Escherichia coli MutY and Fpg utilize a processive mechanism for target location

Anthony W. Francis, Sheila S. David

Research output: Contribution to journalArticle

48 Scopus citations

Abstract

MutY and formamidopyrimidine-DNA-glycosylase (Fpg) are base-excision repair (BER) enzymes involved in the 8-oxoguanine repair pathway in Escherichia coli. An impressive feature of these enzymes is the ability to locate 8-oxoguanine lesions among a large excess of undamaged DNA. To provide insight into the mechanism of target location, the ability of these enzyme to utilize a one-dimensional processive search (DNA sliding) or distributive (random diffusion-mediated) mechanism was investigated. Each enzyme was incubated with double-stranded concatemeric polynucleotides containing a site-specific target site at 25-nucleotide (nt) intervals. The products of each reaction were analyzed after further treatment and denaturation. A rapid accumulation of predominantly 25-nt fragments would indicate the utilization of a processive mechanism, whereas oligomeric multiples of 25-nt fragments would form if a distributive mechanism were used. Both Fpg and MutY were found to function processively on concatemers containing 7,8-dihydro-8-oxo-2′-deoxyguanosine (OG)·C and G·A mispairs, respectively. An increase in sodium chloride concentration results in the modulation from a processive to distributive mechanism for both enzymes. Interestingly, processive behavior was not observed in the reaction of MutY with concatemers containing OG·A mispairs. A truncated form of MutY (Stop 225) containing only the N-terminal domain was found to behave in a manner consistent with a processive mechanism with both OG·A- and G·A-containing substrates. This suggests that the C-terminal domain of MutY plays an important role in the mechanism by which the enzyme detects OG·A base pairs in DNA.

Original languageEnglish (US)
Pages (from-to)801-810
Number of pages10
JournalBiochemistry
Volume42
Issue number3
DOIs
StatePublished - Jan 28 2003
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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