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THE BINDING OF NONERYTHROID ALPHA SPECTRIN WITH DNA CONTAINING INTERSTRAND CROSS-LINKS

by
Melissa Baralt
Molecular Pathology and Immunology

B.S., 2001, Montclair State University
M.S., 2003, Montclair State University

Thesis Advisor: Muriel W. Lambert, Ph.D.
Professor
Department: Pathology and Laboratory Medicine

MSB C555

Monday, May 4, 2009
10:00 a.m.


Abstract

The cells of patients with the genetic disorder, Fanconi anemia (FA), are hypersensitive to DNA interstrand cross-linking agents and are defective in their ability to repair damage produced by these agents. Our laboratory has identified a structural protein, nonerythroid á spectrin (áIISp), in the nucleus of normal human cells and has shown that it plays an important role in the repair of DNA interstrand cross-links. We have demonstrated that purified spectrin binds to DNA containing interstrand cross-links. In addition, we have shown that there is a deficiency in levels of áIISp in FA cells, which correlates with the defect in ability of these cells to repair DNA interstrand cross-links. In order to develop a clear understanding of the role of áIISp in DNA repair, studies were undertaken to determine the domain of áIISp that binds to cross-linked DNA. For these studies, human áIISp was divided into 4 overlapping regions: Region1 (M1-E887), Region 2 (A864-R1239), Region 3 (K1089-N1766), and Region 4 (E1661-N2477). Each of these regions was expressed as a GST-fusion protein in E. coli, purified on a glutathione-Sepharose 4B column, and used in binding studies to determine if each bound to DNA containing an interstrand cross-link. For these studies a 98 bp DNA substrate was synthesized that contained a centrally located hotspot for formation of a psoralen interstrand cross-link. The top strand was biotinylated on the 5’ end. For formation of psoralen interstrand cross-links, the DNA was treated with 4,5’,8- trimethylpsoralen (TMP) followed by irradiation with long wavelength ultraviolet (UVA) light. Binding of each of the four regions of áIISp to the cross-linked substrate was carried out using DNA affinity chromatography with Streptadvidin-coated acrylamide beads. The results indicate that Region 1 and Region 2 of aIISp bound to the TMP cross-linked DNA substrate. However, this binding was competed out using a non-damaged competitor DNA. However, region 4 displayed no greater binding to the cross-linked DNA than to undamaged DNA. Region 3 bound to the cross-linked DNA with greater affinity than to undamaged DNA. This binding was not competed out using a non-damaged competitor indicating that there was specificity of binding of region 3 to the damaged DNA. These results thus indicate that the binding of aIISp to cross-linked DNA is specific for a particular region of aIISp. Since this region is different from the region that we have shown binds to a proteins involved in repair of DNA interstrand crosslinks, these results strengthen our model that aIISp binds to cross-linked DNA and acts as a scaffold to help in the recruitment of repair proteins to the site of damage, aiding in their interaction with each other and thus enhancing the efficiency of the repair process.


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