Journal of Molecular Biology
Volume 304, Issue 5, 15 December 2000, Pages 765-778
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Regular Article
Interaction of the E. coli DNA G:T-mismatch Endonuclease (vsr Protein) with Oligonucleotides Containing its Target Sequence,☆☆

https://doi.org/10.1006/jmbi.2000.4248Get rights and content

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 preference for G:T mismatches within a particular sequence context, derived from the recognition site of the E. coli 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 (kst/KD; kst=rate constant for single turnover, KD=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]GG and C5MeC[T/A]GG. Conversion of the mispaired T (underlined) to dU or the d5MeC 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 32P-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.

References (52)

  • S.E. Tsutakawa et al.

    Recognition of a TG mismatch: the crystal structure of very short patch repair endonuclease in complex with a DNA duplex

    Cell

    (1999)
  • W. Cao

    Binding kinetics and footprinting of TaqI endonuclease: effects of metal cofactors on sequence-specific interactions

    Biochemistry

    (1999)
  • S.H. Choi et al.

    Identification of the XorII methyltransferase gene and a vsr homolog from Xanthomonas oryzae pv. oryzae

    Mol. Gen. Genet.

    (1994)
  • B.A. Connolly

    Oligonucleotides containing modified bases

  • M.E. Dar et al.

    Mechanism of expression of DNA repair gene vsr, an Escherichia coli gene that overlaps the DNA cytosine methylase gene

    Mol. Microbiol.

    (1993)
  • K. Drotschmann et al.

    The Escherichia coli MutL protein stimulates binding of VSR and MutS to heteroduplex DNA

    Nucl. Acids Res.

    (1998)
  • B.K. Duncan et al.

    Mutagenic deamination of cytosine residues in DNA

    Nature

    (1980)
  • S. Dzidic et al.

    Genetic requirements for hyper-recombination of very short patch repair: involvement of Escherichia coli DNA polymerase I

    Mol. Gen. Genet.

    (1989)
  • A.R. Fersht

    Enzyme Structure and Mechanism

    (1984)
  • K.R. Fox et al.

    Recognition of G:T mismatches by vsr mismatch endonuclease

    Nucl. Acids Res.

    (2000)
  • S. Gabbara et al.

    A DNA repair process in Escherichia coli corrects U:G and T:G mismatches to C:G at sites of cytosine methylation

    Mol. Gen. Genet.

    (1994)
  • P. Gallinari et al.

    A new class of uracil-DNA glycosylases related to human thymine-DNA glycosylase

    Nature

    (1996)
  • GraFit

    (version 3.09a) Erithacus Software

    (1992)
  • F. Hennecke et al.

    The vsr gene product of E. coli K-12 is a strand and sequence-specific DNA mismatch endonuclease

    Nature

    (1991)
  • L. Jen-Jacobson

    Structural-perturbation approaches to thermodynamics of site-specific protein-DNA interactions: energetics of macromolecules

    Methods Enzymol.

    (1995)
  • L. Jen-Jacobson

    Protein-DNA recognition complexes: conservation of structure and binding energy in the transition state

    Biopolymers

    (1997)
  • Cited by (0)

    Abbreviations used: d5MeC, 5-methyldeoxycytosine; UDGase, uracil-DNA glycosylase; vsr, very short-patch repair

    ☆☆

    Edited by J. Karn

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    Corresponding author

    f2

    E-mail address of the corresponding author: [email protected]

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