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. 2002 Nov 12;99(23):14970-5.
doi: 10.1073/pnas.182557199. Epub 2002 Oct 23.

Repair of topoisomerase I covalent complexes in the absence of the tyrosyl-DNA phosphodiesterase Tdp1

Chunyan Liu  1 , Jeffrey J Pouliot, Howard A Nash
Affiliations

Repair of topoisomerase I covalent complexes in the absence of the tyrosyl-DNA phosphodiesterase Tdp1

Chunyan Liu et al. Proc Natl Acad Sci U S A. .

Abstract

Accidental or drug-induced interruption of the breakage and reunion cycle of eukaryotic topoisomerase I (Top1) yields complexes in which the active site tyrosine of the enzyme is covalently linked to the 3' end of broken DNA. The enzyme tyrosyl-DNA phosphodiesterase (Tdp1) hydrolyzes this protein-DNA link and thus functions in the repair of covalent complexes, but genetic studies in yeast show that alternative pathways of repair exist. Here, we have evaluated candidate genes for enzymes that might act in parallel to Tdp1 so as to generate free ends of DNA. Despite finding that the yeast Apn1 protein has a Tdp1-like biochemical activity, genetic inactivation of all known yeast apurinic endonucleases does not increase the sensitivity of a tdp1 mutant to direct induction of Top1 damage. In contrast, assays of growth in the presence of the Top1 poison camptothecin (CPT) indicate that the structure-specific nucleases dependent on RAD1 and MUS81 can contribute independently of TDP1 to repair, presumably by cutting off a segment of DNA along with the topoisomerase. However, cells in which all three enzymes are genetically inactivated are not as sensitive to the lethal effects of CPT as are cells defective in double-strand break repair. We show that the MRE11 gene is even more critical than the RAD52 gene for double-strand break repair of CPT lesions, and comparison of an mre11 mutant with a tdp1 rad1 mus81 triple mutant demonstrates that other enzymes complementary to Tdp1 remain to be discovered.

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Figures

Fig 1.
Fig 1.
Sensitivity to topoisomerase damage of WT yeast and mutants defective in global repair. (A) Cell death after exposure to CPT. Colony-forming ability of strains of the indicated genotype was assessed just before and after addition of CPT (5 μg/ml) to a midlogarithmic culture. CFU, colony-forming units; wt, wild type. (B) Cell death after induction of a toxic topoisomerase. Strains of the indicated genotype each contained a plasmid bearing a mutant form of Top1 downstream of a galactose-inducible promoter. Colony-forming ability was assessed just before and for several hours after addition of galactose to a midlogarithmic culture. (C) Growth in the presence of CPT. Spots of serial dilutions of saturated cultures of the indicated strains were applied to plates containing the indicated concentration (μg/ml) of CPT. Plates were incubated at 30°C for 4 days and photographed. In all panels, a tdp1 deletion strain is included for comparison; in addition to the indicated genotype, all strains contained a null mutation of the ERG6 gene.
Fig 2.
Fig 2.
Hypothetical avenues for repair of covalent complexes. (A) A duplex DNA (with 5′ ends marked by filled circles) bearing a covalent complex of Top1 (whose active site tyrosine emerges from an irregular proteinaceous blob). The protein moiety is shown as being removed either by hydrolysis of the tyrosyl-DNA phosphodiester (curved arrows) or by endonuclease action at phosphodiesters 5′ to this bond (arrowheads). (B) The DNA portion of the covalent complex has undergone conversion to a more open structure. This might happen after collision with a replication fork or an elongating transcription complex (2) or after the action of a helicase or an exonuclease. Sites of potential removal of the covalent complex are indicated as in A. (C) Branched DNA substrates for structure-specific nucleases. (Left) The preferred site of cleavage by Rad1/Rad10 on a “simple Y flap.” (Right) The same for Mus81/Mms4 on a “duplex flap.” The relationship between these artificial constructs and structures shown in A and B is suggestive but not proven.
Fig 3.
Fig 3.
AP endonucleases and Tdp1. (A) Cleavage of the tyrosyl-DNA phosphodiester bond by purified Apn1. An oligonucleotide substrate synthesized to terminate in a 3′-tyrosyl phosphodiester was either used as a single-strand substrate (S) or annealed with conventional oligonucleotides as indicated to produce a blunt duplex (B), a duplex with a 5′-tail (T), or a nicked duplex (N). These were incubated as described in Materials and Methods with (+) or without (−) purified Apn1 and electrophoresed in a denaturing gel. The positions of the substrate oligonucleotide (Y) and the products terminated by a 3′-phosphate (P) and 3′-hydroxyl (O) are marked. Under our conditions, hydrolysis is largely limited to the terminal phosphodiester; at lower ionic strengths, we observed some exonucleolytic degradation from the 3′ end, as has been recently described for Apn1 action on conventional oligonucleotides (35). (B) Cell death after induction of a toxic topoisomerase. The protocol of Fig. 1B was applied to strains of the indicated genotype. (C) Relative activity of Tdp1 on 3′-tyrosine and 3′-PG substrates. Yeast Tdp1, purified as described (6) and included at the indicated concentrations, was incubated with a mixture containing roughly equimolar amounts of an 18-mer synthetic tyrosine oligonucleotide and a 21-mer synthetic PG oligonucleotide. Reaction mixtures, supplemented with T4 polynucleotide kinase, were assembled as described in Materials and Methods, incubated for the indicated time at 30°C, and electrophoresed on a denaturing polyacrylamide gel. The positions of the substrates (18-Y and 21-PG) and the dephosphorylated products (18-OH and 21-OH) are marked. (D) Cell death after exposure to bleomycin. Cultures were incubated with the indicated concentration of bleomycin for 1 h and then diluted and plated. Survival is calculated relative to the number of colonies obtained after 1 h without drug.
Fig 4.
Fig 4.
Sensitivity to topoisomerase damage of yeast mutated in genes for structure-specific nucleases. (A) Growth in the presence of CPT. The indicated strains were grown on plates containing various concentrations of CPT as in Fig. 1C. (B) Cell death after exposure to CPT. Assays on the indicated strains were carried out and analyzed as in Fig. 1A. CFU, colony-forming unit.
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