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rdf:type |
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lifeskim:mentions |
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pubmed:issue |
3
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pubmed:dateCreated |
2006-1-17
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pubmed:abstractText |
We report the first pre-steady-state kinetic studies of DNA replication in the absence of hydrogen bonds. We have used nonpolar nucleotide analogues that mimic the shape of a Watson-Crick base pair to investigate the kinetic consequences of a lack of hydrogen bonds in the polymerase reaction catalyzed by the Klenow fragment of DNA polymerase I from Escherichia coli. With a thymine isostere lacking hydrogen-bonding ability in the nascent pair, the efficiency (k(pol)/Kd) of the polymerase reaction is decreased by 30-fold, affecting the ground state (Kd) and transition state (k(pol)) approximately equally. When both thymine and adenine analogues in the nascent pair lack hydrogen-bonding ability, the efficiency of the polymerase reaction is decreased by about 1000-fold, with most of the decrease attributable to the transition state. Reactions using nonpolar analogues at the primer-terminal base pair demonstrated the requirement for a hydrogen bond between the polymerase and the minor groove of the primer-terminal base. The R668A mutation of Klenow fragment abolished this requirement, identifying R668 as the probable hydrogen-bond donor. Detailed examination of the kinetic data suggested that Klenow fragment has an extremely low tolerance of even minor deviations of the analogue base pairs from ideal Watson-Crick geometry. Consistent with this idea, some analogue pairings were better tolerated by Klenow fragment mutants having more spacious active sites. In contrast, the Y-family polymerase Dbh was much less sensitive to changes in base pair dimensions and more dependent upon hydrogen bonding between base-paired partners.
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pubmed:grant |
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pubmed:commentsCorrections |
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-1062791,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-10966467,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-11041863,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-11258875,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-11389835,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-11545743,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-11545744,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-11595188,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-11685247,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-11790092,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-11830658,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-12023283,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-12045095,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-12649320,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-12692307,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-12832493,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-12950174,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-14644182,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-15035983,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-15210707,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-15533035,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-1569092,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-16055723,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-16249340,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-1899034,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-2201688,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-2832946,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-7516580,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-8594356,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-9054433,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-9287163,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-9380669,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-9440688,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-9571040,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-9808038,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-9857206,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16411765-9915846
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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 |
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pubmed:status |
MEDLINE
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pubmed:month |
Jan
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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 |
24
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pubmed:volume |
45
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
890-8
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pubmed:dateRevised |
2009-11-18
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pubmed:meshHeading |
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pubmed:year |
2006
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pubmed:articleTitle |
DNA polymerase catalysis in the absence of Watson-Crick hydrogen bonds: analysis by single-turnover kinetics.
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pubmed:affiliation |
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
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pubmed:publicationType |
Journal Article,
Research Support, N.I.H., Extramural
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