Processing of nucleopeptides mimicking the topoisomerase I-DNA covalent complex by tyrosyl-DNA phosphodiesterase

Abstract

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.

Figure 1

Schematic representation of topoisomerase I cleavage intermediates and processing by Tdp1 and PNKP. (A) Reversible topoisomerase I (top1) cleavage complex. (B) Tdp1 hydrolyzes the phosphotyrosyl bond. The resulting DNA break bears a 3′-phosphate and 5′-hydroxyl. PNKP (such as T4 PNKP) has two activities: removal of the phosphate at the 3′-end and phosphorylation of the 5′-end.

Figure 2

Nucleopeptides used as Tdp1 substrates. (Insert) Structure of the tyrosyl–3′-nucleoside phosphodiester bond in the nucleopeptides.

Figure 3

Reaction of Tdp1 with nucleopeptides N15P13 and N15R8. Controls: 15mer oligonucleotide (lanes 1 and 8) and nucleopeptides N15P13 (lane 2) and N15R8 (lane 5). All products were 5′-³²P-radiolabeled. Incubation of 1 pmol nucleopeptide with 11 ng Tdp1 at 30°C for 30 min, followed by reaction with 15 U T4 PNKP at 37°C for 20 min yielded the 15mer oligonucleotides N15P13 (lane 4) and N15R8 (lane 7), whereas the reaction of N15P13 and N15R8 (1 pmol) with Tdp1 (11 ng) resulted in formation of a product with a slightly faster mobility (lanes 3 and 6, respectively). As a result, Tdp1 cleavage of the nucleopeptides generates the corresponding 3′-phosphate oligonucleotide and treatment with T4 PNKP removes the 3′-end phosphate.

Figure 4

Kinetics of Tdp1 processing of nucleopeptides N15P13, N15P4, N4P13, N4P4 and N15Y. (A) An aliquot of 1 pmol of each nucleopeptide (5′-³²P-radiolabeled) was incubated at 30°C with 11 ng Tdp1. Aliquots were taken at 0.33, 0.67, 1, 3, 5, 10 and 30 min and, after addition of a 3-fold excess of Maxam–Gilbert loading buffer to stop the reactions, mixtures were analyzed by gel electrophoresis. (B) Representation of the percentage of Tdp1 cleavage as a function of time.

Figure 5

Structure of the DNA duplex used as substrate for (A) human topoisomerase I and (B) of the topoisomerase I–14mer oligonucleotide suicide complex. Tdp1 processing of the topoisomerase I–14mer oligonucleotide suicide complex: (C) PAGE analysis and (D) graphical representation of suicide complex processing as a function of time.