conserved motif A in the polymerase active site of Pol ␦. Homozygous Pold1 L604G/L604G and Pold1 L604K/L604K mice died in utero. However, heterozygous animals were viable and displayed no overall increase in disease incidence, indicative of efficient compensation for the defective mutant polymerase. The life spans of wild-type and heterozygous Pold1 ؉/L604G mice did not differ, while that of Pold1 ؉/L604K mice was reduced by 18%. Cultured embryonic fibroblasts from the heterozygous strains exhibited comparable increases in both spontaneous mutation rate and chromosome aberrations. We observed no significant increase in cancer incidence; however, Pold1 ؉/L604K mice bearing histologically diagnosed tumors died at a younger median age than wild-type mice. Our results indicate that heterozygous mutation at L604 in the polymerase active site of DNA polymerase ␦ reduces life span, increases genomic instability, and accelerates tumorigenesis in an allele-specific manner, novel findings that have implications for human cancer.Eukaryotic DNA polymerase ␦ (Pol ␦) is an essential, highly conserved enzyme that participates in DNA replication, DNA repair, and genetic recombination. Pol ␦ 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 3Ј35Ј exonucleolytic proofreading (5, 18). Pol ␦ is highly processive in association with PCNA and synthesizes DNA with great accuracy, catalyzing about one error in 10 Ϫ5 to 10 Ϫ6 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.
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