Mutagenic agents generally exert their effects by introducing structural lesions into DNA block DNA replication. Error-prone repair, the major mechanism of damage-induced mutagenesis, occurs when DNA replication bypasses such damaged bases in a poorly- templated (and thus highly mutagenic) manner. Studies are currently underway in this laboratory to investigate this process in a mammalian system. A particular type of lesion will be introduced quantitatively into a DNA substrate at a single site. Its effects on DNA synthesis can then be assessed either by using purified mammalian replication proteins (the SV40 in vitro replication system) or by introducing the lesion-containing DNA into mammalian cells. By introducing the lesion into a gene (beta- galactosidase) at a site where mutations are readily detectable, the mutagenic consequences of the lesion can also be evaluated. Recently, a novel, non-templated nucleotide addition reaction was observed with DNA polymerase I (Klenow fragment) from E. coli. This reaction has now been demonstrated with enzymes from both procaryotic and eucaryotic sources and may be a common property of DNA polymerases. Non-templated DNA synthesis is important in vivo to replicate the ends of linear chromosomes (telomers) and to generate antibody diversity. Reactions of this typs may also represent a new mechanism to generate spontaneous mutations.
