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Structure and Function of Human Tyrosyl-DNA Phosphodiesterase I

出版	University of California, San Diego, and San Diego State University, 2004
URL	http://books.google.com.hk/books?id=jXpmlHYx908C&hl=&source=gbs_api

註釋Tyrosyl-DNA Phosphodiesterase I (Tdp1) is involved in the repair of DNA lesions created by topoisomerase I in vivo. Tdp1 is a member of the phospholipase D (PLD) superfamily of enzymes and hydrolyzes 3 2 phosphotyrosyl bonds to generate 32 phosphate DNA and free tyrosine in vitro. Previous mechanistic and structural studies of Tdp1 and other phosholipase D superfamily members argue that Tdp1 uses a two-step reaction mechanism to cleave 32 phosphotyrosyl bonds, transesterification followed by hydrolysis. Kinetic analysis of hTdp1 shows that the enzyme has nanomolar affinity for synthetic 3'-tyrosine phosphate and 32 tyrosine phosphate analog substrates and the overall in vitro reaction is diffusion limited. Analysis of active site mutants phosphate DNA demonstrates that hTdp1 uses an acid/base catalytic mechanism. The results show that histidine 493 serves as the general acid during the initial transesterification, in agreement with hypotheses based on previous crystal structure models. The results also argue that lysine 495 and asparagine 516 participate in the general acid reaction, and analysis of crystal structures suggests that these residues may function in a proton relay. Together with previous crystal structure data, the new functional data provides a mechanistic understanding of the conserved active site residues found among all PLD family members. Functional studies of Tdp1 in model systems have provided evidence for roles in both double strand break repair and single strand break repair pathways. Kinetic analysis of hTdp1 action with single stranded and variously duplexed 32 phosphotyrosyl substrates under catalysis limited conditions in vitro suggest the role of Tdp1 in DNA repair in vivo. Single stranded and blunt end double stranded DNA oligonucleotides are efficiently cleaved by hTdp1, while nicked and tailed double stranded DNA oligonucleotides are cleaved less readily. Structural modeling of hTdp1 and phylogenetic analysis of implicated catalytic residues are consistent with this functional finding.