Skin cancer is the most prevalent form of cancer and its incidence has been steadily rising over the past 20 years. The most common forms are squamous cell and basal cell carcinomas and will affect about one out of four Americans in their life time. In the past few years, great concern has arisen because of evidence that the ozone layer, which shields us from ultraviolet light, is slowly being depleted. The structural changes of DNA induced by UV, the dimerization of pyrimidines, are responsible in part for these harmful or lethal effects. Unfortunately, the precise structural impact of dimers on local DNA structure is unknown. We propose to identify the alteration in the structure of DNA induced by the formation of pyrimidine dimers, using the techniques of x-ray crystallography. The linkage between mutations in DNA repair genes to some cancers has become increasingly evident. We will study the means of recognition of altered DNA by a repair enzyme. We propose to study the interaction of T4 endonuclease V with several pyrimidine dimers. We also propose to solve the crystal structure of human mitochondria single stranded DNA binding protein, which performs a vital role in repair of DNA. A better understanding of the details of DNA damage and repair will help pave the way to risk assessment based on the mechanics of carcinogenesis.
Our specific aims are as follows. A.
SPECIFIC AIM #1 - Crystal structure determination of photo product- containing DNAs: We propose to synthesize site-specific photo product- containing oligonucleotides on a large scale by utilizing both solution and solid phase phosphoramidite and phosphotriester methodology and will carry out X-ray studies of these oligonucleotides (6-, 8-, 10-, 12-mers). B.
SPECIFIC AIM #2 - Molecular Architecture of Endonuclease V bound at a Cyclobutane Pyrimidine Dimer: We propose to carry out X-ray studies of T4 endonuclease V, an enzyme responsible for the first step of a pyrimidine- dimer-specific excision-repair pathway. We will crystallize a: covalently trapped enzyme-substrate complex, b: a site-directed mutant which has unaltered specific binding to photo damaged DNA but has largely inhibited glycosylase and abasic lyase activities, c: the C-terminal portion (17mer, 35mer) of endonuclease V with a pyrimidine-dimer containing DNA, d: native endonuclease with non-cleavable pyrimidine-dimer analogues. C.
SPECIFIC AIM #3 - Crystal structure determination of Human mitochondrial single-stranded-DNA-binding (HsmtSSB) protein, an enzyme involved in a process that maintains the integrity of the genome including nucleotide excision repair of damaged DNA. We have crystallized the native protein and we prepare to crystallize protein-single stranded DNA complex.
