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. 1996 Oct;178(20):5977–5988. doi: 10.1128/jb.178.20.5977-5988.1996

Molecular cloning and characterization of Saccharomyces cerevisiae RAD28, the yeast homolog of the human Cockayne syndrome A (CSA) gene.

P K Bhatia 1, R A Verhage 1, J Brouwer 1, E C Friedberg 1
PMCID: PMC178455  PMID: 8830695

Abstract

Cockayne syndrome patients exhibit severe developmental and neurological abnormalities. Cells derived from these patients are sensitive to killing by UV radiation and do not support the rapid repair of the transcribed strand of transcriptionally active genes observed in cells from normal individuals. We report the cloning of the Saccharomyces cerevisiae homolog of the Cockayne syndrome A (CSA) gene, which we designate as RAD28. A rad28 null mutant does not manifest increased sensitivity to killing by UV or gamma radiation or to methyl methanesulfonate. Additionally, the rate of repair of the transcribed and nontranscribed strands of the yeast RPB2 gene in the rad28 mutant is identical to that observed in wild-type cells following exposure to UV light. As previously shown for rad7 rad26 and rad16 rad26 double mutants, the rad28 null mutant shows slightly enhanced sensitivity to UV light in the presence of mutations in the RAD7 or RAD16 gene. Both rad28 and rad26 null mutants are hypermutable following exposure to UV light.

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Selected References

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  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Bang D. D., Verhage R., Goosen N., Brouwer J., van de Putte P. Molecular cloning of RAD16, a gene involved in differential repair in Saccharomyces cerevisiae. Nucleic Acids Res. 1992 Aug 11;20(15):3925–3931. doi: 10.1093/nar/20.15.3925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baudin A., Ozier-Kalogeropoulos O., Denouel A., Lacroute F., Cullin C. A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res. 1993 Jul 11;21(14):3329–3330. doi: 10.1093/nar/21.14.3329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bohr V. A., Smith C. A., Okumoto D. S., Hanawalt P. C. DNA repair in an active gene: removal of pyrimidine dimers from the DHFR gene of CHO cells is much more efficient than in the genome overall. Cell. 1985 Feb;40(2):359–369. doi: 10.1016/0092-8674(85)90150-3. [DOI] [PubMed] [Google Scholar]
  5. Bohr V. A., Wassermann K. DNA repair at the level of the gene. Trends Biochem Sci. 1988 Nov;13(11):429–433. doi: 10.1016/0968-0004(88)90216-2. [DOI] [PubMed] [Google Scholar]
  6. Broughton B. C., Thompson A. F., Harcourt S. A., Vermeulen W., Hoeijmakers J. H., Botta E., Stefanini M., King M. D., Weber C. A., Cole J. Molecular and cellular analysis of the DNA repair defect in a patient in xeroderma pigmentosum complementation group D who has the clinical features of xeroderma pigmentosum and Cockayne syndrome. Am J Hum Genet. 1995 Jan;56(1):167–174. [PMC free article] [PubMed] [Google Scholar]
  7. Cleaver J. E. Do we know the cause of xeroderma pigmentosum? Carcinogenesis. 1990 Jun;11(6):875–882. doi: 10.1093/carcin/11.6.875. [DOI] [PubMed] [Google Scholar]
  8. Côté J., Quinn J., Workman J. L., Peterson C. L. Stimulation of GAL4 derivative binding to nucleosomal DNA by the yeast SWI/SNF complex. Science. 1994 Jul 1;265(5168):53–60. doi: 10.1126/science.8016655. [DOI] [PubMed] [Google Scholar]
  9. Eckardt F., Haynes R. H. Kinetics of mutation induction by ultraviolet light in excision-deficient yeast. Genetics. 1977 Feb;85(2):225–247. doi: 10.1093/genetics/85.2.225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Eckardt F., Siede W. Mutagen testing with yeast. Basic Life Sci. 1985;34:305–322. doi: 10.1007/978-1-4684-4976-1_19. [DOI] [PubMed] [Google Scholar]
  11. Friedberg E. C., Henning K. A. The conundrum of xeroderma pigmentosum--a rare disease with frequent complexities. Mutat Res. 1993 Sep;289(1):47–53. doi: 10.1016/0027-5107(93)90129-4. [DOI] [PubMed] [Google Scholar]
  12. Friedberg E. C. Xeroderma pigmentosum, Cockayne's syndrome, helicases, and DNA repair: what's the relationship? Cell. 1992 Dec 11;71(6):887–889. doi: 10.1016/0092-8674(92)90384-o. [DOI] [PubMed] [Google Scholar]
  13. Hanawalt P. C. Preferential DNA repair in expressed genes. Environ Health Perspect. 1987 Dec;76:9–14. doi: 10.1289/ehp.87769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hanawalt P. C. Transcription-coupled repair and human disease. Science. 1994 Dec 23;266(5193):1957–1958. doi: 10.1126/science.7801121. [DOI] [PubMed] [Google Scholar]
  15. Henning K. A., Li L., Iyer N., McDaniel L. D., Reagan M. S., Legerski R., Schultz R. A., Stefanini M., Lehmann A. R., Mayne L. V. The Cockayne syndrome group A gene encodes a WD repeat protein that interacts with CSB protein and a subunit of RNA polymerase II TFIIH. Cell. 1995 Aug 25;82(4):555–564. doi: 10.1016/0092-8674(95)90028-4. [DOI] [PubMed] [Google Scholar]
  16. Hoeijmakers J. H. Nucleotide excision repair I: from E. coli to yeast. Trends Genet. 1993 May;9(5):173–177. doi: 10.1016/0168-9525(93)90164-d. [DOI] [PubMed] [Google Scholar]
  17. Hoeijmakers J. H. Nucleotide excision repair. II: From yeast to mammals. Trends Genet. 1993 Jun;9(6):211–217. doi: 10.1016/0168-9525(93)90121-w. [DOI] [PubMed] [Google Scholar]
  18. Kraemer K. H., Levy D. D., Parris C. N., Gozukara E. M., Moriwaki S., Adelberg S., Seidman M. M. Xeroderma pigmentosum and related disorders: examining the linkage between defective DNA repair and cancer. J Invest Dermatol. 1994 Nov;103(5 Suppl):96S–101S. doi: 10.1111/1523-1747.ep12399329. [DOI] [PubMed] [Google Scholar]
  19. Kwon H., Imbalzano A. N., Khavari P. A., Kingston R. E., Green M. R. Nucleosome disruption and enhancement of activator binding by a human SW1/SNF complex. Nature. 1994 Aug 11;370(6489):477–481. doi: 10.1038/370477a0. [DOI] [PubMed] [Google Scholar]
  20. Lambright D. G., Sondek J., Bohm A., Skiba N. P., Hamm H. E., Sigler P. B. The 2.0 A crystal structure of a heterotrimeric G protein. Nature. 1996 Jan 25;379(6563):311–319. doi: 10.1038/379311a0. [DOI] [PubMed] [Google Scholar]
  21. Lawrence C. W. Mutagenesis in Saccharomyces cerevisiae. Adv Genet. 1982;21:173–254. doi: 10.1016/s0065-2660(08)60299-0. [DOI] [PubMed] [Google Scholar]
  22. Leadon S. A., Cooper P. K. Preferential repair of ionizing radiation-induced damage in the transcribed strand of an active human gene is defective in Cockayne syndrome. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10499–10503. doi: 10.1073/pnas.90.22.10499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Leadon S. A., Lawrence D. A. Strand-selective repair of DNA damage in the yeast GAL7 gene requires RNA polymerase II. J Biol Chem. 1992 Nov 15;267(32):23175–23182. [PubMed] [Google Scholar]
  24. Lehmann A. R. Three complementation groups in Cockayne syndrome. Mutat Res. 1982 Dec;106(2):347–356. doi: 10.1016/0027-5107(82)90115-4. [DOI] [PubMed] [Google Scholar]
  25. Lorenz M. C., Muir R. S., Lim E., McElver J., Weber S. C., Heitman J. Gene disruption with PCR products in Saccharomyces cerevisiae. Gene. 1995 May 26;158(1):113–117. doi: 10.1016/0378-1119(95)00144-u. [DOI] [PubMed] [Google Scholar]
  26. Luria S. E., Delbrück M. Mutations of Bacteria from Virus Sensitivity to Virus Resistance. Genetics. 1943 Nov;28(6):491–511. doi: 10.1093/genetics/28.6.491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Manivasakam P., Weber S. C., McElver J., Schiestl R. H. Micro-homology mediated PCR targeting in Saccharomyces cerevisiae. Nucleic Acids Res. 1995 Jul 25;23(14):2799–2800. doi: 10.1093/nar/23.14.2799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mellon I., Spivak G., Hanawalt P. C. Selective removal of transcription-blocking DNA damage from the transcribed strand of the mammalian DHFR gene. Cell. 1987 Oct 23;51(2):241–249. doi: 10.1016/0092-8674(87)90151-6. [DOI] [PubMed] [Google Scholar]
  29. Morrow D. M., Tagle D. A., Shiloh Y., Collins F. S., Hieter P. TEL1, an S. cerevisiae homolog of the human gene mutated in ataxia telangiectasia, is functionally related to the yeast checkpoint gene MEC1. Cell. 1995 Sep 8;82(5):831–840. doi: 10.1016/0092-8674(95)90480-8. [DOI] [PubMed] [Google Scholar]
  30. Nance M. A., Berry S. A. Cockayne syndrome: review of 140 cases. Am J Med Genet. 1992 Jan 1;42(1):68–84. doi: 10.1002/ajmg.1320420115. [DOI] [PubMed] [Google Scholar]
  31. Neer E. J., Schmidt C. J., Nambudripad R., Smith T. F. The ancient regulatory-protein family of WD-repeat proteins. Nature. 1994 Sep 22;371(6495):297–300. doi: 10.1038/371297a0. [DOI] [PubMed] [Google Scholar]
  32. Neer E. J., Smith T. F. G protein heterodimers: new structures propel new questions. Cell. 1996 Jan 26;84(2):175–178. doi: 10.1016/s0092-8674(00)80969-1. [DOI] [PubMed] [Google Scholar]
  33. Otsuka F., Tarone R. E., Cayeux S., Robbins J. H. Use of lymphoblastoid cell lines to evaluate the hypersensitivity to ultraviolet radiation in Cockayne syndrome. J Invest Dermatol. 1984 May;82(5):480–484. doi: 10.1111/1523-1747.ep12260999. [DOI] [PubMed] [Google Scholar]
  34. Parris C. N., Kraemer K. H. Ultraviolet-induced mutations in Cockayne syndrome cells are primarily caused by cyclobutane dimer photoproducts while repair of other photoproducts is normal. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7260–7264. doi: 10.1073/pnas.90.15.7260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Perozzi G., Prakash S. RAD7 gene of Saccharomyces cerevisiae: transcripts, nucleotide sequence analysis, and functional relationship between the RAD7 and RAD23 gene products. Mol Cell Biol. 1986 May;6(5):1497–1507. doi: 10.1128/mcb.6.5.1497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Peterson C. L., Tamkun J. W. The SWI-SNF complex: a chromatin remodeling machine? Trends Biochem Sci. 1995 Apr;20(4):143–146. doi: 10.1016/s0968-0004(00)88990-2. [DOI] [PubMed] [Google Scholar]
  37. Quinn J., Fyrberg A. M., Ganster R. W., Schmidt M. C., Peterson C. L. DNA-binding properties of the yeast SWI/SNF complex. Nature. 1996 Feb 29;379(6568):844–847. doi: 10.1038/379844a0. [DOI] [PubMed] [Google Scholar]
  38. Sancar A. Mechanisms of DNA excision repair. Science. 1994 Dec 23;266(5193):1954–1956. doi: 10.1126/science.7801120. [DOI] [PubMed] [Google Scholar]
  39. Schaeffer L., Moncollin V., Roy R., Staub A., Mezzina M., Sarasin A., Weeda G., Hoeijmakers J. H., Egly J. M. The ERCC2/DNA repair protein is associated with the class II BTF2/TFIIH transcription factor. EMBO J. 1994 May 15;13(10):2388–2392. doi: 10.1002/j.1460-2075.1994.tb06522.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Schaeffer L., Roy R., Humbert S., Moncollin V., Vermeulen W., Hoeijmakers J. H., Chambon P., Egly J. M. DNA repair helicase: a component of BTF2 (TFIIH) basic transcription factor. Science. 1993 Apr 2;260(5104):58–63. doi: 10.1126/science.8465201. [DOI] [PubMed] [Google Scholar]
  41. Schild D., Glassner B. J., Mortimer R. K., Carlson M., Laurent B. C. Identification of RAD16, a yeast excision repair gene homologous to the recombinational repair gene RAD54 and to the SNF2 gene involved in transcriptional activation. Yeast. 1992 May;8(5):385–395. doi: 10.1002/yea.320080506. [DOI] [PubMed] [Google Scholar]
  42. Sherman F. Getting started with yeast. Methods Enzymol. 1991;194:3–21. doi: 10.1016/0076-6879(91)94004-v. [DOI] [PubMed] [Google Scholar]
  43. Sondek J., Bohm A., Lambright D. G., Hamm H. E., Sigler P. B. Crystal structure of a G-protein beta gamma dimer at 2.1A resolution. Nature. 1996 Jan 25;379(6563):369–374. doi: 10.1038/379369a0. [DOI] [PubMed] [Google Scholar]
  44. Sweder K. S., Hanawalt P. C. Preferential repair of cyclobutane pyrimidine dimers in the transcribed strand of a gene in yeast chromosomes and plasmids is dependent on transcription. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10696–10700. doi: 10.1073/pnas.89.22.10696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tanaka K., Kawai K., Kumahara Y., Ikenaga M., Okada Y. Genetic complementation groups in cockayne syndrome. Somatic Cell Genet. 1981 Jul;7(4):445–455. doi: 10.1007/BF01542989. [DOI] [PubMed] [Google Scholar]
  46. Troelstra C., van Gool A., de Wit J., Vermeulen W., Bootsma D., Hoeijmakers J. H. ERCC6, a member of a subfamily of putative helicases, is involved in Cockayne's syndrome and preferential repair of active genes. Cell. 1992 Dec 11;71(6):939–953. doi: 10.1016/0092-8674(92)90390-x. [DOI] [PubMed] [Google Scholar]
  47. Tugendreich S., Bassett D. E., Jr, McKusick V. A., Boguski M. S., Hieter P. Genes conserved in yeast and humans. Hum Mol Genet. 1994;3(Spec No):1509–1517. doi: 10.1093/hmg/3.suppl_1.1509. [DOI] [PubMed] [Google Scholar]
  48. Venema J., Mullenders L. H., Natarajan A. T., van Zeeland A. A., Mayne L. V. The genetic defect in Cockayne syndrome is associated with a defect in repair of UV-induced DNA damage in transcriptionally active DNA. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4707–4711. doi: 10.1073/pnas.87.12.4707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Verhage R. A., van Gool A. J., de Groot N., Hoeijmakers J. H., van de Putte P., Brouwer J. Double mutants of Saccharomyces cerevisiae with alterations in global genome and transcription-coupled repair. Mol Cell Biol. 1996 Feb;16(2):496–502. doi: 10.1128/mcb.16.2.496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Verhage R., Zeeman A. M., de Groot N., Gleig F., Bang D. D., van de Putte P., Brouwer J. The RAD7 and RAD16 genes, which are essential for pyrimidine dimer removal from the silent mating type loci, are also required for repair of the nontranscribed strand of an active gene in Saccharomyces cerevisiae. Mol Cell Biol. 1994 Sep;14(9):6135–6142. doi: 10.1128/mcb.14.9.6135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Vermeulen W., van Vuuren A. J., Chipoulet M., Schaeffer L., Appeldoorn E., Weeda G., Jaspers N. G., Priestley A., Arlett C. F., Lehmann A. R. Three unusual repair deficiencies associated with transcription factor BTF2(TFIIH): evidence for the existence of a transcription syndrome. Cold Spring Harb Symp Quant Biol. 1994;59:317–329. doi: 10.1101/sqb.1994.059.01.036. [DOI] [PubMed] [Google Scholar]
  52. Wall M. A., Coleman D. E., Lee E., Iñiguez-Lluhi J. A., Posner B. A., Gilman A. G., Sprang S. R. The structure of the G protein heterotrimer Gi alpha 1 beta 1 gamma 2. Cell. 1995 Dec 15;83(6):1047–1058. doi: 10.1016/0092-8674(95)90220-1. [DOI] [PubMed] [Google Scholar]
  53. Wang G., Seidman M. M., Glazer P. M. Mutagenesis in mammalian cells induced by triple helix formation and transcription-coupled repair. Science. 1996 Feb 9;271(5250):802–805. doi: 10.1126/science.271.5250.802. [DOI] [PubMed] [Google Scholar]
  54. Wang Z., Svejstrup J. Q., Feaver W. J., Wu X., Kornberg R. D., Friedberg E. C. Transcription factor b (TFIIH) is required during nucleotide-excision repair in yeast. Nature. 1994 Mar 3;368(6466):74–76. doi: 10.1038/368074a0. [DOI] [PubMed] [Google Scholar]
  55. van Gool A. J., Verhage R., Swagemakers S. M., van de Putte P., Brouwer J., Troelstra C., Bootsma D., Hoeijmakers J. H. RAD26, the functional S. cerevisiae homolog of the Cockayne syndrome B gene ERCC6. EMBO J. 1994 Nov 15;13(22):5361–5369. doi: 10.1002/j.1460-2075.1994.tb06871.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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