Context-dependent mutagenesis by DNA lesions

Detailed analyses of mutational hotspots following DNA damage provide an understanding of oncogene activation and tumor suppressor gene inactivation, and hence provide an insight into the earliest steps in the induction of cancer. A mutational hotspot might be created by preferential lesion 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.

As a prelude to addressing the contribution of all possible nearest-neighbor contexts on the replication past O6-methylguanine (m6G) and repair of m6G in vivo, we have devised a mutation frequency (MF) detection strategy on the basis of the properties of type IIs restriction enzymes. We also report a method for constructing site-specific single-stranded viral DNA genomes that should yield identical ligation efficiencies regardless of the lesion or its surrounding sequence context. Using repair-deficient Escherichia coli, we discovered that m6G in three sequence contexts was nearly 100% mutagenic in vivo, showing that the DNA polymerase holoenzyme almost always placed a thymine base opposite m6G during replication. In partially repair-proficient cells, the Ada O6-methylguanine-DNA methyltransferase repair protein was twice as efficient on m6G when a guanine base rather than an adenine base was 5' to the lesion.

The system allows the mutagenic potential of, theoretically, any DNA lesion that exhibits point mutations, in any varied local sequence context, to be rapidly determined. The assay demonstrates low background, high throughput, and does not require phenotypic selection, making it possible to discern the effects of sequence context on the processing of m6G.