Human uracil-initiated base-excision DNA repair constitutes a major cellular defense mechanism for avoiding DNA damage. Uracil residues are frequently introduced in the human genome as a consequence of dUMP incorporation during DNA synthesis or by spontaneous and chemically induced deamination of cytosine residue in DNA. If unrepaired, the accumulation of uracil residues may provoke cytotoxic, mutagenic and lethal consequences. The long-term objective of this research is focused on defining the biochemical and molecular mechanisms used by human cells to conduct uracil-DNA repair. In this continuation application, we shift our emphasis to focus primarily on human uracil-initiated DNA repair systems. An integrated series of experiments with four specific alms is set forth to further advance this objective. (1) In the first phase of this project, the fidelity and mutational specificity associated with both short parch and long patch uracil-initiated base excision repair will be investigated using human fibroblast and mouse fibroblast (Pol beta +/+ and -/-) cells. Using an M13mp2 lacZalpha DNA-based reversion assay to detect mutations, the contribution of various DNA polymerases (beta, delta, and epsilon) and DNA repair synthesis patch size will be evaluated. (2) The mode of action of an alternative uracil-initiated DNA repair pathway recognized in human glioblastoma U251 mugi-17 cells that express the uracil-DNA glycosylase inhibitor (Ugi) protein be investigated. In addition, the Ugi-insensitive uracil-DNA glycosylase that initiates this back-up uracil-DNA repair pathway will be purified and characterized. (3) The tertiary structure of full-length human uracil-DNA glycosylase which contains a putative binding domain for the p32 subunit of human RPA protein will be determined by x-ray crystallography through collaborative efforts. (4) The role of individual amino acids located in three defined structural elements (beta-zipper, omega loop, Leu loop) of human UDG1 and UDG2 will be examined using regio-specific site-directed mutagenesis. The effects of mutations on DNA-binding, processivity, substrate specificity and catalysis will be elucidated. It is anticipated that the information gained from these investigations will prove to be fundamental and relevant to understanding the human biochemical pathways that prevent mutagenesis and carcinogenesis.
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