Escherichia coli MutY and Fpg utilize a processive mechanism for target location.
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.
Francis
Anthony W
AW
Department of Chemistry, University of Utah, 315 S. 1400 E., Salt Lake City, Utah 84112, USA.
David
Sheila S
SS
D001665:Binding Sites; D002384:Catalysis; D045647:DNA Glycosylases; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D043382:DNA-Formamidopyrimidine Glycosylase; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D015183:Restriction Mapping; D012965:Sodium Chloride / Q000737:chemistry; D013379:Substrate Specificity
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D012965:Sodium Chloride; C024829:8-hydroxyguanine; D006147:Guanine; D015246:Deoxyribonuclease EcoRI; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C061738:mutY adenine glycosylase; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli