Requirement of yeast SGS1 and SRS2 genes for replication and transcription

Science

Volumn 286, Issue 5448, 1999, Pages 2339-2342

  Lee, Sung Keun ^a   Johnson, Robert E ^a   Yu, Sung Lim ^a   Prakash, Louise ^a   Prakash, Satya ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

DNA DIRECTED RNA POLYMERASE; HELICASE;

ARTICLE; BLOOM SYNDROME; DNA REPLICATION; DNA SYNTHESIS; DNA TRANSCRIPTION; GENE MUTATION; NONHUMAN; PRIORITY JOURNAL; RNA SYNTHESIS; SACCHAROMYCES CEREVISIAE; WERNER SYNDROME;

BLOOM SYNDROME; CODON; DNA HELICASES; DNA REPLICATION; DNA, FUNGAL; FUNGAL PROTEINS; GENE DELETION; GENES, FUNGAL; HUMANS; MUTATION; RNA POLYMERASE I; RNA POLYMERASE II; RNA POLYMERASE III; RNA, FUNGAL; RNA, MESSENGER; RNA, RIBOSOMAL; RNA, TRANSFER, AMINO ACID-SPECIFIC; SACCHAROMYCES CEREVISIAE; SACCHAROMYCES CEREVISIAE PROTEINS; TRANSCRIPTION, GENETIC; WERNER SYNDROME;

SACCHAROMYCES CEREVISIAE;

EID: 0033579343     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.286.5448.2339     Document Type: Article

References (34)

1. J. German, Dermatol. Clin. 13, 7 (1995); N. A. Ellis, Nature 381, 110 (1996).
2. J. German, Dermatol. Clin. 13, 7 (1995); N. A. Ellis, Nature 381, 110 (1996).
3. S. Gangloff, J. P. McDonald, C. Bendixen, L. Arthur, R. Rothstein, Mol. Cell. Biol. 14, 8391 (1994); P. M. Watt, E. J. Louis, R. H. Borts, I. D. Hickson, Cell 81, 253 (1995); P. M. Watt, I. D. Hickson, R. H. Borts, E. J. Louis, Genetics 144, 935 (1996).
4. S. Gangloff, J. P. McDonald, C. Bendixen, L. Arthur, R. Rothstein, Mol. Cell. Biol. 14, 8391 (1994); P. M. Watt, E. J. Louis, R. H. Borts, I. D. Hickson, Cell 81, 253 (1995); P. M. Watt, I. D. Hickson, R. H. Borts, E. J. Louis, Genetics 144, 935 (1996).
5. S. Gangloff, J. P. McDonald, C. Bendixen, L. Arthur, R. Rothstein, Mol. Cell. Biol. 14, 8391 (1994); P. M. Watt, E. J. Louis, R. H. Borts, I. D. Hickson, Cell 81, 253 (1995); P. M. Watt, I. D. Hickson, R. H. Borts, E. J. Louis, Genetics 144, 935 (1996).
6. N. A. Ellis et al., Cell 83, 655 (1995); C.-E. Yu et al., Science 272, 258 (1996).
7. N. A. Ellis et al., Cell 83, 655 (1995); C.-E. Yu et al., Science 272, 258 (1996).
8. J. Lu et al., Nature 383, 678 (1996); R. J. Bennett, J. A. Sharp, J. C. Wang, J. Biol. Chem. 273, 9644 (1998); M. D. Gray et al., Nature Genet. 17, 100 (1997); J. K. Karow, R. K. Chakraverty, I. D. Hickson, J. Biol. Chem. 272, 30611 (1997).
9. J. Lu et al., Nature 383, 678 (1996); R. J. Bennett, J. A. Sharp, J. C. Wang, J. Biol. Chem. 273, 9644 (1998); M. D. Gray et al., Nature Genet. 17, 100 (1997); J. K. Karow, R. K. Chakraverty, I. D. Hickson, J. Biol. Chem. 272, 30611 (1997).
10. J. Lu et al., Nature 383, 678 (1996); R. J. Bennett, J. A. Sharp, J. C. Wang, J. Biol. Chem. 273, 9644 (1998); M. D. Gray et al., Nature Genet. 17, 100 (1997); J. K. Karow, R. K. Chakraverty, I. D. Hickson, J. Biol. Chem. 272, 30611 (1997).
11. J. Lu et al., Nature 383, 678 (1996); R. J. Bennett, J. A. Sharp, J. C. Wang, J. Biol. Chem. 273, 9644 (1998); M. D. Gray et al., Nature Genet. 17, 100 (1997); J. K. Karow, R. K. Chakraverty, I. D. Hickson, J. Biol. Chem. 272, 30611 (1997).
12. S. Huang et al., Nature Genet. 20, 114 (1998); A. S. Kamath-Loeb, J.-C. Shen, L. A. Loeb, M. Fry, J. Biol. Chem. 273, 34145 (1998).
13. S. Huang et al., Nature Genet. 20, 114 (1998); A. S. Kamath-Loeb, J.-C. Shen, L. A. Loeb, M. Fry, J. Biol. Chem. 273, 34145 (1998).
14. D. A. Sinclair, K. Mills, L. Guarente, Science 277, 1313 (1997).
15. R. A. Marciniak, D. B. Lombard, F. B. Johnson, L. Guarente, Proc. Natl. Acad. Sci. U.S.A. 95, 6887 (1998).
16. D. A. Sinclair and L. Guarente, Cell 91, 1033 (1997).
17. L. Rong and H. L. Klein, J. Biol. Chem. 268, 1252 (1993); L. Rong, F. Palladino, A. Aguilera, H. L. Klein, Genetics 127, 75 (1991).
18. L. Rong and H. L. Klein, J. Biol. Chem. 268, 1252 (1993); L. Rong, F. Palladino, A. Aguilera, H. L. Klein, Genetics 127, 75 (1991).
19. To construct the sgs1Δ-generating plasmid, 1005-base pair (bp) and 919-bp polymerase chain reaction (PCR) products corresponding to the 5′ and 3′ regions of the SGS1 gene, respectively, were amplified from total yeast genomic DNA and cloned into pUC19. The URA3 geneblaster fragment [E. Alani, L. Cao, N. Kleckner, Genetics 116, 541 (1987)] or the LEU2 gene was then cloned between the 5′ and 3′ PCR products, generating plasmids pPM658 and pPM668, respectively. These plasmids, when digested with the restriction enzyme Sph I and transformed into yeast, delete nucleotides from +171 to +4106 of the 4341-bp SGS1 open reading frame. To generate the srs2Δ-generating ptasmid, a 5.8-kb PCR product encompassing the entire SRS2 gene was amplified from yeast genomic DNA and cloned into pUC19. The internal 3.4-kb Pst I-Cla I fragment of SRS2 was then replaced with the URA3 geneblaster fragment, generating pPM690. This plasmid, when digested with the restriction enzymes Sph I and Sal I, releases a linear DNA fragment that, when transformed into yeast, deletes nucleotides from +90 to +3515 of the 3520-bp SRS2 gene. The generation of deletions was confirmed by PCR analysis of genomic DNA. All mutant yeast strains used here were derived from EMY74.7 (MATa his3-Δ1 leu2-3 leu2-112 trp1Δ ura3-52).
20. - transformants were then analyzed for temperature-sensitive growth. One of the isolates lacked the ability to grow at 39°C and was analyzed further. The entire sgs1-ts mutant gene from the plasmid conferring the ts phenotype was cloned into another plasmid that had not been treated with hydroxylamine and was shown to confer the ts phenotype in the srs2Δ sgs1Δ strain. The sgs1-ts mutant gene in plasmid pPM980 was then sequenced with the Thermo Sequenace kit (Amersham Pharmacia Biotech).
21. H. Qiu, E. Park, L. Prakash, S. Prakash, Genes Dev. 7, 2161 (1993); S. N. Guzder, P. Sung, V. Bailly, L. Prakash, S. Prakash, Nature 367, 91 (1994).
22. H. Qiu, E. Park, L. Prakash, S. Prakash, Genes Dev. 7, 2161 (1993); S. N. Guzder, P. Sung, V. Bailly, L. Prakash, S. Prakash, Nature 367, 91 (1994).
23. The incorporation of radioactivity into DNA and RNA in continuous labeling experiments, pulse-labeling of total RNA, electrophoresis and fluorographic detection of rRNA, and Northern blot analyses to examine the transcription of constitutive and inducible mRNAs and tRNAs were done as described in (12), except that total cellular RNA was isolated with the hot phenol method as described in Current Protocols in Molecular Biology, Wiley, New York, 1993), vol. 2, unit 13.12.
24. S.-K. Lee, R. E. Johnson, S.-L. Yu, L. Prakash, S. Prakash, unpublished data.
25. H. J. Himmelfarb, E. M. Simpson, J. D. Friesen, Mol. Cell. Biol. 7, 2155 (1987).
26. C. Mann et al., Mol. Cell. Biol. 12, 4314 (1992).
27. The amounts of TRP3, RAD23, and CDC9 mRNAs also showed no decline in the srs2Δ sgs1-ts strain at 39°C.
28. G. Knapp, J. S. Beckmann, P. F. Johnson, S. A. Fuhrman, J. Abelson, Cell 14, 221 (1978); B. P. Cormack and K. Struhl, Cell 69, 685 (1992).
29. G. Knapp, J. S. Beckmann, P. F. Johnson, S. A. Fuhrman, J. Abelson, Cell 14, 221 (1978); B. P. Cormack and K. Struhl, Cell 69, 685 (1992).
30. 1 under the microscope, α factor was washed away and the cells were resuspended in fresh YPD medium. The cultures were then split (time O). Half of the culture was incubated at 25°C and the other half at 39°C. DNA content was determined as described [R. Nash, G. Tokiwa, S. Anand, K. Erickson, A. B. Futcher, EMBO J. 7, 4335 (1988)]. Samples were taken at the indicated time points and fixed overnight at 4°C in 1 ml of 70% ethanol. The fixed cells were washed twice, resuspended in 1 ml of 50 mM sodium citrate (pH 7.0), and then treated with ribonuclease A (0.25 mg/ml) for 3 hours at 50°C. Cells were then washed and resuspended in 1 ml of 50 mM sodium citrate (pH 7.0), propidium iodide (16 μg/ml) was added and cells were incubated overnight at 4°C. The DNA content of cells was analyzed by a Becton-Dickinson FACS-can flow cytometer.
31. 3H]uracil. The cultures were then split and placed at 25° or at 39°C, and DNA synthesis was measured by the incorporation of radioactivity into DNA. Upon transfer to 39°C, DNA synthesis ceased rapidly in the srs2Δ sgs1-ts strain but was not affected in the srs2Δ SGS1 strain.
32. S. J. Brill, S. DiNardo, K. Voelkel-Meiman, R. Sternglanz, Nature 326, 414 (1987).
33. H. Yan, C.-Y. Chen, R. Kobayashi, J. Newport, Nature Genet. 19, 375 (1998).
34. Supported by NIH grants GM19261 and CA80882. We thank S. J. Brill for the SGS1 gene.