Context-dependent mutagenesis by DNA lesions

Background: Detailed analyses of mutational hotspots following DNA damage p rovide an understanding of oncogene activation and tumor suppressor gene in activation, and hence provide an insight into the earliest steps in the ind uction of cancer. A mutational hotspot might be created by preferential les ion formation, decreased lesion repair, or increased misinsertion past the lesion during DNA replication. The respective contribution of these factors might be influenced by the DNA sequence context of the hotspot. Results: As a prelude to addressing the contribution of all possible neares t-neighbor contexts on the replication past O-6-methylguanine (m(6)G) and r epair of m(6)G in vivo, we have devised a mutation frequency (MF) detection strategy on the basis of the properties of type Ils restriction enzymes. W e also report a method for constructing site-specific single-stranded viral DNA genomes that should yield identical ligation efficiencies regardless o f the lesion or its surrounding sequence context. Using repair-deficient Es cherichia coil we discovered that m(6)G in three sequence contexts was near ly 100% mutagenic in vivo, showing that the DNA polymerase holoenzyme almos t always placed a thymine base opposite m(6)G during replication. In partia lly repair-proficient cells, the Ada O-6-methylguanine-DNA methyltransferas e repair protein was twice as efficient on m(6)G when a guanine base rather than an adenine base was 5' to the lesion. Conclusions: The system allows the mutagenic potential of, theoretically, a ny DNA lesion that exhibits point mutations, in any varied local sequence c ontext, to be rapidly determined, The assay demonstrates low background, hi gh throughput, and does not require phenotypic selection, making it possibl e to discern the effects of sequence context on the processing of m(6)G.