Pathways for repair of topoisomerase I covalent complexes in Saccharomyces cerevisiae.
BACKGROUND
The covalent linkage between DNA and the active site tyrosine of topoisomerase I can be stabilized by chemotherapeutic agents, adjacent DNA lesions, or mutational defects in the topoisomerase itself. Following collision with a replication fork, the covalent complex can be converted to a double-strand break. Tdp1, an enzyme that can hydrolyse the bond between topoisomerase I and DNA, is thought to be involved in the repair of these lesions, but little is known about how such repair is accomplished.


RESULTS
Reaction kinetics with model substrates reveal that the catalytic efficiency of Saccharomyces cerevisiae Tdp1 is relatively poor when the scissile bond is located in the middle of a duplex, but much better when it is located at the end of a structure. Survival of yeast after induction of a toxic topoisomerase is substantially reduced by inactivation of the TDP1 gene. Comparison of survival of single and double mutants places TDP1 and RAD52 in the same epistasis group but TDP1 and RAD9 in different epistasis groups. In the absence of RAD9, inactivation of TDP1 has a significant effect on the survival of cells following exposure to camptothecin but is without consequence for the survival of agents that do not target topoisomerase I.


CONCLUSIONS
Tdp1 acts as a specific repair enzyme for topoisomerase I lesions. Rather than working at their earliest occurrence, the enzyme acts after covalent complexes have been converted to DSBs. A second repair pathway also exists that functions independently of Tdp1 but requires RAD9 function to efficiently repair topoisomerase I-linked DSBs. The efficiency of these pathways differs for complexes induced with the chemotherapeutic agent camptothecin vs. those accumulated by mutant forms of topoisomerase I.
Pouliot
J J
JJ
Laboratory of Molecular Biology, National Institute of Mental Health, Building 36, Room 1B08, Bethesda, MD 20892-4034, USA.
Robertson
C A
CA
Nash
H A
HA
D002166:Camptothecin / Q000494:pharmacology; D002384:Catalysis; D004260:DNA Repair; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000378:metabolism*; D004271:DNA, Fungal / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005838:Genotype; D007700:Kinetics; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism*; D051721:Rad52 DNA Repair and Recombination Protein; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C497197:RAD52 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; D029701:Saccharomyces cerevisiae Proteins; D010727:Phosphoric Diester Hydrolases; C122147:tyrosyl-DNA phosphodiesterase; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin