Making Mutations is an Active Process: Methods to Examine DNA Polymerase Errors

Endogenous metabolic processes and environmental exposures result in the formation of DNA lesions. The conversion of lesions into mutations is controlled by DNA repair efficiency and by the accuracy of DNA polymerases. The failure to complete lesion repair prior to replication may require DNA synthesis to proceed in the presence of DNA damage - a process we will refer to as translesion synthesis (TLS). When DNA polymerase error discrimination is compromised during TLS, mutations are introduced into an organism's genome. In this chapter, we describe genetic and biochemical approaches to examine damage-induced DNA polymerase errors in vitro. Genetic assays use circular single-stranded DNA templates containing a reporter gene, and score polymerase-induced errors as mutations after transfection of bacteria. Forward mutation assays are advantageous, in that most types of errors within multiple sequence contexts can be scored. Biochemical methods to study polymerase errors use chemically synthesized, site-specific lesion-containing oligonucleotides as DNA templates. Quantitation of reaction products is used to measure the extent of lesion inhibition and the efficiency of TLS. In this way, distinct steps in the TLS reaction can be analyzed. When both approaches use the same DNA sequence as a template for synthesis, the genetic and biochemical assays provide complementary data regarding DNA polymerase error discrimination mechanisms.