Linked Life Data

9915846

Source:http://linkedlifedata.com/resource/pubmed/id/9915846

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
5
pubmed:dateCreated
1999-3-3
pubmed:abstractText
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 enzyme-substrate 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 T.G mismatch under the conditions of the fidelity assay, providing one explanation for the anti-mutator phenotypes of mutants such as R754A and Q849A.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
29
pubmed:volume
274
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
3067-75
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1999
pubmed:articleTitle
Side chains that influence fidelity at the polymerase active site of Escherichia coli DNA polymerase I (Klenow fragment).
pubmed:affiliation
Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.