Mapping and Quantification of Bulky-Chemical-Induced DNA Damage Using UvrABC Nucleases

Abstract

Nucleotide excision repair (NER) is one of the most versatile and conservative repair systems in the biological kingdom. This repair pathway repairs DNA damage caused by a variety of agents, including ultraviolet light (UV), benzo[a]pyrene diol epoxide (BPDE), N-acetoxy-2-acetyl-aminofluorene (NAAAF), N-hydroxy-2-aminofluorene (N-OH-AF), dimethylbenzanthracene diol epoxide (DMBA-DE), and therapeutic drugs (cis-platinum, CC-1065, anthramycin, mitomycin C., and psoralen) in both prokaryotes and eukaryotes (for a review see Friedberg et al., 1995; Sancar and Tang, 1993; van Houten, 1990). The initial step of NER involves the recognition of the damaged bases and dual incisions at the 5′ and 3′ sides of the damaged base(s). In E. coli cells these steps are controlled by three gene products—the UvrA, UvrB, and UvrC proteins. The uvrA, uvrB, and uvrC genes have been cloned into expression vectors allowing relatively large quantities of these proteins to be readily purified without elaborate procedures. Consequently, the biochemical nature of the recognition of DNA damage and of the dual incisions 5′ and 3′ of the damaged base(s) by the coordinative mechanisms of these three proteins have been extensively studied. The details of these reactions can be found in several review articles (Sancar, 1994; Sancar and Tang, 1993; Grossman and Yeung, 1990; van Houten, 1990), and the following four steps represent a brief synopsis of the reactions of the Uvr proteins: 1. 2 UvrA → (UvrA)2 In solution two UvrA proteins may form a dimer. 2. (UvrA)2 + UvrB → (UvrA)2·UvrB The dimeric form of UvrA may bind to damaged DNA or form a complex with UvrB. 3. (UvrA)2·UvrB locates and binds to the damaged base(s) and UvrA is released from the complex. 4. UvrC joins the UvrB-damaged base(s) complex, resulting in 5′ and 3′ incisions.