The major goal of this project is to analyse the mechanisms by which single site DNA lesions located either in the minor groove or major groove of DNA are repaired. In order to achieve this goal we will construct a single site DNA interstrand crosslink (ISC) induced within the minor groove of DNA by the cyclopropylpyroloindole analog U-77,779 (U-77). This agent can induce DNA ISC's which span two adenine N3 positions, 6 nucleotide or 7 nucleotides apart within either a 5'-TAAAAA-3' or 5'- TAAAAAA-3' sequences. DNA ISC located in the major groove within 5'-GC-3', 5'-GNC-3' and 5'-GNNC-3' sequences will be synthesized by incubating a defined DNA oligonucleotide with two distinct aziridinylbenzoquinones in the presence of the two electron reductase DT-diaphorase (DTD). DNA ISC will be isolated and purified by gel electrophoresis and single site DNA ISC can be constructed within a defined DNA template. The biochemical mechanisms by which human tumor cell extracts can repair such lesions will then be assessed in three distinct ways. 1) The ability of damage recognition proteins (DRP's) to associate with each individual lesion will be determined. Such proteins will then be isolated, purified and identified. 2) The ability of either cell extracts or purified enzymes to incise such defined lesions will be determined. This will allows us to assess the importance of either base excision or nucleotide excision to these distinct group of DNA adducts. 3) The ability of cell extracts to support repair synthesis upon our defined damaged DNA templates will also be assessed. The advantages of the latter two DNA repair assays are that complementation experiments can be performed. That is the mixture of a repair proficient cell extract, purified protein or partially purified extract may complement the repair activity of a cell extract deficient in DNA repair. Such experiments will be important for identifying and purifying proteins essential to the repair of DNA ISC located in either the minor or major groove of DNA. This work could have important implication in terms of enhancing our knowledge of the mechanisms by which human tumor cells are resistant to DNA reactive antitumor agents.
