Yeast Gene for a Tyr-DNA Phosphodiesterase that Repairs Topoisomerase I Complexes
10.1126/science.286.5439.552
Crossref journal-article
American Association for the Advancement of Science (AAAS)
Science (221)
Abstract

Covalent intermediates between topoisomerase I and DNA can become dead-end complexes that lead to cell death. Here, the isolation of the gene for an enzyme that can hydrolyze the bond between this protein and DNA is described. Enzyme-defective mutants of yeast are hypersensitive to treatments that increase the amount of covalent complexes, indicative of enzyme involvement in repair. The gene is conserved in eukaryotes and identifies a family of enzymes that has not been previously recognized. The presence of this gene in humans may have implications for the effectiveness of topoisomerase I poisons, such as the camptothecins, in chemotherapy.

Bibliography

Pouliot, J. J., Yao, K. C., Robertson, C. A., & Nash, H. A. (1999). Yeast Gene for a Tyr-DNA Phosphodiesterase that Repairs Topoisomerase I Complexes. Science, 286(5439), 552–555.

Authors 4
  1. Jeffrey J. Pouliot (first)
  2. Kevin C. Yao (additional)
  3. Carol A. Robertson (additional)
  4. Howard A. Nash (additional)
References 27 Referenced 313
  1. For an overview see
  2. 10.1146/annurev.bi.65.070196.003223
  3. Chen A. Y., Liu L. F., Annu. Rev. Pharmacol. Toxicol. 34, 191 (1994). (10.1146/annurev.pa.34.040194.001203) / Annu. Rev. Pharmacol. Toxicol. by Chen A. Y. (1994)
  4. Pommier Y., Pourquier P., Fan Y., Strumberg D., Biochim. Biophys. Acta 1400, 83 (1998). (10.1016/S0167-4781(98)00129-8) / Biochim. Biophys. Acta by Pommier Y. (1998)
  5. E. C. Friedberg G. C. Walker W. Siede DNA Repair and Mutagenesis (American Society for Microbiology Washington DC 1995);
  6. Kanaar R., Hoeijmakers J. H. J., van Gent D. C., Trends Cell Biol. 8, 483 (1998). (10.1016/S0962-8924(98)01383-X) / Trends Cell Biol. by Kanaar R. (1998)
  7. Yang S.-w., et al., Proc. Natl. Acad. Sci. U.S.A. 93, 11534 (1996). (10.1073/pnas.93.21.11534) / Proc. Natl. Acad. Sci. U.S.A. by Yang S.-w. (1996)
  8. One-step gene disruptions (16) of RAD9 RAD52 and TOP1 used plasmids originally constructed in the laboratories of L. Prakash J. Nitiss and R. Sternglanz respectively. The RAD17 gene was disrupted with a PCR protocol (13)
  9. J. J. Pouliot C. A. Robertson H. A. Nash unpublished observations.
  10. A MATα leu2 ura3 ise 1 strain designated here as HNY102 was obtained from J. Nitiss. The ise 1 mutation inactivates the ERG6 gene and thereby renders the strain permeable to CPT (17). HNY102 was treated with ethylmethane sulfonate and colonies were chosen that showed poor growth when replicated to YPD plates (containing yeast extract peptone and dextrose) doped with CPT (3 μg/ml; Sigma). Midlogarithmic cultures of ∼60 candidates were extracted and assayed for removal of the tyrosine moiety from oligonucleotide oHN279Y (5). A single colony was found that reproducibly yielded very low TDP activity. The mutant line KYY337 was back crossed to HNY115 a MATa derivative of HNY102 made by transient introduction of a GAL-HO plasmid (18). Haploid segregants from the resulting diploid were picked at random and assayed as above; one low-activity segregant was used for a subsequent back cross. After four such rounds hypersensitivity to growth on CPT-containing plates was lost as was the hypersensitivity to cycloheximide and slow growth on YPD that also characterized KYY337. These phenotypes presumably reflected adventitious mutations that were unlinked to the one denoted enz causing low TDP activity. The enz mutation does not affect phosphatase activity. This is evidenced by similar amounts of hydrolysis of terminal processing when extracts of HNY102 KYY337 and the back-crossed derivatives are presented with a substrate oligonucleotide synthesized to have a 3′-phosphate (7). Standard protocols were used for yeast growth mutagenesis mating and sporulation (19).
  11. Because preliminary experiments showed that mitochondrial status influences killing by CPT (7) cells were grown to near saturation in medium with glycerol in place of dextrose (YPG) to ensure a starting population with few or no petite derivatives (19). These cells were resuspended at optical density at 650 nm (OD 650 ) = 0.4 in YPD grown for 2 hours and diluted again in YPD to OD 650 = 0.4. Drug was then added and samples were withdrawn immediately and after 24 hours at 30°C. After dilution and plating on YPD surviving colonies were counted after 3 to 4 days of growth. When plasmid-containing strains were assessed for CPT sensitivity YPD was replaced throughout by uracil-deficient minimal medium (19) to ensure plasmid retention.
  12. 10.1093/emboj/17.19.5679
  13. Megonigal M. D., Fertala J., Bjornsti M.-A., J. Biol. Chem. 272, 12801 (1997). (10.1074/jbc.272.19.12801) / J. Biol. Chem. by Megonigal M. D. (1997)
  14. The cloning scheme was based on our assessment that (i) the signal-to-noise ratio of the TDP assay would permit us to detect one positive colony in a group of 5 to 10 mutants and (ii) one cycle of CPT killing would enrich a positive colony by ∼10-fold. Accordingly strain HNY244 was transformed by electroporation with a genomic library that had been made in a low–copy number vector (20). Transformants were picked from uracil-selective plates and pooled in groups of ∼50. Each pool was separately grown and treated with CPT for 24 hours (9); the survivors were amplified by growth in YPD. An extract was made from a portion of the resulting cells and assayed for TDP activity. From 30 such pools one was identified that had increased activity. Growth and assay of 15 colonies from this pool identified a single clone L10-13 with amounts of activity nearly equal to those of wild type. DNA sequence from the insert of the plasmid in L10-13 placed its ends at coordinates 666257 and 673926 of chromosome II (21). Plasmid pNS2 was made from pL10-13 by elimination of a Not I–Sal I fragment; elimination of an Aat II–Xba I fragment from pNS2 yielded pAXb which has a 3.2-kb insert. Transformation of HNY244 with pNS2 or pAXb restored TDP activity and improved CPT resistance. A control plasmid pX1 that failed to restore TDP activity was made by removal of the central Xba I fragment from pL10-13.
  15. 10.1093/nar/21.14.3329
  16. 10.1002/(SICI)1097-0061(19980130)14:2<115::AID-YEA204>3.0.CO;2-2
  17. A PCR fragment containing the entire ORF YBR223c was cloned into the Bam H1 site of pET15b; the resulting plasmid pHN1856 was transformed into strain BL21(DE3) (Novagen Madison WI). Bacterial pellets from 3 liters of a culture that had been induced for 2 hours were resuspended in 100 ml of disruption buffer (5) sonicated (seven times for 3 min) clarified by centrifugation at 20 000 g and assayed as described above.
  18. Larsen A. K., Skladanowski A., Biochim. Biophys. Acta 1400, 257 (1998). (10.1016/S0167-4781(98)00140-7) / Biochim. Biophys. Acta by Larsen A. K. (1998)
  19. 10.1016/0076-6879(91)94022-5
  20. Nitiss J., Wang J. C., Proc. Natl. Acad. Sci. U.S.A. 85, 7501 (1988); (10.1073/pnas.85.20.7501) / Proc. Natl. Acad. Sci. U.S.A. by Nitiss J. (1988)
  21. Graham T. R., Scott P. A., Emr S. D., EMBO J. 12, 869 (1993). (10.1002/j.1460-2075.1993.tb05727.x) / EMBO J. by Graham T. R. (1993)
  22. Herskowitz I., Jensen R. E., Methods Enzymol. 194, 132 (1991). (10.1016/0076-6879(91)94011-Z) / Methods Enzymol. by Herskowitz I. (1991)
  23. F. Sherman ibid. p. 3; D. A. Treco and V. Lundblad in Current Protocols in Molecular Biology F. M. Ausubel et al. Eds. (Wiley New York 1991) vol. 2 pp. 13.1.1–13.1.7.
  24. Rose M. D., Novick P., Thomas J. H., Botstein D., Fink G. R., Gene 60, 237 (1987). (10.1016/0378-1119(87)90232-0) / Gene by Rose M. D. (1987)
  25. Feldmann H., et al., EMBO J. 13, 5795 (1994). (10.1002/j.1460-2075.1994.tb06923.x) / EMBO J. by Feldmann H. (1994)
  26. The 5′- and 3′-RACE products of the human TDP1 homolog have been deposited as GenBank and respectively.
  27. We thank M. Lichten for advice about yeast manipulations S.-w. Yang for early TDP assays A. Hinnebusch for the yeast genomic library and L. Rasmussen and R. Sugarek for DNA sequencing. K.C.Y. was supported by the Research Scholars Program of the Howard Hughes Medical Institute.
Dates
Type When
Created 23 years, 1 month ago (July 27, 2002, 5:49 a.m.)
Deposited 1 year, 7 months ago (Jan. 13, 2024, 5:08 a.m.)
Indexed 1 month, 1 week ago (July 16, 2025, 8:31 a.m.)
Issued 25 years, 10 months ago (Oct. 15, 1999)
Published 25 years, 10 months ago (Oct. 15, 1999)
Published Print 25 years, 10 months ago (Oct. 15, 1999)
Funders 0

None

@article{Pouliot_1999, title={Yeast Gene for a Tyr-DNA Phosphodiesterase that Repairs Topoisomerase I Complexes}, volume={286}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.286.5439.552}, DOI={10.1126/science.286.5439.552}, number={5439}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Pouliot, Jeffrey J. and Yao, Kevin C. and Robertson, Carol A. and Nash, Howard A.}, year={1999}, month=oct, pages={552–555} }