Yeast DNA Polymerases KAREN C. SITNEY? MARTIN E. BUDD, AND JUDITH L. CAMPBELL Divisions of Biology and Chemistry California Institute of Technology Pasadena, California 91125 Eukaryotes contain at least three nuclear DNA polymerases, known as a,E, and 6. DNA polymerase a is a nonprocessive enzyme with a tightly associated primase activity. Polymerases 6 and E are both processive enzymes, with polymerase 6 requiring the association of another protein, proliferating cell nuclear antigen (PCNA), as a processivity factor. Both polymerases 6 and E also possess 3' to 5' exonuclease activities, whereas polymerase a has no associated proofreading activity.'-3 An argument for the involvement of two of these three polymerases in chromosomal replication can be made on the basis of these properties alone. As both DNA polymerases a and 6, along with PCNA, have been shown to be required for SV40 viral origin-directed DNA replication in in vitro reactions containing human cell extracts,"8 polymerase a has long been thought to serve as the lagging strand polymerase with polymerase 6 then replicating the leading strand. However, genetic evidence from the yeast Saccharomyces cerevisiae supports the dependence of replication on these two polymerases, known as polymerases I (a)and I11 (6) in y e a ~ t , ~ -and l ~ has recently implicated polymerase I1 (E) as a third replicative p01ymerase.l~A model incorporating all three polymerases has been proposed in which the DNA polymerase I-primase complex initiates DNA synthesis on both strands and primes and initiates each Okazaki fragment on the lagging strand. Polymerases I1 and I11 are then both called upon for e10ngation.l~This model is appealing in that only polymerases possessing proofreading activity, I1 and 111, carry out extensive DNA synthesis. In order to actually assess the mechanistic roles of each of these three polymerases in eukaryotic DNA replication, we and others have undertaken a systematic study of yeast nuclear DNA polymerases. This has involved purifying each of these enzymes and identifying and cloning the genes encoding each protein?.11-18Temperature-sensitive alleles of POL1 and POL3 are currently available.11-'2*1"21 The ease with which such mutations can be obtained in yeast makes it likely that conditional POL2 alleles will also be available soon and that the individual roles of each DNA polymerase can be addressed. Yeast DNA polymerase I holoenzyme has been immunoaffinity-purified and shown to have the four-subunit structure typical of DNA polymerase a's from higher e u k a r y o t e ~ . ~ The ~ - * ~structural gene for the yeast polymerase catalytic subunit has been cloned and shown to be an essential gene.9.15A set of temperature-sensitive poll mutants was isolated by the plasmid shuffle technique and characterized.l0Sz0 One of these mutants, poll-17, was completely defective in DNA synthesis at the restrictive temperature. This mutant was selected for further study. Homozygous 'Current address: Amgen, Incorporated,Thousand Oaks, California 91320. 52
SITNEY et al.: YEAST DNA POLYMERASES
53
poll-17 diploids did not undergo premeiotic DNA synthesis.I0 DNA polymerase I is then required for both meiotic and mitotic DNA replication, with no other cellular polymerase being able to substitute when polymerase I is lacking. DNA sequence analysis of the yeast POLl gene reveals a high degree of homology to the primary sequence of the human polymerase a gene.2s.26Sequencing of the temperaturesensitive poll alleles (K. Sitney, M. Budd, and J. Campbell, unpublished data) has shown that each of the base changes occurred within a region of homology shared by several viral and eukaryotic DNA polymerases.2s Biochemical analysis of poll-17 extracts revealed two residual DNA polymerase activities, polymerases I1 and 111, which together comprised roughly one-third of the polymerase activity in wild-type cells.11J7Both of these enzymes had very similar biochemical properties, including a 3' to 5' proofreading exonuclease.' 1~16.17Neither activity was thermolabile in poll-I7 extracts, indicating that neither protein was encoded by the POL1 gene. The two polymerases can be distinguished by their template preferences. DNA polymerase I1 is a more processive enzyme than polymerase 111 and thus is better able to utilize substrates such as poly dA-oligo dT with a relatively low primer-to-template ratio.I7 DNA polymerase 111 is more active on substrates such as nuclease-activated DNA.I6 Addition of the processivity factor PCNA from either yeast or mammalian sources to purified polymerase 111 enables this enzyme to carry out processive s y n t h e ~ i s . ~ ~ ~ ~ ~ We have shown DNA polymerase I11 to be encoded by the CDC2 gene.11J2The cdc2 mutant was first isolated as part of a collection of temperature-sensitive mutants blocked in various stages of the cell cycle.IYLikepoll-17, cdc2 mutant cells arrest at S phase with a large bud, a phenotype common to replication mutants. Whereas cdc2 mutants were able to replicate fully 70% of their DNA at the restrictive temperat ~ r epermeabilized , ~ ~ cdc2 mutant cells were completely defective in the elongation of preformed replication forks at 37 0C.30Cloning and sequencing of the CDC2 gene suggested that it might encode a DNA polymerase as it too shared extensive sequence homology with the active site regions of other DNA polymerases.lz~zsIn fact, DNA polymerase 111 activity was absent from extracts prepared from cdc2 mutant cells, indicating that CDC2 encoded DNA polymerase III."J2 Again, DNA polymerase I1 activity was not thermolabile in cdc2 extracts. FIGURE 1 shows chromatograms of both wild-type (panel A) and cdc2-1 mutant (panel B) cell extracts. Polymerase 111 activity is clearly absent from the mutant extract. The 3' to 5' exonuclease activity was also deficient in the cdc2-1 mutant extracts (data not shown), indicating that both activities are contained in the same polypeptide.'IJ2 The last detectable DNA polymerase activity, polymerase 11, was purified to near homogeneity from poll-l7 mutant cells.I7 Polymerase I1 comprises roughly 10% of the yeast polymerase activity when assayed with activated DNA as substrate." FIGURE 2 shows residual polymerase activities in poll-17 extracts assayed on both activated DNA (panel A) and poly dAm-oligo dTlo (panel B). FIGURE2C shows polymerase I1 activity in apoll-l7cdc2-I double mutant strain. Two forms of DNA polymerase I1 can be purified from both wild-type and poll-1 7 ~ t r a i n s . ~The ~J~ two forms are biochemically indistinguishable except for subunit composition and chromatographic behavior on hydroxylapatite (FIGURE3). Both have 3' to 5' exonuclease activity, are resistant to the nucleotide analogue Nz-( p,n-butylphenyl)-deoxyguanosine 5' triphosphate, and have identical substrate
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chromatographed in parallel on DEAE silica HPLC. The identities of the three DNA polymerases were established by chromatographic behavior and drug resistance profiles.
FIGURE 1. DEAE silica chromatography of wild-type and cdc2-1 extracts. Extracts from isogenic wild-type (A) and cdc2-1 (B) cells were
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FIGURE 2. Residual polymerase activities inpoll-17 mutant cells. An extract frompoll-17 cells was chromatographed on DEAE cellulose and assayed using both activated DNA (A) and poly d & H l i g o dTlo (B). A similar extract from apoZ1-17 cdc2-1 double mutant strain was chromatographed on DEAE cellulose and assayed with activated DNA (C). Note that the scale is different in panel C.
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ANNALS NEW YORK ACADEMY OF SCIENCES
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~pecificities.1~ Both forms of polymerase I1 are immunoprecipitated by antibody prepared from “peak 2” DNA polymerase 11, suggesting that they are derived from the same protein (K. Sitney, M. Budd, and J. Campbell, unpublished data). DNA polymerase I1 is relatively unstable when purified. Two factors that stimulated DNA polymerase I1 activity, designated stimulatory factor I (SF I) and SF 11, were identified during the course of polymerase I1 purification and facilitated assaying DNA polymerase I1 activity in the final stages of p~rification.’~ SF I has been purified and shown to comprise three subunits, the largest of which (66 kDa) is a single-stranded DNA binding protein.31 It is specific for DNA polymerase I1 and increases the activity of this enzyme on poly dA-oligo dT, but not on activated DNA. 500
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FRACTION NUMBER FIGURE 3. Hydroxylapatite chromatography of yeast DNA polymerase 11. An extract from poll-1 7 mutant cells was subjected to chromatography on phosphocellulose, DEAE cellulose, heptyCSepharose, and heparin agarose. Active fractions containing polymerase I1 were then fractionated on hydroxylapatite. Polymerase I1 activity resolved into two peaks, designated “peak 1” (early fractions) and “peak 2” (later fractions).
Its mechanism of interaction with DNA polymerase I1 is as yet unknown, but its cloning and characterization may be of use in determining the cellular functions of DNA polymerase 11. The gene encoding DNA polymerase I1 (POL2) has recently been cloned and sequenced.13 The nucleotide sequence predicts a protein of a molecular weight of 256 kDa, although it has not yet been possible to identify an active polymerase I1 species of M,greater than 200 kDa during conventional purifi~ation.*~J~ Disruption of the chromosomal POL2 gene is lethal to the cell, indicating that POL2 encodes an essential function and suggesting that DNA polymerase I1 is required for replication.13
SITNEY el al.: YEAST DNA POLYMERASES
51
Perhaps the most striking result obtained from these biochemical studies of yeast
DNA replication proteins is the degree of conservation between the replication machineries of yeast and higher eukaryotes. Mammalian cells contain functional homologues to each of these three yeast polymerases. One of the three mammalian polymerases (a)has been cloned and sequenced and shows considerable sequence homology to the yeast POL1 gene.25,26It is likely, based on the similar sizes and subunit structures of the yeast and mammalian enzymes, that (i) yeast polymerase I1 and human polymerase E and (ii) yeast D N A polymerase I11 and human polymerase 6 will show a similar degree of interspecies homology. Because functional homologues for all of the replication proteins shown to be essential for DNA replication in the SV40 in vitro system, with the exception of an origin-specific initiation protein, have been identified and purified from yeast, it appears that results from the yeast system can be extended directly to human D N A replication. It is thus probable that three DNA polymerases are required for mammalian chromosomal replication. It has recently been demonstrated that polymerase a is indispensable for initiation of DNA synthesis in the SV40 in vitro replication system, but that various polymerase holoenzymes, including E. coli polymerase I11 and bacteriophage T4 polymerase, could extend the primed chains.32 As both polymerase E (11) and polymerase 6 (HI) have proofreading exonuclease activities, whereas polymerase a (I) does not, the first two enzymes presumably do the bulk of DNA synthesis with polymerase a carrying out all initiation. Support for this model is expected to be provided by studies with yeast mutants defective in each of the enzymes.
REFERENCES
1. 2. 3. 4.
5.
10. 11. 12. 13. 14. 15. 16. 17.
SYVAOJA, J. & S. LINN.1989. J. Biol. Chem. 264: 2489-2497. BURGERS, P. M. J. 1989. Prog. Nucleic Acid Res. Mol. Biol. 37: 235-280. STILLMAN, B. 1989. Annu. Rev. Cell Biol. 5: 197-245. MURAKAMI, Y., T. EKI,M-A. YAMADA, C. PRivEs & J. HURWITZ. 1986. Proc. Natl. Acad. Sci. U.S.A. 83: 6347-6351. TAN.C. K., C. CASTILLO, A. G . So & K. M. DOWNEY. 1986. J. Biol. Chem. 261: 1 2 3 1 c 12316. M. B. MATHEWS, A. G. SO, K. M. DOWNEY & B. PRELICH, G., C-K. TAN, M. KOSTURA, STILLMAN. 1987. Nature 326 517-520. PRELICH, G., M. KOSTURA, D. R. MARSHAK, M. B. MATHEWS & B. STILLMAN. 1987. Nature 3 2 6 471-475. PRELICH, G. & B. STILLMAN. 1988. Cell 53: 117-126. JOHNSON, L. M., M. SNYDER, L. M. S. CHANG, R. W. DAVIS & J. L. CAMPBELL. 1985. Cell 43: 369-377. BUDD,M. E., K. D. WI'ITRUP,J. E. BAILEY & J. L. CAMPBELL. 1989. Mol. Cell. Biol. 9 365-376. SITNEY, K. C., M. E. BUDD& J. L. CAMPBELL. 1989. Cell 56: 599-605. BOULET,A., M. SIMON,G. FAYE,G. A. BAUER& P. M. J. BURGERS.1989. EMBO J. 8 1849-1854. MORRISON, A., H. ARAKI,A. B. CLARK,R. K. HAMATAKE & A. SUGINO.1990. Cell 62: 1143-1151. CHANG,L. M. S. 1977.J. Biol. Chem. 252: 1873-1880. LUCCHINI, G . , A. BRANDAZZA, G . BADARACCO, M. BIANCHI & P. PLEVANI. 1985. Curr. Genet. 10 245-252. 1988. J. Biol. Chem. 263: 917-924. BAUER,G . A., H. M. HELLER& P. M. J. BURGERS. & J. L. CAMPBELL. 1989. J. Biol. Chem. 264: 6557-6565. BUDD,M. E., K. C. SITNEY
58
ANNALS NEW YORK ACADEMY OF SCIENCES
18. HAMATAKE, R. K., H. HASEGAWA, A. B. CLARK, K. BEBENEK, T. KUNKEL & A. SUGINO. 1990. J. Biol. Chem. 265: 4072-4083. 19. HARTWELL, L. H. 1967. J. Bacteriol. 93: 1662-1670. 1987. Proc. Natl. Acad. Sci. U.S.A. 8 4 2838-2842. 20. BUDD,M. E. & J. L. CAMPBELL. 21. LUCCHINI, G., C. MAZZA,E. SCACHERE & P. PLEVANI. 1988. Mol. Gen. Genet. 212: 459465. C. AUGL& L. M. S. CHANG. 1984. J. Biol. Chem. 2 5 9 753222. PLEVANI, P., G. BADAKACCO, 7539. 23. WANG,T. S-F., S-Z. HU& D. KORN.1984. J. Biol. Chem. 259: 1854-1865. R. C. CONAWAY, G. R. BANKS & 1. R. LEHMAN. 1983. J . 24. KAGUNI, L. S., J-M. ROSSIGNOL, Biol. Chem. 2 5 8 9037-9039. 25. WONG,S. W.,A. F. WAHL,P-M.YuAN,N.ARAI,B. E. PEARSON, K. ARAI,D. KORN,M. W. HUNKAPILLER & T. S-F. WANG.1988. EMBO J. 7: 37-47. 26. PIZZAGALI, A., P. VALSASNINI, P. PLEVANI & G. LUCCHINI. 1988. Proc. Natl. Acad. Sci. U.S.A. 85: 3772-3776. 27. BAUER, G. A. & P. M. J. BURGERS. 1988. Proc. Natl. Acad. Sci. U.S.A. 85: 7506-7510. 28. BURGERS, P. M. J. 1988. Nucleic Acids Res. 1 6 6297-6307. 29. CONRAD, M. W. & C. S. NEWLON.1983. Mol. Cell. Biol. 3: 1000-1012. 1983. Proc. Natl. Acad. Sci. U.S.A. 30. Kuo, C. L., N. H. HUANG& J. L. CAMPBELL. 8 0 6465-6469. & J . L. CAMPBELL. 1989. Proc. Natl. Acad. Sci. U.S.A. 31. BROWN, W. C., J. K. SMILEY 87: 677-681. T. EKI& J. HURWITZ. 1990. Proc. Natl. Acad. Sci. U.S.A. 87: 9712-9716. 32. MATSUMOTO,T.,
SITNEY et al.: YEAST DNA POLYMERASES
53
poll-17 diploids did not undergo premeiotic DNA synthesis.I0 DNA polymerase I is then required for both meiotic and mitotic DNA replication, with no other cellular polymerase being able to substitute when polymerase I is lacking. DNA sequence analysis of the yeast POLl gene reveals a high degree of homology to the primary sequence of the human polymerase a gene.2s.26Sequencing of the temperaturesensitive poll alleles (K. Sitney, M. Budd, and J. Campbell, unpublished data) has shown that each of the base changes occurred within a region of homology shared by several viral and eukaryotic DNA polymerases.2s Biochemical analysis of poll-17 extracts revealed two residual DNA polymerase activities, polymerases I1 and 111, which together comprised roughly one-third of the polymerase activity in wild-type cells.11J7Both of these enzymes had very similar biochemical properties, including a 3' to 5' proofreading exonuclease.' 1~16.17Neither activity was thermolabile in poll-I7 extracts, indicating that neither protein was encoded by the POL1 gene. The two polymerases can be distinguished by their template preferences. DNA polymerase I1 is a more processive enzyme than polymerase 111 and thus is better able to utilize substrates such as poly dA-oligo dT with a relatively low primer-to-template ratio.I7 DNA polymerase 111 is more active on substrates such as nuclease-activated DNA.I6 Addition of the processivity factor PCNA from either yeast or mammalian sources to purified polymerase 111 enables this enzyme to carry out processive s y n t h e ~ i s . ~ ~ ~ ~ ~ We have shown DNA polymerase I11 to be encoded by the CDC2 gene.11J2The cdc2 mutant was first isolated as part of a collection of temperature-sensitive mutants blocked in various stages of the cell cycle.IYLikepoll-17, cdc2 mutant cells arrest at S phase with a large bud, a phenotype common to replication mutants. Whereas cdc2 mutants were able to replicate fully 70% of their DNA at the restrictive temperat ~ r epermeabilized , ~ ~ cdc2 mutant cells were completely defective in the elongation of preformed replication forks at 37 0C.30Cloning and sequencing of the CDC2 gene suggested that it might encode a DNA polymerase as it too shared extensive sequence homology with the active site regions of other DNA polymerases.lz~zsIn fact, DNA polymerase 111 activity was absent from extracts prepared from cdc2 mutant cells, indicating that CDC2 encoded DNA polymerase III."J2 Again, DNA polymerase I1 activity was not thermolabile in cdc2 extracts. FIGURE 1 shows chromatograms of both wild-type (panel A) and cdc2-1 mutant (panel B) cell extracts. Polymerase 111 activity is clearly absent from the mutant extract. The 3' to 5' exonuclease activity was also deficient in the cdc2-1 mutant extracts (data not shown), indicating that both activities are contained in the same polypeptide.'IJ2 The last detectable DNA polymerase activity, polymerase 11, was purified to near homogeneity from poll-l7 mutant cells.I7 Polymerase I1 comprises roughly 10% of the yeast polymerase activity when assayed with activated DNA as substrate." FIGURE 2 shows residual polymerase activities in poll-17 extracts assayed on both activated DNA (panel A) and poly dAm-oligo dTlo (panel B). FIGURE2C shows polymerase I1 activity in apoll-l7cdc2-I double mutant strain. Two forms of DNA polymerase I1 can be purified from both wild-type and poll-1 7 ~ t r a i n s . ~The ~J~ two forms are biochemically indistinguishable except for subunit composition and chromatographic behavior on hydroxylapatite (FIGURE3). Both have 3' to 5' exonuclease activity, are resistant to the nucleotide analogue Nz-( p,n-butylphenyl)-deoxyguanosine 5' triphosphate, and have identical substrate
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chromatographed in parallel on DEAE silica HPLC. The identities of the three DNA polymerases were established by chromatographic behavior and drug resistance profiles.
FIGURE 1. DEAE silica chromatography of wild-type and cdc2-1 extracts. Extracts from isogenic wild-type (A) and cdc2-1 (B) cells were
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FIGURE 2. Residual polymerase activities inpoll-17 mutant cells. An extract frompoll-17 cells was chromatographed on DEAE cellulose and assayed using both activated DNA (A) and poly d & H l i g o dTlo (B). A similar extract from apoZ1-17 cdc2-1 double mutant strain was chromatographed on DEAE cellulose and assayed with activated DNA (C). Note that the scale is different in panel C.
10
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ANNALS NEW YORK ACADEMY OF SCIENCES
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~pecificities.1~ Both forms of polymerase I1 are immunoprecipitated by antibody prepared from “peak 2” DNA polymerase 11, suggesting that they are derived from the same protein (K. Sitney, M. Budd, and J. Campbell, unpublished data). DNA polymerase I1 is relatively unstable when purified. Two factors that stimulated DNA polymerase I1 activity, designated stimulatory factor I (SF I) and SF 11, were identified during the course of polymerase I1 purification and facilitated assaying DNA polymerase I1 activity in the final stages of p~rification.’~ SF I has been purified and shown to comprise three subunits, the largest of which (66 kDa) is a single-stranded DNA binding protein.31 It is specific for DNA polymerase I1 and increases the activity of this enzyme on poly dA-oligo dT, but not on activated DNA. 500
400
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100 0 0
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FRACTION NUMBER FIGURE 3. Hydroxylapatite chromatography of yeast DNA polymerase 11. An extract from poll-1 7 mutant cells was subjected to chromatography on phosphocellulose, DEAE cellulose, heptyCSepharose, and heparin agarose. Active fractions containing polymerase I1 were then fractionated on hydroxylapatite. Polymerase I1 activity resolved into two peaks, designated “peak 1” (early fractions) and “peak 2” (later fractions).
Its mechanism of interaction with DNA polymerase I1 is as yet unknown, but its cloning and characterization may be of use in determining the cellular functions of DNA polymerase 11. The gene encoding DNA polymerase I1 (POL2) has recently been cloned and sequenced.13 The nucleotide sequence predicts a protein of a molecular weight of 256 kDa, although it has not yet been possible to identify an active polymerase I1 species of M,greater than 200 kDa during conventional purifi~ation.*~J~ Disruption of the chromosomal POL2 gene is lethal to the cell, indicating that POL2 encodes an essential function and suggesting that DNA polymerase I1 is required for replication.13
SITNEY el al.: YEAST DNA POLYMERASES
51
Perhaps the most striking result obtained from these biochemical studies of yeast
DNA replication proteins is the degree of conservation between the replication machineries of yeast and higher eukaryotes. Mammalian cells contain functional homologues to each of these three yeast polymerases. One of the three mammalian polymerases (a)has been cloned and sequenced and shows considerable sequence homology to the yeast POL1 gene.25,26It is likely, based on the similar sizes and subunit structures of the yeast and mammalian enzymes, that (i) yeast polymerase I1 and human polymerase E and (ii) yeast D N A polymerase I11 and human polymerase 6 will show a similar degree of interspecies homology. Because functional homologues for all of the replication proteins shown to be essential for DNA replication in the SV40 in vitro system, with the exception of an origin-specific initiation protein, have been identified and purified from yeast, it appears that results from the yeast system can be extended directly to human D N A replication. It is thus probable that three DNA polymerases are required for mammalian chromosomal replication. It has recently been demonstrated that polymerase a is indispensable for initiation of DNA synthesis in the SV40 in vitro replication system, but that various polymerase holoenzymes, including E. coli polymerase I11 and bacteriophage T4 polymerase, could extend the primed chains.32 As both polymerase E (11) and polymerase 6 (HI) have proofreading exonuclease activities, whereas polymerase a (I) does not, the first two enzymes presumably do the bulk of DNA synthesis with polymerase a carrying out all initiation. Support for this model is expected to be provided by studies with yeast mutants defective in each of the enzymes.
REFERENCES
1. 2. 3. 4.
5.
10. 11. 12. 13. 14. 15. 16. 17.
SYVAOJA, J. & S. LINN.1989. J. Biol. Chem. 264: 2489-2497. BURGERS, P. M. J. 1989. Prog. Nucleic Acid Res. Mol. Biol. 37: 235-280. STILLMAN, B. 1989. Annu. Rev. Cell Biol. 5: 197-245. MURAKAMI, Y., T. EKI,M-A. YAMADA, C. PRivEs & J. HURWITZ. 1986. Proc. Natl. Acad. Sci. U.S.A. 83: 6347-6351. TAN.C. K., C. CASTILLO, A. G . So & K. M. DOWNEY. 1986. J. Biol. Chem. 261: 1 2 3 1 c 12316. M. B. MATHEWS, A. G. SO, K. M. DOWNEY & B. PRELICH, G., C-K. TAN, M. KOSTURA, STILLMAN. 1987. Nature 326 517-520. PRELICH, G., M. KOSTURA, D. R. MARSHAK, M. B. MATHEWS & B. STILLMAN. 1987. Nature 3 2 6 471-475. PRELICH, G. & B. STILLMAN. 1988. Cell 53: 117-126. JOHNSON, L. M., M. SNYDER, L. M. S. CHANG, R. W. DAVIS & J. L. CAMPBELL. 1985. Cell 43: 369-377. BUDD,M. E., K. D. WI'ITRUP,J. E. BAILEY & J. L. CAMPBELL. 1989. Mol. Cell. Biol. 9 365-376. SITNEY, K. C., M. E. BUDD& J. L. CAMPBELL. 1989. Cell 56: 599-605. BOULET,A., M. SIMON,G. FAYE,G. A. BAUER& P. M. J. BURGERS.1989. EMBO J. 8 1849-1854. MORRISON, A., H. ARAKI,A. B. CLARK,R. K. HAMATAKE & A. SUGINO.1990. Cell 62: 1143-1151. CHANG,L. M. S. 1977.J. Biol. Chem. 252: 1873-1880. LUCCHINI, G . , A. BRANDAZZA, G . BADARACCO, M. BIANCHI & P. PLEVANI. 1985. Curr. Genet. 10 245-252. 1988. J. Biol. Chem. 263: 917-924. BAUER,G . A., H. M. HELLER& P. M. J. BURGERS. & J. L. CAMPBELL. 1989. J. Biol. Chem. 264: 6557-6565. BUDD,M. E., K. C. SITNEY
58
ANNALS NEW YORK ACADEMY OF SCIENCES
18. HAMATAKE, R. K., H. HASEGAWA, A. B. CLARK, K. BEBENEK, T. KUNKEL & A. SUGINO. 1990. J. Biol. Chem. 265: 4072-4083. 19. HARTWELL, L. H. 1967. J. Bacteriol. 93: 1662-1670. 1987. Proc. Natl. Acad. Sci. U.S.A. 8 4 2838-2842. 20. BUDD,M. E. & J. L. CAMPBELL. 21. LUCCHINI, G., C. MAZZA,E. SCACHERE & P. PLEVANI. 1988. Mol. Gen. Genet. 212: 459465. C. AUGL& L. M. S. CHANG. 1984. J. Biol. Chem. 2 5 9 753222. PLEVANI, P., G. BADAKACCO, 7539. 23. WANG,T. S-F., S-Z. HU& D. KORN.1984. J. Biol. Chem. 259: 1854-1865. R. C. CONAWAY, G. R. BANKS & 1. R. LEHMAN. 1983. J . 24. KAGUNI, L. S., J-M. ROSSIGNOL, Biol. Chem. 2 5 8 9037-9039. 25. WONG,S. W.,A. F. WAHL,P-M.YuAN,N.ARAI,B. E. PEARSON, K. ARAI,D. KORN,M. W. HUNKAPILLER & T. S-F. WANG.1988. EMBO J. 7: 37-47. 26. PIZZAGALI, A., P. VALSASNINI, P. PLEVANI & G. LUCCHINI. 1988. Proc. Natl. Acad. Sci. U.S.A. 85: 3772-3776. 27. BAUER, G. A. & P. M. J. BURGERS. 1988. Proc. Natl. Acad. Sci. U.S.A. 85: 7506-7510. 28. BURGERS, P. M. J. 1988. Nucleic Acids Res. 1 6 6297-6307. 29. CONRAD, M. W. & C. S. NEWLON.1983. Mol. Cell. Biol. 3: 1000-1012. 1983. Proc. Natl. Acad. Sci. U.S.A. 30. Kuo, C. L., N. H. HUANG& J. L. CAMPBELL. 8 0 6465-6469. & J . L. CAMPBELL. 1989. Proc. Natl. Acad. Sci. U.S.A. 31. BROWN, W. C., J. K. SMILEY 87: 677-681. T. EKI& J. HURWITZ. 1990. Proc. Natl. Acad. Sci. U.S.A. 87: 9712-9716. 32. MATSUMOTO,T.,
