Heterogeneous repair of N-methylpurines at the nucleotide level in normal human cells.
Base excision repair rates of dimethyl sulfate-induced 3-methyladenine and 7-methylguanine adducts were measured at nucleotide resolution along the PGK1 gene in normal human fibroblasts. Rates of 7-methylguanine repair showed a 30-fold dependence on nucleotide position, while position-dependent repair rates of 3-methyladenine varied only sixfold. Slow excision rates for 7-methylguanine bases afforded the opportunity to study their excision in vitro as a model for base excision repair. A two-component in vitro excision system, composed of human N-methylpurine-DNA glycosylase (MPG protein) and dimethyl sulfate-damaged DNA manifested sequence context-dependent rate differences for 7-methylguanine of up to 185-fold from position to position. This in vitro system reproduced both the global repair rate, and for the PGK1 coding region, the position-dependent repair patterns observed in cells. The equivalence of in vivo repair and in vitro excision data indicates that removal of 7-methylguanine by the MPG protein is the rate-limiting step in base excision repair of this lesion. DNA "repair rate footprints" associated with DNA glycosylase accessibility were observed only in a region with bound transcription factors. The "repair rate footprints" represent a rare chromatin component of 7-meG base excision repair otherwise dominated by sequence-context dependence. Comparison of in vivo repair rates to in vitro rates for 3-methyladenine, however, shows that the rate-limiting step determining position-dependent repair for this adduct is at one of the post-DNA glycosylase stages. In conclusion, this study demonstrates that a comparison of sequence context-dependent in vitro reaction rates to in vivo position-dependent repair rates permits the identification of steps responsible for position-dependent repair. Such analysis is now feasible for the different steps and adducts repaired via the base excision repair pathway.
Ye
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Department of Biology, Beckman Research Institute, City of Hope National Medical Center, 1450 East Duarte Road, Duarte, CA, 91010, USA.
Holmquist
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GP
O'Connor
T R
TR
D000225:Adenine / Q000031:analogs & derivatives* / Q000378:metabolism; D001483:Base Sequence; D002843:Chromatin / Q000378:metabolism; D018736:DNA Adducts / Q000378:metabolism*; D004249:DNA Damage; D018983:DNA Footprinting; D045647:DNA Glycosylases; D004260:DNA Repair; D005091:Exons; D005347:Fibroblasts / Q000166:cytology; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006207:Half-Life; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases; D010735:Phosphoglycerate Kinase / Q000235:genetics*; D011401:Promoter Regions, Genetic; D013463:Sulfuric Acid Esters / Q000494:pharmacology
D002843:Chromatin; D018736:DNA Adducts; D013463:Sulfuric Acid Esters; C025946:3-methyladenine; D006147:Guanine; C008450:7-methylguanine; D010735:Phosphoglycerate Kinase; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D000225:Adenine; C007482:dimethyl sulfate