title: Effects of DNA and protein size on substrate cleavage by human tyrosyl-DNA phosphodiesterase 1.
abstract: TDP (tyrosyl-DNA phosphodiesterase) 1 catalyses the hydrolysis of phosphodiester linkages between a DNA 3' phosphate and a tyrosine residue as well as a variety of other DNA 3' substituents, and has been implicated in the repair of covalent complexes involving eukaryotic type IB topoisomerases. To better understand the substrate features that are recognized by TDP1, the size of either the DNA or protein component of the substrate was varied. Competition experiments and gel-shift analyses comparing a series of substrates with DNA lengths increasing from 6 to 28 nt indicated that, contrary to predictions based on the crystal structure of the protein, the apparent affinity for the substrate increased as the DNA length was increased over the entire range tested. It has been found previously that a substrate containing the full-length native form of human topoisomerase I protein is not cleaved by TDP1. Protein-oligonucleotide complexes containing either a 53 or 108 amino acid topoisomerase I-derived peptide were efficiently cleaved by TDP1, but similar to the full-length protein, a substrate containing a 333 amino acid topoisomerase I fragment was resistant to cleavage. Consistent with these results, evidence is presented that processing by the proteasome is required for TDP1 cleavage in vivo.
authors.affiliation: Department of Microbiology, School of Medicine, Box 357242, University of Washington, Seattle, WA 98195, USA.
authors.forename: Heidrun
authors.initials: H
authors.lastname: Interthal
authors.forename: James J
authors.initials: JJ
authors.lastname: Champoux
mesh_terms: D004247:DNA / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D006801:Humans; D010446:Peptide Fragments / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D010446:Peptide Fragments; D004247:DNA; D010727:Phosphoric Diester Hydrolases; C122147:tyrosyl-DNA phosphodiesterase; D004264:DNA Topoisomerases, Type I; C528909:TOP1 protein, human