DESCRIPTION (provided by applicant): Chromatin structure plays a critical role in modulation of cellular response to DNA damage. The mechanism utilized by repair proteins to locate target sites on damaged nucleosomal DNA is critical to understanding these repair processes. The present proposal will examine the effect of nucleosome structure on the ability of human repair proteins to interact with and incise DNA containing interstrand cross-links, produced by potentially mutagenic and carcinogenic environmental agents such as psoralen plus UVA light, and to ascertain whether there is a relationship between the processive mechanism of action shown by the endonucleases involved in this process and their ability to incise damaged nucleosomal DNA. We have isolated a protein complex from the nuclei of normal human cells that contains all the proteins needed for recognition and incision of DNA containing interstrand cross-links. In cells from patients with the cancer-prone, repair deficient genetic disease, xeroderma pigmentosum, complementation group A (XPA), this same complex has normal levels of activity on cross-linked naked DNA but is defective in ability to incise damaged nucleosomal DNA. This defect correlates with the distributive mechanism of action utilized by the endonucleases present in the XPA complex, in contrast to the processive mechanism of action of the normal endo-nucleases, and is related to the loss of ability of the XPA protein to act as a processivity factor. The present proposal will focus on this new role of XPA as a processivity factor and on which domain in XPA is responsible for its mechanism of action. Site-directed mutagenesis will be used to create selected mutations in XPA. Recombinant mutant proteins will be produced and examined to ascertain which domain(s) is needed for its action as a processivity factor on damaged naked DNA and the importance of this domain for its action on damaged nucleosomal DNA. DNA substrates will be constructed that contain a positioned nucleosome and a site-specific 4,5',8-trimethyl-psoralen (TMP) interstrand cross-link in either the core or linker region. This will allow evaluation of the effect of nucleosome structure and cross-link location on the influence of the mutant and wild type XPA proteins on incisions produced by the normal and XPA complexes on damaged DNA. Whether defects in ability of the XPA complex to incise damaged nucleosomal DNA can be corrected by the wild type or mutant XPA proteins will also be examined. These studies will provide important insights into the complex mechanism by which interstrand cross-links are repaired in nucleosomal DNA, the importance of a processive mechanism of action by repair proteins on damaged nucleosomal DNA, and the severe consequences that occur in individuals when this mechanism is defective.
|Effective start/end date||9/1/03 → 5/31/06|
- Cancer Research
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