Processing of nucleopeptides mimicking the topoisomerase I-DNA covalent complex by tyrosyl-DNA phosphodiesterase.
Tyrosyl-DNA phosphodiesterase-1 (Tdp1) is the only known enzyme to remove tyrosine from complexes in which the amino acid is linked to the 3'-end of DNA fragments. Such complexes can be produced following DNA processing by topoisomerase I, and recent studies in yeast have demonstrated the importance of TDP1 for cell survival following topoisomerase I-mediated DNA damage. In the present study, we used synthetic oligodeoxynucleotide-peptide conjugates (nucleopeptides) and recombinant yeast Tdp1 to investigate the molecular determinants for Tdp1 activity. We find that Tdp1 can process nucleopeptides with up to 13 amino acid residues but is poorly active with a 70 kDa fragment of topoisomerase I covalently linked to a suicide DNA substrate. Furthermore, Tdp1 was more effective with nucleopeptides with one to four amino acids than 15 amino acids. Tdp1 was also more effective with nucleopeptides containing 15 nt than with homolog nucleopeptides containing 4 nt. These results suggest that DNA binding contributes to the activity of Tdp1 and that Tdp1 would be most effective after topoisomerase I has been proteolyzed in vivo.
Debéthune
Laurent
L
Department of Organic Chemistry, Faculty of Chemistry, University of Barcelona, Martí i Franquès 1-11, E-08028 Barcelona, Spain.
Kohlhagen
Glenda
G
Grandas
Anna
A
Pommier
Yves
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008956:Models, Chemical; D009698:Nucleoproteins / Q000737:chemistry / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity
D046911:Macromolecular Substances; D009698:Nucleoproteins; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D004247:DNA; D010727:Phosphoric Diester Hydrolases; C122147:tyrosyl-DNA phosphodiesterase; D004264:DNA Topoisomerases, Type I