Yeast gene for a Tyr-DNA phosphodiesterase that repairs topoisomerase I complexes

Science

Volumn 286, Issue 5439, 1999, Pages 552-555

  Pouliot, Jeffrey J ^a   Yao, Kevin C ^a   Robertson, Carol A ^a   Nash, Howard A ^a  

^a NATIONAL INSTITUTE OF MENTAL HEALTH   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CAMPTOTHECIN; DNA TOPOISOMERASE; PHOSPHODIESTERASE;

ARTICLE; COVALENT BOND; DNA DAMAGE; DNA REPAIR; FUNGUS MUTANT; GENE ISOLATION; NONHUMAN; NUCLEOTIDE SEQUENCE; PRIORITY JOURNAL; SACCHAROMYCES CEREVISIAE;

AMINO ACID SEQUENCE; ANIMALS; CAMPTOTHECIN; DNA REPAIR; DNA TOPOISOMERASES, TYPE I; DNA, FUNGAL; EXPRESSED SEQUENCE TAGS; GENES, FUNGAL; HUMANS; MOLECULAR SEQUENCE DATA; MUTATION; PHOSPHORIC DIESTER HYDROLASES; SACCHAROMYCES CEREVISIAE; SEQUENCE ALIGNMENT;

EUKARYOTA; FUNGI; SACCHAROMYCES CEREVISIAE;

EID: 0033569666     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.286.5439.552     Document Type: Article

References (27)

1. For an overview, see J. C. Wang, Annu. Rev. Biochem. 65, 635 (1996).
2. A. Y. Chen and L. F. Liu, Annu. Rev. Pharmacol. Toxicol. 34, 191 (1994).
3. Y. Pommier, P. Pourquier, Y. Fan, D. Strumberg, Biochim. Biophys. Acta 1400, 83 (1998).
4. E. C. Friedberg, G. C. Walker, W. Siede, DNA Repair and Mutagenesis (American Society for Microbiology, Washington, DC, 1995); R. Kanaar, J. H. J. Hoeijmakers, D. C. van Gent, Trends Cell Biol. 8, 483 (1998).
5. E. C. Friedberg, G. C. Walker, W. Siede, DNA Repair and Mutagenesis (American Society for Microbiology, Washington, DC, 1995); R. Kanaar, J. H. J. Hoeijmakers, D. C. van Gent, Trends Cell Biol. 8, 483 (1998).
6. S.-w. Yang et al., Proc. Natl. Acad. Sci. U.S.A. 93, 11534 (1996).
7. 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)
8. J. J. Pouliot, C. A. Robertson, H. A. Nash, unpublished observations.
9. A MATα leu2 ura3 ise1 strain, designated here as HNY102, was obtained from J. Nitiss. The ise1 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 CAL-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).
10. 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.
11. J. E. Vialard, C. S. Gilbert, C. M. Green, N. F. Lowndes, EMBO J. 17, 5679 (1998).
12. M. D. Megonigal, J. Fertala, M.-A. Bjornsti, J. Biol. Chem. 272, 12801 (1997).
13. 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 (27). 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.
14. A. Baudin, O. Ozier-Kalogeropoulos, A. Denouel, F. Lacroute, C. Cullin, Nucleic Acids Res. 21, 3329 (1993); C. Brachmann et al., Yeast 14, 115 (1998).
15. A. Baudin, O. Ozier-Kalogeropoulos, A. Denouel, F. Lacroute, C. Cullin, Nucleic Acids Res. 21, 3329 (1993); C. Brachmann et al., Yeast 14, 115 (1998).
16. 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 20,000g, and assayed as described above.
17. A. K. Larsen and A. Skladanowski, Biochim. Biophys. Acta 1400, 257 (1998).
18. R. Rothstein, Methods Enzymol. 194, 281 (1991).
19. J. Nitiss and J. C. Wang, Proc. Natl. Acad. Sci. U.S.A. 85, 7501 (1988); T. R. Graham, P. A. Scott, S. D. Emr, EMBO J. 12, 869 (1993).
20. J. Nitiss and J. C. Wang, Proc. Natl. Acad. Sci. U.S.A. 85, 7501 (1988); T. R. Graham, P. A. Scott, S. D. Emr, EMBO J. 12, 869 (1993).
21. I. Herskowitz and R. E. Jensen, Methods Enzymol. 194, 132 (1991).
22. 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.
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. M. D. Rose, P. Novick, J. H. Thomas, D. Botstein, G. R. Fink, Gene 60, 237 (1987).
25. H. Feldmann et al., EMBO J. 13, 5795 (1994).
26. The 5′-and 3′-RACE products of the human TDP1 homolog have been deposited as GenBank AF182002 and AF182003, 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.