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rdf:type |
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lifeskim:mentions |
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pubmed:issue |
9
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pubmed:dateCreated |
2005-3-1
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pubmed:abstractText |
To investigate the molecular basis for the selective utilization of nucleoside triphosphates complementary to templating bases, by RB69 DNA polymerase (RB69 pol), we constructed a set of mutants that we predicted would perturb the "floor" of the nascent base-pairing interface in the enzyme. We then determined the pre-steady-state kinetic parameters for the incorporation of complementary and noncomplementary dNTPs by the exo(-) form of RB69 pol and its mutants. We found that the Y567A mutant had the same K(d) and k(pol) values for incorporation of C versus G as the wild-type exo(-) enzyme; however, the k(pol)/K(d) ratio for G versus G incorporation with the Y567A mutant was 10 times higher than the k(pol)/K(d) efficiency of G versus G incorporation using the exo(-) RB69 pol. The reduced level of discrimination by the Y567A mutant against incorporation of mismatched bases was also seen with the Y391A mutant. Stopped-flow fluorescence was also employed to monitor rates of putative conformational changes with the exo(-) RB69 pol and its mutants using a primer-template complex containing 2-aminopurine. The rates of fluorescence changes were equal to or greater than the rates of the rapid chemical quench, indicating that we were monitoring a process occurring before or during the phosphoryl transfer reaction. We have interpreted our results within the context of the crystal structure of the RB69 pol ternary complex [Franklin, M. C., et al. (2001) Cell 105, 657-667].
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pubmed:grant |
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/2'-deoxy-1'-(2,4-difluorotol-5-yl)ri...,
http://linkedlifedata.com/resource/pubmed/chemical/2'-deoxyadenosine triphosphate,
http://linkedlifedata.com/resource/pubmed/chemical/2'-deoxycytidine 5'-triphosphate,
http://linkedlifedata.com/resource/pubmed/chemical/Alanine,
http://linkedlifedata.com/resource/pubmed/chemical/DNA-Directed DNA Polymerase,
http://linkedlifedata.com/resource/pubmed/chemical/Deoxyadenine Nucleotides,
http://linkedlifedata.com/resource/pubmed/chemical/Deoxycytosine Nucleotides,
http://linkedlifedata.com/resource/pubmed/chemical/Deoxyguanine Nucleotides,
http://linkedlifedata.com/resource/pubmed/chemical/Nucleotides,
http://linkedlifedata.com/resource/pubmed/chemical/Phenylalanine,
http://linkedlifedata.com/resource/pubmed/chemical/Thymine Nucleotides,
http://linkedlifedata.com/resource/pubmed/chemical/Toluene,
http://linkedlifedata.com/resource/pubmed/chemical/Tyrosine,
http://linkedlifedata.com/resource/pubmed/chemical/Viral Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/bacteriophage RB69 DNA polymerase,
http://linkedlifedata.com/resource/pubmed/chemical/deoxyguanosine triphosphate,
http://linkedlifedata.com/resource/pubmed/chemical/thymidine 5'-triphosphate
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pubmed:status |
MEDLINE
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pubmed:month |
Mar
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pubmed:issn |
0006-2960
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pubmed:author |
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pubmed:issnType |
Print
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pubmed:day |
8
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pubmed:volume |
44
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
3338-46
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pubmed:dateRevised |
2007-11-14
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pubmed:meshHeading |
pubmed-meshheading:15736944-Alanine,
pubmed-meshheading:15736944-Amino Acid Substitution,
pubmed-meshheading:15736944-Base Pair Mismatch,
pubmed-meshheading:15736944-Binding Sites,
pubmed-meshheading:15736944-DNA-Directed DNA Polymerase,
pubmed-meshheading:15736944-Deoxyadenine Nucleotides,
pubmed-meshheading:15736944-Deoxycytosine Nucleotides,
pubmed-meshheading:15736944-Deoxyguanine Nucleotides,
pubmed-meshheading:15736944-Enterobacter,
pubmed-meshheading:15736944-Hydrogen Bonding,
pubmed-meshheading:15736944-Kinetics,
pubmed-meshheading:15736944-Nucleotides,
pubmed-meshheading:15736944-Phenylalanine,
pubmed-meshheading:15736944-Substrate Specificity,
pubmed-meshheading:15736944-Thymine Nucleotides,
pubmed-meshheading:15736944-Toluene,
pubmed-meshheading:15736944-Tyrosine,
pubmed-meshheading:15736944-Viral Proteins
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pubmed:year |
2005
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pubmed:articleTitle |
Base selectivity is impaired by mutants that perturb hydrogen bonding networks in the RB69 DNA polymerase active site.
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pubmed:affiliation |
Department of Molecular Biophysics and Biochemistry, Yale University, 333 Cedar Street, New Haven, Connecticut 06520, USA.
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pubmed:publicationType |
Journal Article,
Research Support, U.S. Gov't, P.H.S.
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