Substrate specificity of tyrosyl-DNA phosphodiesterase I (Tdp1)

J Biol Chem. 2005 Jun 10;280(23):22029-35. doi: 10.1074/jbc.M502148200. Epub 2005 Apr 4.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Crystallography, X-Ray
  • DNA / chemistry
  • DNA Damage
  • DNA Repair
  • DNA, Single-Stranded / chemistry
  • Dose-Response Relationship, Drug
  • Humans
  • Hydrogen-Ion Concentration
  • Kinetics
  • Models, Molecular
  • Phosphoric Diester Hydrolases / chemistry*
  • Phosphoric Diester Hydrolases / physiology*
  • Protein Binding
  • Spectrometry, Fluorescence
  • Substrate Specificity
  • Time Factors

Substances

  • DNA, Single-Stranded
  • DNA
  • Phosphoric Diester Hydrolases
  • TDP1 protein, human
  • tyrosyl-DNA phosphodiesterase