DNA REPAIR DEFECT IN FANCONI ANEMIA, GROUP A

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DESCRIPTION: The applicant and her colleagues have isolated a DNA endonuclease complex from normal human chromatin that seems to be involved in the initial steps of the repair of DNA interstrand crosslinks. The complex contains the endonuclease and a damage recognition protein as well. In addition, this complex is abnormal in extracts from Fanconi Anemia (FA) cells of the A complementation group. She has found that these cells have absent or reduced activity of damage recognition and have a defect in the ability to endonucleolytically excise damaged DNA. In addition, using a strategy for site-specific trimethyl psoralen monoadducts, the FA complex is specifically defective in its capacity to make the an incision on the 3' side of the cross link while being competent to incise at the 5' side. Moreover, monoclonal antibodies have been raised by the applicant against specific proteins in the complex and have permitted the identification of a 41 Kda protein and a 225 Kda protein. The antibodies inhibit the capacity of normal complexes to create an incision on the 3' side of the crosslink. Finally, Western blot analysis shows that the FA cells are deficient in the 225 Kda protein. Based upon these observations the Applicant proposes to: (1) isolate DNA damage recognition protein and endonuclease involved in repair of interstrand crosslinks and to prepare tryptic peptide digests from each protein, (2) isolate and sequence CDNAS encoding these proteins and subsequently express these CDNAS in FA cells of the A complementation group to confirm their role in the repair process, (3) determine the chromosomal location of the gene and express recombinant proteins will be expressed in E.coli, yeast, and baculovirus to determine whether the recombinant molecules have functional activity in the cell-free system, (4) analyze the mutations in homologous CDNAS isolated from FA cells, (5) characterize the damage recognition protein and endonuclease in normal and FA cells and study their interactions on psoralen plus UVA light irradiated naked DNA.