In the crystal structure of a substrate complex, the side chains of residues Asn 279 , Tyr 271, and Arg 283 of DNA polymerase ␤ are within hydrogen bonding distance to the bases of the incoming deoxynucleoside 5-triphosphate (dNTP), the terminal primer nucleotide, and the templating nucleotide, respectively (Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H., and Kraut, J. (1994) Science 264, 1891-1903). We have altered these side chains through individual site-directed mutagenesis. Each mutant protein was expressed in Escherichia coli and was soluble. The mutant enzymes were purified and characterized to probe their role in nucleotide discrimination and catalysis. A reversion assay was developed on a short (5 nucleotide) gapped DNA substrate containing an opal codon to assess the effect of the amino acid substitutions on fidelity. Substitution of the tyrosine at position 271 with phenylalanine or histidine did not influence catalytic efficiency (k cat /K m ) or fidelity. The hydrogen bonding potential between the side chain of Asn 279 and the incoming nucleotide was removed by replacing this residue with alanine or leucine. Although catalytic efficiency was reduced as much as 17-fold for these mutants, fidelity was not. In contrast, both catalytic efficiency and fidelity decreased dramatically for all mutants of Arg 283 (Ala > Leu > Lys). The fidelity and catalytic efficiency of the alanine mutant of Arg 283 decreased 160-and 5000-fold, respectively, relative to wild-type enzyme. Sequence analyses of the mutant DNA resulting from short gap-filling synthesis indicated that the types of base substitution errors produced by the wild-type and R283A mutant were similar and indicated misincorporations resulting in frequent T⅐dGTP and A⅐dGTP mispairing. With R283A, a dGMP was incorporated opposite a template thymidine as often as the correct nucleotide. The x-ray crystallographic structure of the alanine mutant of Arg 283 verified the loss of the mutated side chain. Our results indicate that specific interactions between DNA polymerase ␤ and the template base, but not hydrogen bonding to the incoming dNTP or terminal primer nucleotide, are required for both high catalytic efficiency and nucleotide discrimination.Accurate DNA synthesis during replication and DNA repair is crucial in maintaining genomic integrity. Although DNA polymerases play a central role in these essential processes, the fundamental mechanism by which they select the correct deoxynucleoside 5Ј-triphosphate (dNTP)1 from a pool of structurally similar compounds and substrates to accomplish rapid and efficient polymerization is poorly understood. Vertebrate DNA polymerase ␤ (␤-pol) has been suggested to play a role in both DNA repair (1-5) and replication (6 -8). The x-ray crystal structures of rat and human ␤-pol in complex with substrates have suggested a detailed model of the chemical mechanism for the nucleotidyl transfer reaction and also have suggested several protein/substrate interactions that may play a role in nucleotide discrimination (9...

To investigate the interactions that determine DNA polymerase accuracy, we have measured the fidelity of 26 mutants with amino acid substitutions in the polymerase domain of a 3-5-exonuclease-deficient Klenow fragment. Most of these mutant polymerases synthesized DNA with an apparent fidelity similar to that of the wild-type control, suggesting that fidelity at the polymerase active site depends on highly specific enzymesubstrate interactions and is not easily perturbed. In addition to the previously studied Y766A mutator, four novel base substitution mutators were identified; they are R668A, R682A, E710A, and N845A. Each of these five mutator alleles results from substitution of a highly conserved amino acid side chain located on the exposed surface of the polymerase cleft near the polymerase active site. Analysis of base substitution errors at four template positions indicated that each of the five mutator polymerases has its own characteristic error specificity, suggesting that the Arg-668, Arg-682, Glu-710, Tyr-766, and Asn-845 side chains may contribute to polymerase fidelity in a variety of different ways. We separated the contributions of the nucleotide insertion and mismatch extension steps by using a novel fidelity assay that scores base substitution errors during synthesis to fill a single nucleotide gap (and hence does not require mismatch extension) and by measuring the rates of polymerase-catalyzed mismatch extension reactions. The R682A, E710A, Y766A, and N845A mutations cause decreased fidelity at the nucleotide insertion step, whereas R668A results in lower fidelity in both nucleotide insertion and mismatch extension. Relative to wild type, several Klenow fragment mutants showed substantially more discrimination against extension of a T⅐G mismatch under the conditions of the fidelity assay, providing one explanation for the anti-mutator phenotypes of mutants such as R754A and Q849A.

During base excision repair, DNA polymerase ␤ fills 1-6-nucleotide gaps processively, reflecting a contribution of both its 8-and 31-kDa domains to DNA binding. Here we report the fidelity of pol ␤ during synthesis to fill gaps of 1, 5, 6, or >300 nucleotides. Error rates during distributive synthesis by recombinant rat and human polymerase (pol) ␤ with a 390-base gap are similar to each other and to previous values with pol ␤ purified from tissues. The base substitution fidelity of human pol ␤ when processively filling a 5-nucleotide gap is similar to that with a 361-nucleotide gap, but "closely-spaced" substitutions are produced at a rate at least 60-fold higher than for distributive synthesis. Base substitution fidelity when filling a 1-nucleotide gap is higher than when filling a 5-nucleotide gap, suggesting a contribution of the 8-kDa domain to the dNTP binding pocket and/or a difference in base stacking or DNA structure imposed by pol ␤. Nonetheless, 1-nucleotide gap filling is inaccurate, even generating complex substitution-addition errors. Finally, the single-base deletion error rate during processive synthesis to fill a 6-nucleotide gap is indistinguishable from that of distributive synthesis to fill a 390-nucleotide gap. Thus the mechanism of processivity by pol ␤ does not allow the enzyme to suppress template misalignments. DNA polymerase ␤ (pol ␤)1 is the smallest of the mammalian cellular DNA polymerases. Evidence suggests that it can participate in several DNA transactions in vivo, including DNA replication (1, 2), recombination (3, 4), and base excision DNA repair (reviewed in Ref. 5). Base excision repair is needed to replace damaged nucleotides that are depurinated, deaminated, oxidized, or methylated as a result of normal cellular metabolism or environmental insult (reviewed in Ref. 6). There are at least two forms of base excision repair (BER) in mammalian cells (5). The predominant form is "simple" BER (7), involving excision of a single damaged nucleotide and its replacement catalyzed primarily by pol ␤ (8, 9). Alternatively, BER may occur by excision of 2 to 6 nucleotides involving proliferating cell nuclear antigen and flap endonuclease 1, and DNA synthesis may be catalyzed by pol ␤ (10, 11) or by pol ␦ or pol ⑀ (12, 13). This pathway is referred to as "alternate" BER.Early studies of the fidelity of DNA synthesis by pol ␤ employed a long single-stranded template within a 390-nucleotide gap (14). The average pol ␤ error rate for the 12 possible base substitution errors was 7 ϫ 10 Ϫ4 (15), while the average error rate for single-base deletion errors was 3-9 ϫ 10 Ϫ4 (16). These rates are substantially higher than those measured for the other cellular DNA polymerases that perform the bulk of genomic DNA replication (17). These higher error rates seem consistent with the fact that pol ␤ lacks an intrinsic 3Ј 3 5Ј proofreading exonuclease activity. The observations that pol ␤ has low frameshift fidelity and synthesizes DNA in a distributive manner during primer extension to copy long singlestr...

The data presented here demonstrate the robustness of virus inactivation by S/D treatment for a broad spectrum of enveloped test viruses and process variables. Our data substantiate the fact that no transmission of viruses such as human immunodeficiency virus, hepatitis B virus, hepatitis C virus, or of other enveloped viruses was reported for licensed plasma derivatives since the introduction of S/D treatment.