REPAIR OF PROPANODEOXYGUANOSINE BY NUCLEOTIDE EXCISION-REPAIR IN-VIVOAND IN-VITRO

Repair of the exocyclic DNA adduct propanodeoxyguanosine (PdG) was ass essed in both in vivo and in vitro assays. PdG was site specifically i ncorporated at position 6256 of M13MB102 DNA, and the adducted viral g enome was electroporated into repair-proficient and repair-deficient E scherichia coil strains. Comparable frequencies of PdG --> T and PdG - -> A mutations at position 6256 were detected following replication of the adducted genomes in wild-type E. coil strains. A 4-fold increase in the frequencies of transversions and transitions was observed in E. coil strains deficient in Vvr(A)BC-dependent nucleotide excision repa ir. A similar increase in the replication of the adduct containing str and was observed in the repair deficient strains. No change in the fre quency of targeted mutations was observed in strains deficient in one or both of the genes coding for 3-methyladenine glycosylase. Incubatio n of purified E. coil Uvr(A)BC proteins with a duplex 156-mer containi ng a single PdG adduct resulted in removal of a 12-base oligonucleotid e containing the adduct. Incubation of the same adducted duplex with C hinese hamster ovary cell-free extracts also resulted in removal of th e adduct, PdG was a better substrate for repair by the mammalian nucle otide excision repair complex than the bacterial repair complex and wa s approximately equal to a thymine-thymine dimer as a substrate for th e former. The results of these in vivo and in vitro experiments indica te that PdG, a homolog of several endogenously produced DNA adducts, i s repaired by the nucleotide excision repair pathway.