Base excision repair (BER) is one of the cellular defense mechanisms repairing damage to nucleoside 5-monophosphate residues in genomic DNA. This repair pathway is initiated by spontaneous or enzymatic Nglycosidic bond cleavage creating an abasic or apurinicapyrimidinic (AP) site in double-stranded DNA. Class II AP endonuclease, deoxyribonucleotide phosphate (dRP) lyase, DNA synthesis, and DNA ligase activities complete repair of the AP site. In mammalian cell nuclear extract, BER can be mediated by a macromolecular complex containing DNA polymerase ␤ (␤-pol) and DNA ligase I. These two enzymes are capable of contributing the latter three of the four BER enzymatic activities. In the present study, we found that AP site BER can be reconstituted in vitro using the following purified human proteins: AP endonuclease, ␤-pol, and DNA ligase I. Examination of the individual enzymatic steps in BER allowed us to identify an ordered reaction pathway: subsequent to 5 "nicking" of the AP site-containing DNA strand by AP endonuclease, ␤-pol performs DNA synthesis prior to removal of the 5-dRP moiety in the gap. Removal of the dRP flap is strictly required for DNA ligase I to seal the resulting nick. Additionally, the catalytic rate of the reconstituted BER system and the individual enzymatic activities was measured. The reconstituted BER system performs repair of AP site DNA at a rate that is slower than the respective rates of AP endonuclease, DNA synthesis, and ligation, suggesting that these steps are not rate-determining in the overall reconstituted BER system. Instead, the rate-limiting step in the reconstituted system was found to be removal of dRP (i.e. dRP lyase), catalyzed by the amino-terminal domain of ␤-pol. This work is the first to measure the rate of BER in an in vitro reaction. The potential significance of the dRP-containing intermediate in the regulation of BER is discussed. Base excision repair (BER)1 pathways are employed to repair damaged or modified bases in DNA. Because similar BER pathways are found in prokaryotic and eukaryotic cells, the extensive knowledge about prokaryotic BER has facilitated studies of this repair mechanism in mammalian cells. BER has been examined in vitro with crude extracts from Escherichia coli, Saccharomyces cerevisiae, Xenopus laevis oocyte, bovine testis, and various mammalian cells (1-5) and reconstituted using purified proteins from both prokaryotes and eukaryotes (1, 5-8).Mammalian cells can repair abasic sites, an intermediate of BER, using at least two distinct pathways: one involving single nucleotide gap filling by DNA polymerase ␤ ("simple" BER) and an "alternate" pathway that involves proliferating cell nuclear antigen (PCNA). In this latter pathway gap-filling DNA synthesis appears to be catalyzed by DNA polymerase ␦ or ⑀ and results in a repair patch of 2-6 nucleotides (9). In addition, Klungland and Lindahl (10) have described a BER pathway that repairs reduced AP sites. This pathway also generates a repair patch 2-6 nucleotides in length, but in this case gapf...

DNA polymerase fidelity or specificity expresses the ability of a polymerase to select a correct nucleoside triphosphate (dNTP) from a pool of structurally similar molecules. Fidelity is quantified from the ratio of specificity constants (catalytic efficiencies) for alternate substrates (i.e. correct and incorrect dNTPs). An analysis of the efficiency of dNTP (correct and incorrect) insertion for a low fidelity mutant of DNA polymerase ␤ (R283A) and exonuclease-deficient DNA polymerases from five families derived from a variety of biological sources reveals that a strong correlation exists between the ability to synthesize DNA and the probability that the polymerase will make a mistake (i.e. base substitution error). Unexpectedly, this analysis indicates that the difference between low and high fidelity DNA polymerases is related to the efficiency of correct, but not incorrect, nucleotide insertion. In contrast to the loss of fidelity observed with the catalytically inefficient R283A mutant, the fidelity of another inefficient mutant of DNA polymerase ␤ (G274P) is not altered. Thus, although all natural low fidelity DNA polymerases are inefficient, not every inefficient DNA polymerase has low fidelity. Low fidelity polymerases appear to be an evolutionary solution to how to replicate damaged DNA or DNA repair intermediates without burdening the genome with excessive polymerase-initiated errors.The equilibrium between genome stability and instability is tightly regulated since mutations are central to aging, disease, and evolution. Thus, cellular strategies that modulate this equilibrium are of general and immense interest. The structure of DNA was proposed nearly 50 years ago and provided the first clue to how "genetic material" could be replicated faithfully (1). It is now recognized that DNA polymerases play pivotal roles in both genome replication and maintenance (i.e. DNA repair). Polymerases copy the parental (template) strand to generate a new or repaired complementary daughter strand, and accurate DNA synthesis during replication and repair is essential in maintaining genomic integrity. Although DNA polymerases play a central role in these essential processes, the fundamental mechanism(s) by which they select the correct deoxynucleoside 5Ј-triphosphate (dNTP)1 from a pool of structurally similar molecules to accomplish efficient and faithful polymerization is poorly understood. The intrinsic base substitution error frequency for DNA replication and repair polymerases is generally between 10 Ϫ3 and 10 Ϫ6 (2). These frequencies represent one error per thousand or million nucleotides synthesized, respectively. These levels of discrimination are far greater than predicted by free energy differences between matched and mismatched DNA termini (predicted error frequency of ϳ0.4; one error per 3 nucleotides synthesized), indicating that DNA polymerases can enhance fidelity by a large factor (3). However, even this remarkable specificity is inadequate to faithfully replicate a genome of more than 10 9 nucleotides....

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