Minichromosome Maintenance as a Genetic Assay for Defects in DNA Replication

METHODS: A Companion to Methods in Enzymology 18, 329 –334 (1999) Article ID meth.1999.0793, available online at http://www.idealibrary.com on

Minichromosome Maintenance as a Genetic Assay for Defects in DNA Replication Bik K. Tye Section of Biochemistry Molecular and Cell Biology, Cornell University, Ithaca, New York 14853–2703

Minichromosome maintenance (mcm) is an effective genetic assay for mutants defective in DNA replication. Two classes of mcm mutants have been identified using this screen: those that differentially affect the activities of certain autonomously replicating sequences (ARSs) and those that uniformly affect the activities of all ARSs. The ARS-specific MCM genes are essential for the initiation of DNA replication. Among these are members of the MCM2-7 family that encode subunits of the preinitiation complex and MCM10, whose gene product interacts with members of the Mcm2-7 proteins. Among the ARS-nonspecific MCM gene products are chromosome transmission factors. Refinement of this genetic assay as a screening tool and further analysis of existing mcm mutants may reveal new replication initiation proteins. © 1999 Academic Press

Initiation of DNA synthesis in eukaryotes involves a complex, multistep process that requires the participation of a number of proteins. This complex process can be divided into three steps each involving a different set of proteins. The first step involves the binding of the origin recognition proteins to replication origins (1). In the second step, the origin recognition proteins serve as a scaffold for the assembly of another set of proteins which form the preinitiation complex (2). The decision to initiate is then regulated by a third set of proteins, called cyclin-dependent kinases, or Cdks, which are a series of protein kinases that phosphorylate the preinitiation complex (3– 6). Working in series or in combinations, these protein kinases ensure that DNA replication occurs only once at a specific time of the cell cycle. To dissect this complex, multistep process of replication initiation, it is important to identify the protein factors that are involved in each of these steps. While structural protein components that bind tightly to origin DNA can be identified biochemically, regulatory proteins that interact transiently with rep1046-2023/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

lication origins cannot be easily identified without an in vitro system for replication initiation. Genetic approaches provide powerful and sometimes the only alternatives for the identification of regulatory factors. In this regard, the yeasts, especially Saccharomyces cerevisiae, are the organisms of choice in the study of DNA replication.

GENETIC APPROACHES Before delving into the use of minichromosome maintenance as a genetic assay for mutants defective in replication, it is important to survey other methods along with their advantages and disadvantages. There are a number of strategies often used for the identification of mutants defective in DNA replication. However, none of them is specific for replication initiation per se and therefore most of them require secondary or even tertiary screens that involve combinations of strategies to be effective. For example, cell division cycle or cdc mutants that have arrest phenotypes characterized by cell and spindle morphologies provide, only a general idea of when in the cell cycle a defect may exert its effect (7, 8). To identify the execution point of a defect requires temporal ordering with respect to other known mutations or growth inhibitors by reciprocal blocks (9 –11). To confirm that the defect is in replication requires yet a different set of tests such as nucleotide incorporation (12). Replication initiation proteins that have multiple functions and therefore execution points outside of S phase would be missed if cell division cycle arrest phenotype is used as the primary criterion for replication defects. An example is Cdc14, a putative protein tyrosine phosphatase (13). cdc14 mutants arrest at a late stage of nuclear division (7), yet Cdc14 is required for the initiation of plasmid replication (14). There are other relevant examples. 329

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CDC46 and CDC47, which encode the replication initiation factors Mcm5 and Mcm7, respectively (15–17), were initially identified as genes required for nuclear division because of their medial nuclear division terminal phenotype (18). Similarly, the Schizosaccharomyces pombe genes nda1 1 and nda4 1, which encode Mcm2 and Mcm5, respectively, were identified for their nuclear division arrest phenotype (19). Defects in nucleotide incorporation can be used to screen for replication mutants (12, 20). This strategy, though tedious, is especially effective in prokaryotes, where “fast stop” phenotypes yielded mutants defective in elongation synthesis and “slow stop” phenotypes yielded mutants defective in initiation (21). In this article, I focus on a third strategy based on minichromosome maintenance (mcm). This strategy has been effective in producing replication initiation mutants both in S. cerevisiae (22) and in S. pombe (19). For a more comprehensive reading of the rationale for this strategy, I refer the reader to our original paper on the isolation of mcm mutants (22).

MUTANTS DEFECTIVE IN MINICHROMOSOME MAINTENANCE (mcm) Mutants that are defective in replication initiation are expected to be inviable. Dead mutants, however, are uninformative about functions. On the other hand, hypomorphs that have a reduced efficiency in the initiation of DNA synthesis may have little effect on the replication of the natural chromosomes which contain multiple replication origins. These viable mutants, however, may have dramatic effects on the replication of minichromosomes that contain a centromere and a single replication origin also known as an ARS. Stability of minichromosomes is therefore a sensitive assay for replication initiation defects. The advantage of this strategy is that it is direct and no assumption is made about other phenotypes that may or may not be associated with replication initiation defects. Stability of plasmids was first used as an indicator for DNA replication defects in the isolation of Escherichia coli mutants defective in F-plasmid replication (23). In general, plasmid instability or plasmid loss could be the result of defects either in plasmid replication or in plasmid segregation (24, 25). Such a screen may yield mutants in both categories. To identify replication factors that regulate the activity of replication origins, a secondary screen is designed to identify mutants that show nonidentical effects on individual replication origins. For example, mcm phenotypes that are enhanced by specific ARSs or suppressed by multiple ARSs on a minichromosome are indications that the defect is directed at replication initiation at ARSs. Finally, a tertiary screen is recommended to identify

temperature conditional lethals among the putative replication initiation mutants. The conditional lethal phenotype is crucial for subsequent cloning of these MCM genes.

DESCRIPTION OF METHOD Choice of Minichromosomes A series of minichromosomes (YCps) have been constructed for the characterization of mcm mutants (see Table 1) (22, 26). YCp1, the minichromosome containing ARS1 (27), is recommended for use in the primary screen because it appears to be the most sensitive to mcm mutations among all the ARSs tested (see Table 1). In contrast, YCp121, which contains ARS121 (28), is the least affected by the known mcm mutations and is recommended for use in the secondary screen. Alternatively, pDK243, a minichromosome carrying ARS1, and pDK368-7, which has seven copies of ARSH4 (29) inserted into pDK243 (14), may be used in the primary and secondary screens, respectively. Procedure for Identifying mcm Mutants 1. Primary Screen for Minichromosome Instability Stationary cultures of yeast strains (e.g., ura3 or leu2) transformed to prototrophy by minichromosomes carrying ARS1, CEN5, and the appropriate markers are mutagenized with ethyl methanesulfonate (EMS) or other mutagens. Mutagenized cultures are plated to single colonies on complete medium lacking leucine or uracil at room temperature without prior regenerative growth. All colonies that grow at room temperature (;23°C) on selective medium should contain minichromosomes. To identify clones that are unable to stably maintain the minichromosomes, colonies growing on selective medium at 23°C are replica plated onto complete or YEPD medium also at 23°C. Residual plasmids in the mutant clones are diluted by repeating this replica plating procedure before a final replica plating on complete and selective medium. Colonies that are Ura 2 (or Leu 2) are examined further. The ideal mutants are hypomorphs that are also conditionally lethal. The serial replica plating at 23°C allows the identification of hypomorphs that may be conditionally lethal at the more extreme temperatures. Although a color sectoring assay has been very effective for the identification of chromosome transmission mutants (30, 31), we do not recommend using this assay for minichromosome instability of mutants defective in replication. Because of the high plasmid loss rates typical of these mutants, without selective pressure, clonal populations of plasmid-containing cells cannot be established to give the sectoring morphology.

Mcm PROTEINS AND REPLICATION INITIATION

2. Secondary Screen for ARS-Dependent Minichromosome Maintenance Defect As mentioned above, the instability of minichromosomes in the mutants isolated in the initial screen could be due to defects either in the replication of the minichromosomes or in their segregation into daughter cells. DNA replication is believed to be regulated at the level of replication initiation rather than replication elongation. Thus, mutants that show specificity for particular ARSs are more likely to be altered in replication initiation than in replication elongation or in plasmid segregation. To determine ARS specificity, mutants are cured of their original minichromosomes, e.g., YCp1, and transformed with tester minichromosomes, such as YCp121. Alternatively, minichromosomes containing one or multiple copies of ARSs may be used in the secondary screen (14). Multiple ARSs on minichromosomes presumably increase the probability of replication initiation and, therefore, increase plasmid stability. Stability assays are then performed. Those that show an ARS-specific minichromosome maintenance defect are analyzed further. 3. Tertiary Screen for Temperature-Sensitive Conditional Lethals One of the greatest obstacles in the analysis of many of the mcm mutants is that without other phenotypes, cloning the MCM genes by complementation is technically difficult. So far, almost all of the MCM genes analyzed have additional phenotypes such as mating defects, as in the case of mcm1-1 (32), or conditional lethality, as in the cases of mcm2-1 (26), mcm3-1 (33), and mcm10-1 (34). For this reason, it is important to screen for additional phenotypes such as temperaturesensitive conditional lethality. Putative mcm mutant strains are streaked on YEPD plates at room temperature and then replica plated on YEPD plates at various temperatures. Those that show growth defects at extreme temperatures are good candidates for cloning the respective MCM genes by complementation.

RESULTS Complementation Groups of mcm Mutants To date, our laboratory has conducted three independent screens for mcm mutants. In each of these screens, a different minichromosome [YCp1, YCpTEL131C (22) or YCpH2B (R. Surosky, unpublished results)] was used. Table 1 includes all of the mutants that have resulted from these screens in addition to replication mutants that were initially obtained from other genetic approaches but subsequently shown to have an ARSspecific mcm defect. These include dbf10 (8), cdc46 (17), cdc47, cdc54 (16), and dna43 (20). MCM6 is a homolog of

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the S. pombe mis5 1 gene which was identified for its minichromosome maintenance defect (19). Mutants in complementation groups 1–7 and 10–12 show ARS specificity in minichromosome maintenance. Mutants in complementation groups 13–23 show no specificity for ARSs. Some of the complementation groups include multiple alleles that resulted from independent screens, such as those in complementation groups 12 (seven alleles), 13 (seven alleles), and 14 (five alleles). Others have single alleles, such as complementation groups 1, 2, 3, and 10. The fact that CDC54, CDC47, and MCM6 were not identified in our mcm screens suggests that we are far from saturation in our search for mcm mutants. ARS-Specific Class of MCM Genes The ARS-specific MCM genes are required for the activity of all ARSs although mutations in these genes may exert different effects on different ARSs. This property of the ARS-specific mcm mutants is best illustrated by the mcm2-1 mutation (see Table 2). At room temperature, the activities of ARS1 and the subtelomeric ARS, ARSTEL131, are exquisitely sensitive to the mcm2-1 mutation, whereas the activities of ARS121, ARSH2B, and ARSTEL120 are hardly affected. However, at a slightly higher temperature, 30°C, the activity of every one of these ARSs is affected by the mcm2-1 mutation. Such discriminatory effects are interpreted as diagnostic of initiation factors that regulate the activities of replication origins. Evidence for a direct role in DNA replication initiation has been established for MCM2-7. MCM2-7 is a family of six conserved proteins that are ubiquitous from Archebacterium to human (26, 35–37). The largest conserved region shared by these six proteins encodes a putative ATPase motif characteristic of helicases (38). Despite their structural and functional similarities, these proteins are not redundant in function, but are each essential for viability (16, 26, 33). Five of the six members were identified by their mcm or cdc phenotype. MCM6 was identified as ORF YGL201c in the Stanford Genome Data Base based on homology to the S. pombe mis5 1 (19, 34). These six proteins interact as a complex in the assembly of the preinitiation complex at replication origins (39 – 42). Their roles in DNA replication may include steps in initiation as well as elongation (43, 44). Mcm10, which shows no homology to Mcm2-7, interacts physically (34) and genetically (L. Homesly and B. Tye, unpublished result) with members of the Mcm2-7 family. Not only is it required for the initiation of DNA synthesis, but it may also be required for disassembly of the preinitiation complex (Y. Kawasaki and B. Tye, unpublished results). A functional homolog of Mcm10 has been identified as Cdc23 in S. pombe (H. Tanaka and H. Okayama, personal communication). Mcm1 is a global transcription factor that regulates the expression of diverse genes including DNA replication genes

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(45, 46). Mcm1 belongs to a family of conserved proteins known as the MADS box transcription factors (47– 49) which includes the serum response factor, SRF (50, 51). A direct role for Mcm1 in DNA replication initiation cannot be ruled out. Non-ARS-Specific Class of MCM Genes The non-ARS-specific mcm mutants equally affect the stability of all minichromosomes independent of which ARS they contain. Analysis of this class of mutants has been hampered by the lack of additional phenotypes that facilitate the cloning of their corresponding genes. The few genes that have been identified are nonessential genes involved in chromosome segregation (52). These include MCM17, also identified as CHL4 or CTF17 (53), and MCM18, which is identical to CTF19 (P. Sinha and P. Hieter, personal communication). An exception may be found in mcm15, which shows supersensitivity to adozelesin. Adozelesin is a DNA-damaging antitumor agent that binds noncovalently to the minor groove of DNA and alkylates the N-3 of adenine (54). Low doses of adozelesin specifically inhibit initiation of cellular DNA replication in S. cerevisiae as well as simian virus 40 DNA replication in

virus-infected mammalian cells. So far, mutant strains of S. cerevisiae tested to be sensitive to adozelesin include orc2-1 and orc5-1, which encode subunits of the origin recognition complex, and mcm15-1 (W. Burhans, personal communication). It is not clear if MCM15 is identical to one of the ORC genes.

CONCLUDING REMARKS A Hierarchy of ARSs Revealed by the ARS-Specific mcm Mutations Two interesting observations have been made with respect to the mcm defect of the ARS-specific class of mutants. First, these mutants affect a similar set of ARSs. For example, ARS1 and ARSTEL131 are sensitive to all mcm mutations while ARS121 and ARSTEL120 are generally less sensitive to these mutations (Table 2). The pattern of ARS specificity shared by the mcm mutants suggests that these MCM gene products may be involved in the same pathway for replication initiation or are subunits of a larger complex that participates in replication initiation (40, 55). The recent

TABLE 1 Complementation Groups of mcm Mutants Minichromosome used in primary screen c MCM a

Alias b

1 2 3 4 5 6* 7 10 11 12 13 14 15 16 17 18 19 20 21 22 23

— —

a

DBF10 CDC54 CDC46 (YGL201c) CDC47 DNA43 — PTC2 d (YER089c) — — ADZ Se (YPR046w) f CHL4/CTF17 g CTF19 (YPL018w) f — — (YDR318w) g (YJR315c) f —

YCp1

YCpTEL131C

YCpH2B

— — — — — — —

— — 2B-61 — — — — 2B-145

131C-11, -30, -33 -41, -58, -63 131C-4, -6, -9, -28, -56 131C-5, -23, -38, -55 — 131C-16 131C-25 131C-29 131C-17, -31 131C-10, -12, -20, -34 131C-39, -62 131C-51 131C-22, -52

2B-67 — — — — — — — — — — —

1–9 — — — 1–4, –8, –11 — — — 1–10, –14 — 1–1, –12 1–3 1–16 — — — — — — — —

131C-46

MCM complementation group. Gene names identified independently or ORF in Stanford Genome Data Base. c Alleles identified in three independent screens. d Identification by complementation only (Y. Kawasaki and B. Tye, unpublished results). e Adozelesin sensitive (W. Burhans, personal communication). f P. Sinha, personal communication. g Reference (52). b

Mcm PROTEINS AND REPLICATION INITIATION

finding by several groups that the Mcm2-7 family of proteins indeed interact to form a complex(es) supports the latter hypothesis (39, 40, 56, 57). Indeed, mutants of Abf1, a well-characterized transcription activator that binds some ARSs but is not part of the MCM complex, show dramatic effects on a different subset of ARSs (58). Second, the varied sensitivities of the different ARSs to the mcm2-7 mutants suggest that the yeast genome contains a hierarchy of ARSs that respond differently to the MCM2-7 complexes. ARSs are typically AT-rich sequences that share no sequence homology except for an 11-bp consensus sequence (59). However, functional dissection of several ARSs indicates that sequence elements within ARSs are organized in functionally conserved modules that are interchangeable between ARSs (27, 28, 60). Furthermore, while all ARSs bind ORC, some bind one or more additional cofactors such as Abf1 (27, 28, 61) and Rap1 (62). Thus, replication origins and their protein scaffolds are functionally conserved but structurally nonidentical. The recent finding that the activity of specific ARSs is significantly TABLE 2 Loss Rates of Minichromosomes in ARS-Specific mcm Mutants a YCP Strains Wild type RT 30°C mcm1-1 (RT) mcm2-1 RT 30°C mcm3-1 RT 30°C cdc46-1 RT 30°C mcm10-1 RT 30°C

1

121

H2B

TEL120

TEL131

0.02 0.05

0.02 0.01

,0.01 ,0.01

0.01 ,0.01

0.04 0.03

0.21

0.06

0.14

0.09

0.22

0.40 0.44

0.02 0.21

0.02 0.20

0.05 0.25

0.36 0.46

0.34 0.33

0.18 0.34

0.36 0.46

0.15 0.27

0.35 0.45

0.09 0.21

0.03 0.03

0.03 0.12

0.02 0.07

0.08 0.13

0.04 0.18

0.03 0.06

0.04 0.19

0.03 0.13

0.05 0.18

a Minichromosomes (YCp) containing different ARSs are transformed into yeast strains. Transformants are streaked on a plate containing selective medium (CM-leu or CM-ura) and grown for 3 days at the appropriate temperature. About 10 4 to 10 5 cells are inoculated into 1 ml of YEPD and grown to saturation (8 –10 generations). Aliquots of this culture are plated out on YEPD plates, and single colonies are then replica plated onto selective medium. Plasmid loss rate is determined as 1-(F/I) 1/N, where I is the initial percentage of plasmid-containing cells, and F is the percentage of plasmid-containing cells after N generations (66). ARS1, ARS121, and ARSH2B (67) are single-copy ARSs. ARSTEL120 and ARSTEL131 are subtelomeric ARSs associated with the repeated X sequences (68). Maximum loss rate per cell division is 0.5.

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influenced by the gene dose of Mcm2 and Mcm6 is consistent with the idea that the differential affinity of individual replication origins for the MCM complex may be a determining factor for origin usage (40, 69) (Y. Kawasaki and B. Tye, unpublished results). ARSs are often classified as early or late firing depending on their time of activation during S phase (63). This temporal hierarchy is believed to be partly encoded in the ARSs and partly influenced by chromosome context (64). Whether the Mcm proteins play a role in specifying this temporal hierarchy in ARSs is unclear. However, no correlation is observed between the activity of ARSs in the mcm mutants and their temporal order of activation during S phase. Both ARS1 and ARS121 are replicated early during S phase (65) (S. Hunt and B. Brewer, personal communication) but they respond very differently to the mcm mutations (Table 2). Similarly, subtelomeric ARSs that replicate late in S phase are affected to different extents by the mcm mutations. We believe that through studies of the mcm mutants, we will learn not only about the replication initiation factors that interact at replication origins but also about the replication origins with which they interact. Further studies of the mcm mutants and exploitation of the genetic screen that produced these mutants will advance our understanding of eukaryotic DNA replication.

ACKNOWLEDGMENTS I thank Sara Sawyer, Ming Lei, and Philip Pian for critical reading of the manuscript. This work is supported by grants from the National Institutes of Health (NIH GM34190) and the American Cancer Society (CB-191).

REFERENCES 1. Stillman, B. (1994) J. Biol. Chem. 269, 7047–7050. 2. Diffley, J. F. X. (1996) Genes Dev. 10, 2819 –2830. 3. Dahmann, C., Diffley, J. F. X., and Nasmyth, K. (1995) Curr. Biol. 5, 1257–1269. 4. Young, M., and Tye, B. K. (1997) Mol. Biol. Cell. 8, 1587–1601. 5. Young, M., Suzuki, K., Yan, H., Gibson, S., and Tye, B. K. (1997) Genes Cells 2, 631– 643. 6. Lei, M., Kawasaki, Y., Young, M. R., Kihara, M., Sugino, A., and Tye, B. K. (1997) Genes Dev. 11, 3365–3374. 7. Pringle, J. R., and Hartwell, L. H. (1981) The Molecular Biology of the Yeast Saccharomyces, pp. 79 –142. 8. Johnston, L. H., and Thomas, A. P. M. (1982) Mol. Gen. Genet. 186, 445– 448. 9. Hereford, L., and Hartwell, L. (1974) J. Mol. Biol. 84, 445– 461. 10. Hartwell, L. H. (1976) J. Mol. Biol. 104, 803– 817. 11. Moir, D., and Botstein, D. (1982) Genetics 100, 565–577.

334

BIK K. TYE

12. Nasmyth, K., and Nurse, P. (1981) Mol. Gen. Genet. 182, 119 – 124. 13. Wan, J., Xu, H., and Grunstein, M. (1992) J. Biol. Chem. 267, 11274 –11280. 14. Hogan, E., and Koshland, D. (1992) Proc. Natl. Acad. Sci. USA 89, 3098 –3102. 15. Hennessy, K. M., Clark, C. D., and Botstein, D. (1990) Genes Dev. 4, 2252–2263. 16. Hennessy, K. M., Lee, A., Chen, E., and Botstein, D. (1991) Genes Dev. 5, 958 –969. 17. Chen, Y., Hennessy, K. M., Botstein, D., and Tye, B. K. (1992) Proc. Natl. Acad. Sci. USA 89, 10459 –10463. 18. Moir, D., Stewart, S. E., Osmond, B. C., and Botstein, D. (1982) Genetics 100, 547–564. 19. Takahashi, K., Yamada, H., and Yanagida, M. (1994) Mol. Biol. Cell. 5, 1145–1158. 20. Solomon, N. A., Wright, M. B., Chang, S., Buckley, A. M., Dumas, L. B., and Gaber, R. F. (1992) Yeast 8, 273–289. 21. Kornberg, A. (1980) DNA Replication, W. H. Freeman, San Francisco. 22. Maine, G. T., Sinha, P., and Tye, B.-K. (1984) Genetics 106, 365–385. 23. Jacob, F., Brenner, S., and Cuzin, F. (1963) Cold Spring Harbor Symp. Quant. Biol. 28, 329 –348. 24. Sinha, P., Chang, V., and Tye, B. K. (1986) J. Mol. Biol. 192, 805– 814. 25. Maiti, A. K., and Sinha, P. (1992) J. Mol. Biol. 224, 545–558. 26. Yan, H., Gibson, S., and Tye, B. K. (1991) Genes Dev. 5, 944 –957. 27. Marahrens, Y., and Stillman, B. (1992) Science 255, 817– 822. 28. Walker, S. S., Malik, A. K., and Eisenberg, S. (1991) Nucleic Acids Res. 19, 6255– 6262. 29. Bouton, A. H., and Smith, M. M. (1986) Mol. Cell. Biol. 6, 2354 – 2363. 30. Koshland, D., Kent, J. C., and Hartwell, L. H. (1985) Cell 40, 393– 403. 31. Spencer, F., Gerring, S. L., Connelly, C., and Hieter, P. (1990) Genetics 124, 237–249. 32. Passmore, S., Maine, G. T., Elble, R., Christ, C., and Tye, B. K. (1988) J. Mol. Biol. 204, 593– 606. 33. Gibson, S. I., Surosky, R. T., and Tye, B. K. (1990) Mol. Cell. Biol. 10, 5707–5720. 34. Merchant, A. M., Kawasaki, Y., Chen, Y., Lei, M., and Tye, B. K. (1997) Mol. Cell. Biol. 17, 3261–3271. 35. Bult, C. J., et al. (1996) Science 273, 1058 –1073. 36. Chong, J. P., Thommes, P., and Blow, J. J. (1995) Trends Biochem. Sci. 21, 102–106. 37. Kearsey, S. E., Maiorano, D., Holmes, E. C., and Todorov, I. (1995) Bioessays 18, 183–189. 38. Koonin, E. V. (1993) Nucleic Acids Res. 21, 2541–2547. 39. Chong, J., Mahbubani, H. M., Khoo, C. Y., and Blow, J. J. (1995) Nature 375, 418 – 421. 40. Lei, M., Kawasaki, Y., and Tye, B. K. (1996) Mol. Cell. Biol. 16, 5081–5090. 41. Kubota, Y., Mimura, S., Nishimoto, S., Masuda, T., Nojima, H., and Takisawa, H. (1997) EMBO J. 16, 3320 –3331.

42. Donovan, S., Harwood, J., Drury, L. S., and Diffley, J. F. X. (1997) Proc. Natl. Acad. Sci. USA 94, 5611–5616. 43. Aparicio, O. M., Weinstein, D. M., and Bell, S. P. (1997) Cell 91, 59 – 69. 44. Ishimi, Y. (1997) J. Biol. Chem. 272, 24508 –24513. 45. Passmore, S., Elble, R., and Tye, B. K. (1989) Genes Dev. 3, 921–935. 46. McInerny, C. J., F., P. J., Mikesell, G. E., Greemer, D. P., and Breeden, L. L. (1997) Genes Dev. 11, 1277–1288. 47. Schwarz-Sommer, Z., Hue, I., Huijser, P., Flor, P. J., Hanwen, R., Tetens, F., Lonnig, W.-E., Saedler, H., and Sommer, H. (1992) EMBO J. 11, 251–263. 48. Shore, P., and Sharrocks, A. D. (1995) Eur. J. Biochem. 229, 1–13. 49. Nurrish, S. J., and Treisman, R. (1995) Mol. Cell. Biol. 15, 4076 – 4085. 50. Mueller, C. G. F., and Nordheim, A. (1991) EMBO J. 10, 4219 – 4229. 51. Wynne, J., and Treisman, R. (1992) Nucleic Acids Res. 20, 3297– 3303. 52. Roy, N., Poddar, A., Lohia, A., and Sinha, P. (1997) Curr. Genet. 32, 182–189. 53. Kouprina, N., Tsouladze, A., Koryabin, M., Hieter, P., Spencer, F., and Larionov, V. (1993) Yeast 9, 11–19. 54. Cobuzzi, R. J. J., Burhans, W. C., and Beerman, T. A. (1996) J. Biol. Chem. 271, 19852–19859. 55. Yan, H., Merchant, A. M., and Tye, B.-K. (1993) Genes Dev. 7, 2149 –2160. 56. Musahl, C., Schulte, D., Burkhart, R., and Knippers, R. (1995) Eur. J. Biochem. 230, 1096 –1101. 57. Adachi, Y., Usukura, J., and Yanagida, M. (1997) Genes Cells 2, 467– 479. 58. Rhode, P. R., Elsasser, S., and Campbell, J. L. (1992) Mol. Cell. Biol. 12, 1064 –1077. 59. Broach, J., Li, Y., Feldman, J., Jayaram, M., Abraham, J., Nasmyth, K., and Hicks, J. (1983) Cold Spring Harbor Symp. Quamt. Biol. 47, 1165–1173. 60. Theis, J. F., and Newlon, C. S. (1994) Mol. Cell Biol. 14, 7652–7659. 61. Diffley, J. F. X., and Stillman, B. (1988) Proc. Natl. Acad. Sci. USA 85, 2120 –2124. 62. Buchman, A. R., Kimmerly, W. J., Rine, J., and Kornberg, R. D. (1988) Mol. Cell. Biol. 8, 210 –225. 63. Fangman, W. L., and Brewer, B. J. (1991) Annu. Rev. Cell Biol. 7, 375– 402. 64. Friedman, K., Diller, J. D., Ferguson, B. M., Nyland, S. V. M., Brewer, B. J., and Fangman, W. L. (1996) Genes Dev. 10, 1595– 1607. 65. Fangman, W. L., Hice, R. H., and Chlebowicz, S. E. (1983) Cell 32, 831– 838. 66. Dani, G. M., and Zakian, V. A. (1983) Proc. Natl. Acad. Sci. USA 80, 3406 –3410. 67. Osley, M. A., and Hereford, L. (1982) Proc. Natl. Acad. Sci. USA 79, 7689 –7693. 68. Chan, C. S. M., and Tye, B.-K. (1983) J. Mol. Biol. 168, 505–523. 69. Tye, B. K. (1999) Annu. Rev. Biochem 68, 649 – 689.