Escherichia coli MutY and Fpg Utilize a Processive Mechanism for Target Location

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• 作者：Anthony W. Francis and Sheila S. David
• 刊名：Biochemistry
• 出版年：2003
• 出版时间：January 28, 2003
• 年：2003
• 卷：42
• 期：3
• 页码：801 - 810
• 全文大小：175K
• 年卷期：v.42,no.3(January 28, 2003)
• ISSN：1520-4995

文摘

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 theseenzymes is the ability to locate 8-oxoguanine lesions among a large excess of undamaged DNA. Toprovide insight into the mechanism of target location, the ability of these enzyme to utilize a one-dimensionalprocessive search (DNA sliding) or distributive (random diffusion-mediated) mechanism was investigated.Each enzyme was incubated with double-stranded concatemeric polynucleotides containing a site-specifictarget site at 25-nucleotide (nt) intervals. The products of each reaction were analyzed after further treatmentand denaturation. A rapid accumulation of predominantly 25-nt fragments would indicate the utilizationof a processive mechanism, whereas oligomeric multiples of 25-nt fragments would form if a distributivemechanism were used. Both Fpg and MutY were found to function processively on concatemers containing7,8-dihydro-8-oxo-2'-deoxyguanosine (OG)·C and G·A mispairs, respectively. An increase in sodiumchloride concentration results in the modulation from a processive to distributive mechanism for bothenzymes. Interestingly, processive behavior was not observed in the reaction of MutY with concatemerscontaining OG·A mispairs. A truncated form of MutY (Stop 225) containing only the N-terminal domainwas 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 themechanism by which the enzyme detects OG·A base pairs in DNA.