Thymine-thymine dimer bypass by yeast DNA polymerase ζ

Science

Volumn 272, Issue 5268, 1996, Pages 1646-1649

  Nelson, John R ^a   Lawrence, Christopher W ^a   Hinkle, David C ^b  

^a UNIVERSITY OF ROCHESTER SCHOOL OF MEDICINE AND DENTISTRY   (United States)

^b UNIVERSITY OF ROCHESTER   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CYCLOBUTANE; DIMER; DNA DIRECTED DNA POLYMERASE ALPHA; DNA POLYMERASE; THYMINE;

ARTICLE; CONTROLLED STUDY; DNA DAMAGE; DNA REPLICATION; FUNGAL GENETICS; MUTAGENESIS; NONHUMAN; PRIORITY JOURNAL; SACCHAROMYCES CEREVISIAE; YEAST;

EID: 0029952294     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.272.5268.1646     Document Type: Article

References (26)

1. For a review, see E. C. Friedberg, G. C. Walker, W. Siede, DNA Repair and Mutagenesis (ASM Press, Washington, DC, 1995).
2. F. W. Larimer, J. R. Perry, A. A. Hardigree, J. Bacteriol. 171, 230 (1989).
3. A. Morrison et al., ibid., p. 5659.
4. L. E. Torpey, P. E. M. Gibbs, J. R. Nelson, C. W. Lawrence, Yeast 10, 1503 (1994).
5. S. Fields and O.-K. Song, Nature 340, 245 (1989).
6. The two-hybrid pairwise test for protein interactions and the library screen for interacting proteins were carried out as described [S. M. Hollenberg, R. Sternglanz, P. F. Cheng, H. Weintraub, Mol. Cell. Biol. 15, 3813 (1995)].
7. J. R. Nelson, C. W. Lawrence, D. C. Hinkle, data not shown.
8. Proofreading 3′ to 5′ exonuclease activity was assayed as described in Fig. 2, except that dNTPs were omitted and the 5′-labeled primer contained a single 3′-terminal nucleotide mismatch. No shortening of the primer was detected (< 1 fmol) with up to 90 ng of Pol ζ or 3 ng of Pol α, nor was shortening observed with several other substrates (including two additional primers with mismatched ends, completely complementary primers, and single-stranded primers in the absence of template). In contrast, incubation of 200 fmol of one of these mismatched substrates with 0.1, 1, and 10 ng of Pol I Klenow fragment (which contains a 3′ to 5′ exonuclease) respectively shortened 2.5%, 25%, and 89% of the primers by 1 to 2 nt.
9. S. D. Rabkin, P. D. Moore, B. S. Strauss, Proc. Natl. Acad. Sci. U.S.A. 80, 1541 (1983); G. L. Chan, P. W. Doetsch, W. A. Haseltine, Biochemistry 24, 5723 (1985); K. L. Larson and B. S. Strauss, ibid. 26, 2471 (1987).
10. S. D. Rabkin, P. D. Moore, B. S. Strauss, Proc. Natl. Acad. Sci. U.S.A. 80, 1541 (1983); G. L. Chan, P. W. Doetsch, W. A. Haseltine, Biochemistry 24, 5723 (1985); K. L. Larson and B. S. Strauss, ibid. 26, 2471 (1987).
11. S. D. Rabkin, P. D. Moore, B. S. Strauss, Proc. Natl. Acad. Sci. U.S.A. 80, 1541 (1983); G. L. Chan, P. W. Doetsch, W. A. Haseltine, Biochemistry 24, 5723 (1985); K. L. Larson and B. S. Strauss, ibid. 26, 2471 (1987).
12. M. Rajagopalan et al., Proc. Natl. Acad. Sci. U.S.A. 89, 10777 (1992).
13. J.-S. Taylor and C. L. O'Day, Biochemistry 29, 1624 (1990).
14. C. L. O'Day, P. M. J. Burgers, J.-S. Taylor, Nucleic Acids Res. 20, 5403 (1992).
15. J. R. Nelson, C. W. Lawrence, D. C. Hinkle, in preparation.
16. At each time and with both enzymes, 55 to 60% of the products were 16- to 18-nt oligomers, ∼25% of the products were 19- to 32-nt oligomers, and 15 to 20% of the products were 33- to 200-nt oligomers.
17. DNA polymerase activity was determined as described in Fig. 2, except that the concentration of the competing nucleotide [deoxycytidine triphosphate (dCTP) for aphidicolin, deoxyguanosine triphosphate (dGTP) for butylphenyl-dGTP, deoxythymidine triphosphate (dTTP) for ddTTP, and dCTP for ddCTP) was lowered to 10 μM. Activity values are relative to activity without inhibitor.
18. K. Shimizu et al., J. Biol. Chem. 268, 27148 (1993).
19. P. Hovland, J. Flick, M. Johnston, R. A. Sclafani, Gene 83, 57 (1989).
20. The plasmid pGST was created by moving a 1330-base pair (bp) Xba I-Sal I fragment containing the GAL1 promoter and the gene encoding Gst from pRD56 [H.-O. Park, J. Chant, I. Herskowitz, Nature 365, 269 (1993)] into YEplac195 [R. D. Geitz and A. Sugino, Gene 74, 527 (1988)], which contains the 2μ replication origin and URA3 for selection.
21. The plasmid pGST was created by moving a 1330-base pair (bp) Xba I-Sal I fragment containing the GAL1 promoter and the gene encoding Gst from pRD56 [H.-O. Park, J. Chant, I. Herskowitz, Nature 365, 269 (1993)] into YEplac195 [R. D. Geitz and A. Sugino, Gene 74, 527 (1988)], which contains the 2μ replication origin and URA3 for selection.
22. The plasmid pGST-REV3 was created by ligating a 4515-bp Eco RI-Sal I fragment containing REV3 into pGST.
23. The plasmid pREV7 was constructed by ligating a 430-bp fragment containing the copper metallothionine gene promoter and a 747-bp fragment containing REV7 into YEplac181, which contains the 2μ replication origin and LEU2 for selection.
24. Rabbit polyclonal antibodies were generated against an SDS-PAGE-purified Rev3-TrpE fusion or Rev7p expressed in E. coli. Immunoblots were developed with goat antibody to horseradish peroxidase-conjugated rabbit secondary antibodies (Bio-Rad) and chemiluminescence (DuPont NEN).
25. The concentration of Gst-Rev3p was estimated by scanning a Coomassie blue-stained SDS-polyacrylamide gel and comparing the band intensities to the intensities of known amounts of bovine serum albumin (BSA).
26. We thank R. Bambara, M. Goodman, and R. Woodgate for comments; P. Ingles for initial work on the project; E. DiMuzio, M. Langer, and W. Sun for help with plasmid constructions; and M. Liskay and members of his lab for their help with the yeast two-hybrid screen. Supported by NIH grants GM21858 and GM29686.