DNA polymerase ␤ (pol ␤) is an error-prone polymerase that plays a central role in mammalian base excision repair. To better characterize the mechanisms governing rat pol ␤ activity, we examined polymerization on synthetic primer-templates of different structure. Steady-state kinetic analyses revealed that the catalytic efficiency of pol ␤ (k cat /K m,dNTP app ) is strongly influenced by gap size and the presence of a phosphate group at the 5-margin of the gap. pol ␤ exhibited the highest catalytic efficiency on 5-phosphorylated 1-nucleotide gapped DNA. This efficiency was >500 times higher than on non-phosphorylated 1-nucleotide and 6-nucleotide (with or without PO 4 ) gapped DNAs and 2,500 times higher than on primer-template with no gaps. The nucleotide insertion fidelity of pol ␤, as judged by its ability to form G-N mispairs, was also higher (10 -100 times) on 5-phosphorylated single-nucleotide gapped DNA compared with the other DNA substrates studied. These data suggest that a primary function of mammalian pol ␤ is to fill 5-phosphorylated 1-nucleotide gaps. DNA polymerase ␤ (pol ␤)1 plays a central role in mammalian base excision repair (BER (1-5)). pol ␤ is a monomeric 39-kDa enzyme organized into a carboxyl-terminal 31-kDa domain that includes the polymerase active site and an aminoterminal 8-kDa domain that participates in DNA binding and harbors 5Ј-deoxyribose phosphodiesterase (lyase) activity (6, 7). The presence of both polymerase and lyase activities suggests that pol ␤ catalyzes two steps in the "short-patch" BER pathway: removal of a 5Ј-deoxyribose phosphate intermediate and subsequent filling of the resultant 1-nt gap (4, 6). pol ␤ has also been implicated in "long-patch" BER (4) and may function in meiosis (8) and nucleotide excision repair (9, 10).The biochemical activities of purified pol ␤ are consistent with a role in gap-filling DNA synthesis. Early studies showed that pol ␤ is non-processive on single-stranded DNA templates, prefers short-gapped DNA substrates, and is capable of filling gaps to completion (11-16). More recently, Wilson and colleagues (17) observed that pol ␤ fills short gaps (2-6 nt) by a processive mechanism that requires a PO 4 group at the 5Ј-margin of the gap. Binding of pol ␤ to these short-gapped substrates is also strongly enhanced by the presence of a 5Ј-PO 4 (18). These experiments, together with recent structural data, suggest a model in which pol ␤ binding to gapped DNA is mediated by interactions between the 8-kDa domain of pol ␤ and the 5Ј-PO 4 at the downstream margin of the gap (7, 18). Processive DNA synthesis on short (2-6-nt) gaps is consistent with roles for pol ␤ in long-patch BER (4) and in the completion of gap-filling synthesis initiated by other cellular DNA polymerases (9, 10, 14 -16).Although DNA polymerization by pol ␤ on single-stranded and short-gapped DNAs is understood in some detail, much less is known about pol ␤ activity on its short-patch BER substrate, 1-nt gapped DNA. The model of pol ␤ binding through its 8-kDa domain to the 5Ј-PO 4 ...

DNA polymerase ␤ (pol ␤) is a 39-kDa protein that functions in DNA repair processes in mammalian cells. As a first step toward understanding mechanisms of polymerase fidelity, we developed a genetic method to identify mammalian pol ␤ mutator mutants. This screen takes advantage of a microbial genetics assay and the ability of rat pol ␤ to substitute for Escherichia coli DNA polymerase I in DNA replication in vivo. Using this screen, we identified 13 candidate pol ␤ mutator mutants. Three of the candidate mutator mutants were further characterized in vivo and shown to confer an increased spontaneous mutation frequency over that of wild-type pol ␤ to our bacterial strain. Purification and subsequent analysis of one of our putative mutator proteins, the pol ␤-14 protein, showed that it possesses intrinsic mutator activity in four different assays that measure the fidelity of DNA synthesis. Therefore, residue 265, which is altered in pol ␤-14 and another of our mutant proteins, pol ␤-166, is probably critical for accurate DNA synthesis by pol ␤. Thus, our genetic method of screening for pol ␤ mutator mutants is useful in identifying active mammalian DNA polymerase mutants that encode enzymes that catalyze DNA synthesis with altered fidelity compared with the wild-type pol ␤ enzyme.