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

Kristin Eckert, Erin E. Gestl

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Scopus citations

Abstract

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.

Original languageEnglish (US)
Title of host publicationThe Handbook of Plant Mutation Screening: Mining of Natural and Induced Alleles
PublisherWiley-VCH
Pages55-82
Number of pages28
ISBN (Print)9783527326044
DOIs
StatePublished - Mar 30 2010

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)

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