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Titolo:
Interaction of the E-coli DNA G : T-mismatch endonuclease (vsr protein) with oligonucleotides containing its target sequence
Autore:
Turner, DP; Connolly, BA;
Indirizzi:
Univ Newcastle Upon Tyne, Dept Biochem & Genet, Newcastle Upon Tyne NE2 4HH, Tyne & Wear, England Univ Newcastle Upon Tyne Newcastle Upon Tyne Tyne & Wear England NE2 4HH
Titolo Testata:
JOURNAL OF MOLECULAR BIOLOGY
fascicolo: 5, volume: 304, anno: 2000,
pagine: 765 - 778
SICI:
0022-2836(200012)304:5<765:IOTEDG>2.0.ZU;2-X
Fonte:
ISI
Lingua:
ENG
Soggetto:
SHORT PATCH REPAIR; ECORV RESTRICTION-ENDONUCLEASE; CYTOSINE METHYLASE GENE; ESCHERICHIA-COLI; BINDING SPECIFICITY; EXCISION-REPAIR; CALCIUM-IONS; RECOGNITION; RESIDUES; RECOMBINATION;
Keywords:
protein-DNA recognition; vsr endonuclease; DNA G : T mismatches; mismatch repair;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
52
Recensione:
Indirizzi per estratti:
Indirizzo: Connolly, BA Univ Newcastle Upon Tyne, Dept Biochem & Genet, Newcastle Upon Tyne NE2 4HH, Tyne & Wear, England Univ Newcastle Upon Tyne Newcastle Upon Tyne Tyne & Wear England NE2 4HH
Citazione:
D.P. Turner e B.A. Connolly, "Interaction of the E-coli DNA G : T-mismatch endonuclease (vsr protein) with oligonucleotides containing its target sequence", J MOL BIOL, 304(5), 2000, pp. 765-778

Abstract

The Escherichia coli vsr endonuclease recognises G:T base-pair mismatches in double-stranded DNA and initiates a repair pathway by hydrolysing the phosphate group 5' to the incorrectly paired T. The enzyme shows a preferencefor G:T mismatches within a particular sequence context, derived from the recognition site of the E. coil dcm DNA-methyltransferase (CC[A/T]GG). Thus, the preferred substrate for the vsr protein is (CT[A/T]GG), where the underlined T is opposed by a dG base. This paper provides quantitative data for the interaction of the vsr protein with a number of oligonucleotides containing G:T mismatches. Evaluation of specificity constant (k(st)/K-D; k(st)= rate constant for single turnover, K-D = equilibrium dissociation constant) confirms vsr's preference for a G:T mismatch within a hemi-methylated dcm sequence, i.e. the best substrate is a duplex (both strands written in the 5'-3' orientation) composed of CT[A/T]CG and C-5Me[T/A]GG. Conversion ofthe mispaired T (underlined) to dU or the d(5Me)C to dC gave poorer substrates. No interaction was observed with oligonucleotides that lacked a G:T mismatch or did not possess a dcm sequence. An analysis of the fraction of active protein, by "reverse-titration" (i.e. adding increasing amounts of DNA to a fixed amount of protein followed by gel-mobility shift analysis) showed that less than 1% of the vsr endonuclease was able to bind to the substrate. This was confirmed using "competitive titrations" (where competitor oligonucleotides are used to displace a P-32-labelled nucleic acid from the vsr protein) and burst kinetic analysis. This result is discussed in the light of previous in vitro and in vivo data which indicate that the MutL protein may be needed for full vsr activity. (C) 2000 Academic Press.

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Documento generato il 29/09/20 alle ore 00:44:37