Abstract
Tyrosyl-DNA phosphodiesterase I (Tdp1) hydrolyzes 3'-phosphotyrosyl bonds to generate 3'-phosphate DNA and tyrosine in vitro. Tdp1 is involved in the repair of DNA lesions created by topoisomerase I, although the in vivo substrate is not known. Here we study the kinetic and binding properties of human Tdp1 (hTdp1) to identify appropriate 3'-phosphotyrosyl DNA substrates. Genetic studies argue that Tdp1 is involved in double and single strand break repair pathways; however, x-ray crystal structures suggest that Tdp1 can only bind single strand DNA. Separate kinetic and binding experiments show that hTdp1 has a preference for single-stranded and blunt-ended duplex substrates over nicked and tailed duplex substrate conformations. Based on these results, we present a new model to explain Tdp1/DNA binding properties. These results suggest that Tdp1 only acts upon double strand breaks in vivo, and the roles of Tdp1 in yeast and mammalian cells are discussed.
Highlights
DNA topoisomerases are ubiquitous enzymes that catalyze changes in DNA topology by altering the linkage of DNA strands [1]
Genetic studies argue that Tdp1 is involved in double and single strand break repair pathways; x-ray crystal structures suggest that Tdp1 can only bind single strand DNA
The results are consistent with genetic data indicating that Tdp1 could function in double strand break repair (DSBR) and single strand break repair (SSBR) after the topoisomerase I-induced damage is converted to a double strand break
Summary
DNA topoisomerases are ubiquitous enzymes that catalyze changes in DNA topology by altering the linkage of DNA strands [1]. TopI-DNA adducts can accumulate in the presence of naturally occurring DNA damage such as nicks [3], abasic sites [4], modified bases [5], or modified sugars [6] or as a result of exposure to a variety of chemotherapeutic drugs such as camptothecin [7, 8] These lesions can be converted to more permanent single strand breaks (SSBs) or double strand. No substrate or mechanism has been proposed to support this function, Tdp is a member of the phospholipase D (PLD) superfamily of enzymes [21], and other members of this superfamily (PLD1) participate in membrane vesicle trafficking by hydrolyzing phosphatidylcholine to generate phosphatidic acid [22,23,24] These results emphasize the importance of understanding the in vitro binding properties and. The results are consistent with genetic data indicating that Tdp could function in DSBR and SSBR after the topoisomerase I-induced damage is converted to a double strand break
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