On the Role of DNA Polymerase in Base Selection

Speyer, Joseph F.; Karam, J.D.; Lenny, Albert B.

doi:10.1101/sqb.1966.031.01.088

Cited by 103 publications

(22 citation statements)

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“…New strains are described in Results. The tsL56 strain is a classical T4 DNA polymerase mutator strain (1,10). The tsL56 DNA polymerase has less 3' -> 5' exonuclease activity compared to wild type (3).…”

Section: Methodsmentioning

confidence: 99%

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DNA polymerization in the absence of exonucleolytic proofreading: in vivo and in vitro studies.

Reha-Krantz

Stocki

Nonay

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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Classical genetic selection was combined with site-directed mutagenesis to study bacteriophage T4 DNA polymerase 3' -S 5' exonuclease activity. A mutant DNA polymerase with very little (<1%) 3' -*5' exonuclease activity was generated. In vivo, the 3' -*5' exonuclease-deficient DNA polymerase produced the highest level ofspontaneous mutation observed in T4, 500-to 1800-fold above that of wild type. The large reduction in 3' -* 5' exonuclease activity appears to be due to two amino acid substitutions: clease motif. Therefore, extrapolation from E. coli DNA polymerase I sequence and structure to other DNA polymerases for which there is no structural information may not be valid.Bacteriophage T4 DNA polymerase is one of the best experimental systems for studying the role of DNA polymerase exonucleolytic proofreading in enhancing DNA replication fidelity (1-10). Wild-type T4 DNA polymerase has a potent 3' 5' exonuclease activity (11), which is important for accurate DNA replication. Mutant DNA polymerases with reduced 3' -* 5' exonuclease activity produce more DNA replication errors (mutator phenotype), whereas mutants with elevated 3' 5' exonuclease activity, relative to polymerizing activity, increase DNA replication accuracy (antimutator phenotype) (3). The mutator phenotype was used to select mutant DNA polymerases with reduced 3' -* 5' exonuclease activity; amino acid substitutions in the mutants were clustered'between T4 DNA polymerase residues 255 and 363 (8)(9)(10)12). Although amino acid changes within this region decreased 3' -* 5' exonuclease activity, the' mutant DNA polymerases still retained significant residual proofreading activity, which suggests that these particular residues do not function catalytically.In the case of Escherichia coli DNA polymerase I (pol I), residues essential for 3' 5' exonuclease activity have been identified. They include four metal ion binding residues; The hypothesis drawn from these sequence comparisons was that many eukaryotic, viral, and bacteriophage DNA polymerases have a conserved 3' -* 5' exonuclease domain similar to that of E. coli pol 1. T4 genetic studies are consistent with this proposal because mutations that reduce 3' -* 5' exonuclease activity are located near the proposed conserved metal ion binding residues. The hypothesis was tested directly in phage 429'DNA polymerase by substituting alanine residues for proposed conserved metal ion binding residues, which resulted in a 1000-fold reduction in 3' -* 5' exonuclease activity without affecting polymerization activity (23). The 429 DNA polymerase result compares favorably with the 105-fold reduction observed when alanine residues were substituted for E. coli pol I residues .We present here in vitro mutagenesis and genetic studies that were designed to test if proposed T4 DNA' polymerase metal ion binding residues, identified on the basis of sequence similarities to those of E. coli pol l (8, 23), are required for 3' 5' exonuclease activity. In 'contrast to the 429 DNA polymerase'studies, alanine subs...

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Section: Methodsmentioning

confidence: 99%

“…Bacteriophage T4 DNA polymerase is one of the best experimental systems for studying the role of DNA polymerase exonucleolytic proofreading in enhancing DNA replication fidelity (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). Wild-type T4 DNA polymerase has a potent 3 ' 5' exonuclease activity (11), which is important for accurate DNA replication.…”

mentioning

confidence: 99%

DNA polymerization in the absence of exonucleolytic proofreading: in vivo and in vitro studies.

Reha-Krantz

Stocki

Nonay

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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“…Biochemical support for this concept was obtained by Brutlag and Kornberg (2), who demonstrated that the 3'--5' exonuclease of Escherichia coli DNA polymerase I (Pol I) preferentially removes mismatched bases at primer termini before initiation of polymerization. Support for proofreading in vivo comes from studies with certain mutator (3) and antimutator (4) bacteriophage T4 DNA polymerases. These studies (5-8) correlated spontaneous mutation rates of bacteriophage T4 with the ratio between the polymerization reaction and the excision of a noncomplementary nucleotide at the primer terminus.…”

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confidence: 99%

Deoxynucleoside [1-thio]triphosphates prevent proofreading during in vitro DNA synthesis.

Kunkel

Eckstein

Mildvan

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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The contribution of proofreading to the fidelity ofcatalysis by DNA polymerases has been determined with deoxyribonucleoside [1-thioltriphosphate substrates. These analogues, which contain a sulfur in place of an oxygen on the ar phosphorus, are incorporated into DNA by DNA polymerases at rates similar to those of the corresponding unmodified deoxynucleoside triphosphates. The fidelity of DNA synthesis was measured with *X174 am3 DNA; reversion to wild type occurs most frequently by a single base substitution, a C for a T at position 587. By using avian myeloblastosis virus DNA polymerase and DNA polymerase (3 (enzymes without a proofreading 3'-*5' exonucleolytic activity), substitution ofdeoxycytidine thiotriphosphate in the reaction mixture did not alter fidelity. In contrast, with DNA polymerases from E. coli (DNA polymerase I) and bacteriophage T4 (enzymes containing a proofreading activity), fidelity was markedly reduced with deoxycytidine [1-thio]triphosphate. DNA containing phosphorothioate nucleotides is insensitive to hydrolysis by the exonuclease associated with these prokapyotic DNA polymerases. These combined results indicate that the deoxynucleoside [1-thio]triphosphates have normal base-pairing properties; however, once misinserted by a polymerase, they are not excised by proofreading. Proofreading ofa C:A mismatch at position 587 is thereby found to contribute 20-fold to the fidelity of E. coli DNA polymerase I and a greater amount to the fidelity of bacteriophage T4 DNA polymerase.The ability to correct errors during DNA replication has long been recognized as one important mechanism by which an organism can potentially achieve the highly accurate replication of its genetic information. This concept stems from the observation that prokaryotic DNA polymerases contain an integrally associated 3'--5' exonuclease activity, which can selectively remove mistakes as they occur during polymerization (1). Biochemical support for this concept was obtained by Brutlag and Kornberg (2), who demonstrated that the 3'--5' exonuclease of Escherichia coli DNA polymerase I (Pol I) preferentially removes mismatched bases at primer termini before initiation of polymerization. Support for proofreading in vivo comes from studies with certain mutator (3) and antimutator (4) bacteriophage T4 DNA polymerases. These studies (5-8) correlated spontaneous mutation rates of bacteriophage T4 with the ratio between the polymerization reaction and the excision of a noncomplementary nucleotide at the primer terminus. Also, differences in discrimination between adenosine and its base analogue 2-aminopurine by mutant T4 DNA polymerase (9) can be accounted for by proofreading. Based on the kinetic data with substrate analogues, a number of mathematic models for proofreading have been proposed (10-13).Our continuing interest in determining the relative importance of the several mechanisms available to the cell to achieve high fidelity (14) has led us to assess the contribution of proofreading to accuracy by direct measuremen...

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“…The mutator or antimutator phenotype has been extensively studied in T4 phage and Escherichia coli (1,2,13), and these phenotypes have been shown to be genetically related to structural genes for DNA polymerase and other replication proteins (3,6). In eukaryotic systems, it has been reported that an aphidicolinresistant Chinese hamster cell mutant which has an altered DNA polymerase exhibits an increase in the rate of spontaneous mutagenesis (8).…”

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Drug‐Resistant Mutants of Herpes Simplex Virus Type 2 with a Mutator or Antimutator Phenotype

Nishiyama

Yoshida²,

Tsurumi

et al. 1985

Microbiology and Immunology

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The mutator or antimutator phenotype has been extensively studied in T4 phage and Escherichia coli (1, 2, 13), and these phenotypes have been shown to be genetically related to structural genes for DNA polymerase and other replication proteins (3, 6). In eukaryotic systems, it has been reported that an aphidicolinresistant Chinese hamster cell mutant which has an altered DNA polymerase exhibits an increase in the rate of spontaneous mutagenesis (8). In addition, Hall et al (4) have shown that phosphonoacetic acid (PAA)-resistant mutants of herpes simplex virus (HSV) type 1 have lower spontaneous mutation rates than the wild-type and that the antimutator phenotype is mapped in the viral DNA polymerase locus. We have recently isolated an aphidicolin-resistant mutant (Aphr) of HSV type 2 ; the mutant was more sensitive to PAA, and its viral DNA polymerase was characterized by lower Kms for both deoxycytidine 5'-triphosphate (dCTP) and deoxythymidine (dTTP) than those of the wild-type polymerase. In the present communication, we show

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On the Role of DNA Polymerase in Base Selection

Cited by 103 publications

References 0 publications

DNA polymerization in the absence of exonucleolytic proofreading: in vivo and in vitro studies.

DNA polymerization in the absence of exonucleolytic proofreading: in vivo and in vitro studies.

Deoxynucleoside [1-thio]triphosphates prevent proofreading during in vitro DNA synthesis.

Drug‐Resistant Mutants of Herpes Simplex Virus Type 2 with a Mutator or Antimutator Phenotype

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