SEQUENCE--SPECIFIC REPAIR OF ALKYLATED CELLULAR DNA

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The long term objectives of the research are to examine the removal of alkylated lesions from specific regions of the genome. This will be done to discern if preferential removal of DNA adducts from active genes is linked to a particular lesion's ability to block RNA synthesis or to the repair pathway responsible for its clearance. It has been documented that the removal of replication and transcripton blocking cyclobutane pyrimidine dimers from the dihydrofolate reductase locus of Chinese hamster ovary B11 cells is limited essentially to the transcribed strand of that gene. 7-Methylguanine, an innocuous lesion that does not block nucleic acid synthesis, is not removed preferentially from this locus. The specific aims of the research are: (1) to determine if the removal of 3-methyladenine, an alkylated base known to block nucleic acid synthesis in vitro, is preferential from actively transcribed genes; (2) to test for preferential removal of 3-methyladenine from the transcribed strand of active loci; (3) to determine if the removal of 3-methyladenine from expressed genes is dependent on nucleotideexcision or base-excision repair; (4) to determine if the repair of purines damaged with longer chain alkyl groups, a process that appears to be dependent on intact nucleotide-excision repair, occurs preferentially in transcribed genes; and (5) to study alkylphosphotriester lesions, which inhibit nucleic acid synthesis but are not well repaired in total cellular DNA, assessing their removal from active genetic loci. Data obtained in this project will aid in determining the generality of preferental repair and help to elucidate the mechanism governing the phenomenon. Two important techniques will be used in these investigations: (1) methoxyamine reduction of apurinic sites generated from N-methyepurine removal will be used to supply controls, and (2) denaturing pulsed-field gel electrophoresis will be employed to examine repair in large segments of DNA (i. e. >25kb). The latter technique will help to prevent the need for toxic doses of alkylating agents and also enhance the versatility of preferential DNA repair methods.