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Characterizing the N terminus of the Escherichia coli DNA repair protein Vsr endonuclease

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Characterizing the N terminus of the Escherichia coli DNA repair protein Vsr endonuclease

Monastiriakos, Stavroula (2002) Characterizing the N terminus of the Escherichia coli DNA repair protein Vsr endonuclease. PhD thesis, Concordia University.

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Abstract

Spontaneous deamination of 5-methylcytosine in Escherichia coli and in human cells causes T/G mismatches. In E. coli , these mismatches are corrected by very short patch (VSP) repair, which requires the product of the vsr gene. Vsr nicks 5 ' of the mismatched T in a sequence dependent manner (C (T/G) WGG where W = A or T). DNA polymerase I removes the T, along with several bases on its 3 ' side, and resynthesizes the DNA, restoring the correct C.G base pair. Overexpression of vsr is mutagenic, causing a genome-wide increase in frameshifts and base substitutions. This mutational spectrum is consistent with saturation of mismatch repair. The published structure of Vsr bound to DNA shows that the N terminus penetrates the minor groove of the DNA, possibly acting as a clamp to aid binding, while three aromatic residues intercalate the DNA from the major groove side. We have analysed five Vsr mutants that alter these above-mentioned areas that bind to the DNA: two N terminus deletion mutants, x14 and x19, along with three alanine amino acid substitutions for Phe67, Trp68 and Trp86. The alanine amino acid substitutions for F67, W68 and W86 are not active either in vivo or in vitro . The loss of activity may be due to conformational changes that alter the metal binding capabilities required for DNA binding. Both N terminus mutants show decreased activity in vitro and in vivo , and decreased DNA binding in bandshift and fluorescence assays. In contrast to the wildtype Vsr, MutL does not stimulate the endonuclease activity of these mutants, indicating that an N terminus function is necessary for endonucleolytic stimulation. While the x14 mutant is half as mutagenic as Vsr when overproduced in vivo , the x19 mutant is considerably less mutagenic than the wild-type protein. The two N terminus mutants are no longer growth phase dependent indicating a requirement for the N terminus for growth phase regulation. Furthermore, overexpression of MutL in vivo was found to stabilize Vsr levels in log phase cells.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (PhD)
Authors:Monastiriakos, Stavroula
Pagination:xiv, 136 leaves : ill. ; 29 cm.
Institution:Concordia University
Degree Name:Theses (Ph.D.)
Program:Chemistry and Biochemistry
Date:2002
Thesis Supervisor(s):Cupples, Claire
ID Code:2136
Deposited By:Concordia University Libraries
Deposited On:27 Aug 2009 13:25
Last Modified:08 Dec 2010 10:24
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