Skip to main content
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1983 Sep;80(18):5680–5684. doi: 10.1073/pnas.80.18.5680

Isolation and characterization of the RAD3 gene of Saccharomyces cerevisiae and inviability of rad3 deletion mutants

David R Higgins *, Satya Prakash *, Paul Reynolds , Renata Polakowska , Shane Weber , Louise Prakash
PMCID: PMC384322  PMID: 16593371

Abstract

The RAD3 gene of Saccharomyces cerevisiae is required for nicking of DNA containing pyrimidine dimers or interstrand crosslinks. We have cloned the RAD3 gene and physically mapped it to 2.6 kilobase of DNA. A DNA segment of the cloned RAD3 insert was ligated into plasmid YIp5, which transforms yeast by homologous integration, and shown to integrate at the RAD3 site in chromosome V, thus verifying the cloned DNA segment to be the RAD3 gene and not a suppressor. The RAD3 gene encodes a 2.5-kilobase mRNA, extending between the Kpn I site and the Sau3A1/BamHI fusion junction in plasmid pSP10, and the direction of transcription has been determined. The 2.5-kilobase transcript could encode a protein of about 90,000 daltons. We also show the deletions of the RAD3 gene to be recessive lethals, indicating that the RAD3 gene plays an important role in other cellular processes in addition to incision of damaged DNA.

Keywords: DNA repair, excision, pyrimidine dimers, DNA crosslinks, cloning

Full text

PDF
5680

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Botstein D., Falco S. C., Stewart S. E., Brennan M., Scherer S., Stinchcomb D. T., Struhl K., Davis R. W. Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene. 1979 Dec;8(1):17–24. doi: 10.1016/0378-1119(79)90004-0. [DOI] [PubMed] [Google Scholar]
  2. Broach J. R., Atkins J. F., McGill C., Chow L. Identification and mapping of the transcriptional and translational products of the yeast plasmid, 2mu circle. Cell. 1979 Apr;16(4):827–839. doi: 10.1016/0092-8674(79)90098-9. [DOI] [PubMed] [Google Scholar]
  3. Carlson M., Botstein D. Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase. Cell. 1982 Jan;28(1):145–154. doi: 10.1016/0092-8674(82)90384-1. [DOI] [PubMed] [Google Scholar]
  4. Clewell D. B., Helinski D. R. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. doi: 10.1073/pnas.62.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fogliano M., Schendel P. F. Evidence for the inducibility of the uvrB operon. Nature. 1981 Jan 15;289(5794):196–198. doi: 10.1038/289196a0. [DOI] [PubMed] [Google Scholar]
  6. Hinnen A., Hicks J. B., Fink G. R. Transformation of yeast. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1929–1933. doi: 10.1073/pnas.75.4.1929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  8. Hu N., Messing J. The making of strand-specific M13 probes. Gene. 1982 Mar;17(3):271–277. doi: 10.1016/0378-1119(82)90143-3. [DOI] [PubMed] [Google Scholar]
  9. Ish-Horowicz D., Burke J. F. Rapid and efficient cosmid cloning. Nucleic Acids Res. 1981 Jul 10;9(13):2989–2998. doi: 10.1093/nar/9.13.2989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kenyon C. J., Walker G. C. Expression of the E. coli uvrA gene is inducible. Nature. 1981 Feb 26;289(5800):808–810. doi: 10.1038/289808a0. [DOI] [PubMed] [Google Scholar]
  12. Loening U. E. The fractionation of high-molecular-weight ribonucleic acid by polyacrylamide-gel electrophoresis. Biochem J. 1967 Jan;102(1):251–257. doi: 10.1042/bj1020251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Messing J., Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. doi: 10.1016/0378-1119(82)90016-6. [DOI] [PubMed] [Google Scholar]
  14. Miller R. D., Prakash L., Prakash S. Defective excision of pyrimidine dimers and interstrand DNA crosslinks in rad7 and rad23 mutants of Saccharomyces cerevisiae. Mol Gen Genet. 1982;188(2):235–239. doi: 10.1007/BF00332681. [DOI] [PubMed] [Google Scholar]
  15. Miller R. D., Prakash L., Prakash S. Genetic control of excision of Saccharomyces cerevisiae interstrand DNA cross-links induced by psoralen plus near-UV light. Mol Cell Biol. 1982 Aug;2(8):939–948. doi: 10.1128/mcb.2.8.939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mortimer R. K., Schild D. Genetic map of Saccharomyces cerevisiae. Microbiol Rev. 1980 Dec;44(4):519–571. doi: 10.1128/mr.44.4.519-571.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Naumovski L., Friedberg E. C. Molecular cloning of eucaryotic genes required for excision repair of UV-irradiated DNA: isolation and partial characterization of the RAD3 gene of Saccharomyces cerevisiae. J Bacteriol. 1982 Oct;152(1):323–331. doi: 10.1128/jb.152.1.323-331.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Norgard M. V., Emigholz K., Monahan J. J. Increased amplification of pBR322 plasmid deoxyribonucleic acid in Escherichia coli K-12 strains RR1 and chi1776 grown in the presence of high concentrations of nucleoside. J Bacteriol. 1979 Apr;138(1):270–272. doi: 10.1128/jb.138.1.270-272.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Prakash L. Defective thymine dimer excision in radiation-sensitive mutants rad10 and rad16 of Saccharomyces cerevisiae. Mol Gen Genet. 1977 Apr 29;152(3):125–128. doi: 10.1007/BF00268808. [DOI] [PubMed] [Google Scholar]
  20. Prakash L., Prakash S. Isolation and characterization of MMS-sensitive mutants of Saccharomyces cerevisiae. Genetics. 1977 May;86(1):33–55. doi: 10.1093/genetics/86.1.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Prakash L., Prakash S. Three additional genes involved in pyrimidine dimer removal in Saccharomyces cerevisiae: RAD7, RAD14 and MMS19. Mol Gen Genet. 1979 Nov;176(3):351–359. doi: 10.1007/BF00333097. [DOI] [PubMed] [Google Scholar]
  22. Prakash L. Repair of pyrimidine dimers in nuclear and mitochondrial DNA of yeast irradiated with low doses of ultraviolet light. J Mol Biol. 1975 Nov 15;98(4):781–795. doi: 10.1016/s0022-2836(75)80010-6. [DOI] [PubMed] [Google Scholar]
  23. Prakash L. Repair of pyrimidine dimers in radiation-sensitive mutants rad3, rad4, rad6 and rad9 of Saccharomyces cerevisiae. Mutat Res. 1977 Oct;45(1):13–20. doi: 10.1016/0027-5107(77)90038-0. [DOI] [PubMed] [Google Scholar]
  24. Resnick M. A., Setlow J. K. Repair of pyrimidine dimer damage induced in yeast by ultraviolet light. J Bacteriol. 1972 Mar;109(3):979–986. doi: 10.1128/jb.109.3.979-986.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Reynolds R. J., Friedberg E. C. Molecular mechanisms of pyrimidine dimer excision in Saccharomyces cerevisiae: incision of ultraviolet-irradiated deoxyribonucleic acid in vivo. J Bacteriol. 1981 May;146(2):692–704. doi: 10.1128/jb.146.2.692-704.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Reynolds R. J. Removal of pyrimidine dimers from Saccharomyces cerevisiae nuclear DNA under nongrowth conditions as detected by a sensitive, enzymatic assay. Mutat Res. 1978 Apr;50(1):43–56. doi: 10.1016/0027-5107(78)90059-3. [DOI] [PubMed] [Google Scholar]
  27. Sancar A., Clarke N. D., Griswold J., Kennedy W. J., Rupp W. D. Identification of the uvrB gene product. J Mol Biol. 1981 May 5;148(1):63–76. doi: 10.1016/0022-2836(81)90235-7. [DOI] [PubMed] [Google Scholar]
  28. Sancar A., Wharton R. P., Seltzer S., Kacinski B. M., Clarke N. D., Rupp W. D. Identification of the uvrA gene product. J Mol Biol. 1981 May 5;148(1):45–62. doi: 10.1016/0022-2836(81)90234-5. [DOI] [PubMed] [Google Scholar]
  29. Scherer S., Davis R. W. Replacement of chromosome segments with altered DNA sequences constructed in vitro. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4951–4955. doi: 10.1073/pnas.76.10.4951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Shortle D., Haber J. E., Botstein D. Lethal disruption of the yeast actin gene by integrative DNA transformation. Science. 1982 Jul 23;217(4557):371–373. doi: 10.1126/science.7046050. [DOI] [PubMed] [Google Scholar]
  31. Silverman S. J., Rose M., Botstein D., Fink G. R. Regulation of HIS4-lacZ fusions in Saccharomyces cerevisiae. Mol Cell Biol. 1982 Oct;2(10):1212–1219. doi: 10.1128/mcb.2.10.1212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  33. Unrau P., Wheatcroft R., Cox B. S. The excision of pyrimidine dimers from DNA of ultraviolet irradiated yeast. Mol Gen Genet. 1971;113(4):359–362. doi: 10.1007/BF00272336. [DOI] [PubMed] [Google Scholar]
  34. Waters R., Moustacchi E. The disappearance of ultraviolet-induced pyrimidine dimers from the nuclear DNA of exponential and stationary phase cells of Saccharomyces cerevisiae following various post-irradiation treatments. Biochim Biophys Acta. 1974 Jul 24;353(4):407–419. doi: 10.1016/0005-2787(74)90048-3. [DOI] [PubMed] [Google Scholar]
  35. Wilcox D. R., Prakash L. Incision and postincision steps of pyrimidine dimer removal in excision-defective mutants of Saccharomyces cerevisiae. J Bacteriol. 1981 Nov;148(2):618–623. doi: 10.1128/jb.148.2.618-623.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yoakum G. H., Grossman L. Identification of E. coli uvrC protein. Nature. 1981 Jul 9;292(5819):171–173. doi: 10.1038/292171a0. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES