PMID:12534293
| Citation |
Francis, AW and David, SS (2003) Escherichia coli MutY and Fpg utilize a processive mechanism for target location. Biochemistry 42:801-10 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1021/bi026375+ |
| Keywords |
Binding Sites; Catalysis; DNA Glycosylases; DNA Repair; DNA-Binding Proteins/chemistry; DNA-Binding Proteins/metabolism; DNA-Formamidopyrimidine Glycosylase; Deoxyribonuclease EcoRI/metabolism; Electrophoresis, Polyacrylamide Gel; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/metabolism; Guanine/analogs & derivatives; Guanine/chemistry; Guanine/metabolism; N-Glycosyl Hydrolases/chemistry; N-Glycosyl Hydrolases/metabolism; Peptide Fragments/chemistry; Peptide Fragments/metabolism; Protein Structure, Tertiary; Restriction Mapping; Sodium Chloride/chemistry; Substrate Specificity |
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