Chapter 42 - DNA Repair Models for Understanding Triplet Repeat Instability

Abstract

This chapter discusses an in vitro model system to understand triplet repeat expansion reflecting events in base excision repair (BER) and single strand (ssDNA) break repair. The model system involves several of the major human BER enzymes and a CAG repeat-containing DNA with an oxidative base lesion, 8-hydroxy-7, 8-dihydro-guanine (8-oxoG). For both the repeat and random sequence substrates, the damaged strand is annealed to its corresponding template strand. The substrates were radiolabeled at the 5' end of the damaged strand. This substrate DNA allows the specific study of Pol j3 gap-filling, reflecting the DNA slippage events occurring downstream of the damage site after the ssDNA break is generated from glycosylase and AP endonuclease reactions. In this system, purified BER proteins and designed substrates can be introduced to study the roles of individual BER proteins, as well as the multiple protein–protein coordination questions in maintaining triplet repeat stability. The system also identifies the roles of DNA base damage and BER specifically in CAG repeat expansion, because this is associated with Huntington's disease.