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J Mol Biol. 1998 Nov 27;284(2):269-85.

Heterogeneous repair of N-methylpurines at the nucleotide level in normal human cells.

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Department of Biology, Beckman Research Institute, City of Hope National Medical Center, 1450 East Duarte Road, Duarte, CA, 91010, USA.


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.

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