Assaying Mutations Associated With Gene Conversion Repair of a Double-Strand Break

Abstract

DNA double-strand break (DSB) is a cytotoxic lesion and needs to be repaired immediately. There are several metabolic pathways evolved to repair a DSB. Gene conversion is one of the least error-prone pathway for repair of a DNA DSB. Despite this there is nearly 1000-fold increase in mutation rate associated with gene conversion. Not only higher mutation rate is associated with gene conversion but also there is a very distinct mutation profile compared to spontaneous mutation events. Gene conversion is characterized by the presence of very high frameshift mutation events and other complex mutations that are not present during regular DNA replication. Another DNA DSB repair pathway widely studied is "break-induced replication" (BIR). BIR has been shown to be highly mutagenic in nature. BIR may lead to chromosomal rearrangement and has potential to cause cluster mutations with serious disease implications. In this chapter, the design of assay systems to study various mutation types and experimental procedures to measure specific mutation frequency associated with gene conversion are discussed.

Keywords: DNA polymerase δ; DSB repair; Double-strand break; Frameshift; Gene conversion; Mutation; Proofreading; Quasipalindrome mutagenesis.