Linked Life Data

8537389

Source:http://linkedlifedata.com/resource/pubmed/id/8537389

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
52
pubmed:dateCreated
1996-2-8
pubmed:abstractText
Amino acids Tyr254 and Tyr390 of phi 29 DNA polymerase belong to one of the most conserved regions in eukaryotic-type DNA polymerases. In this paper we report a mutational study of these two residues to address their role in nucleotide selection. This study was carried out by means of a new kinetic analysis that takes advantage of the competition between DNA polymerization and 3'-->5' exonuclease activity to measure the Km values for correct and incorrect nucleotides in steady-state conditions. This method is valid for any 3'-->5' exonuclease-containing DNA polymerase, without any restriction concerning catalytic rates of nucleotide incorporation. The results showed that the discrimination factor achieved by phi 29 DNA polymerase in the nucleotide binding step of DNA polymerization is 2.4 x 10(3), that is, a wrong nucleotide is bound with a 2.4 x 10(3)-fold lower affinity than the correct one. Mutants Y254F, Y390F, and Y390S showed discrimination values of 7.0 x 10(2), > 1.9 x 10(3), and 2.9 x 10(2), respectively. The reduced accuracy of nucleotide binding produced by mutations Y254F and Y390S lead us to propose that phi 29 DNA polymerase residues Tyr254 and Tyr390, highly conserved in eukaryotic-type DNA polymerases, are involved in nucleotide binding selection, thus playing a crucial role in the fidelity of DNA replication. Comparison of the discrimination factors of mutants Y390S and Y390F strongly suggests that the phenyl ring of Tyr390 is directly involved in checking base-pairing correctness of the incoming nucleotide.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
29
pubmed:volume
270
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
31235-43
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1995
pubmed:articleTitle
A novel kinetic analysis to calculate nucleotide affinity of proofreading DNA polymerases. Application to phi 29 DNA polymerase fidelity mutants.
pubmed:affiliation
Centro de Biología Molecular, Severo Ochoa (Consejo Superior de Investigaciones Ceintíficas-Universidad Autonoma de Madrid), Spain.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't