325 Biotechnology Building
Bik Tye is Professor of Biochemistry, Molecular and Cell Biology. She received her A.B. from Wellesley College in 1969; her M.S. from the University of California, San Francisco in 1971 and her Ph.D. from the Massachusetts Institute of Technology in 1974. She was a Helen Hay Whitney Fellow at Stanford University School of Medicine from 1974 to 1977. She came to Cornell University as assistant professor in 1977.
BioMG 3320 - Principles of Biochemistry: Molecular Biology - Spring Semesters
DNA replication occurs only once in each normal mitotic cell cycle. Our laboratory is interested in understanding the molecular mechanisms that impose this strict regulation. We have identified a number of genes, called MCM (for minichromosome maintenance), whose products play critical roles in the initiation as well as the progression of replication forks  . MCM2-MCM7 encode the six subunits of the replicative helicase that melts origin DNA and unwinds replication forks. The MCM helicase is an integral part of the pre-replication complex (pre-RC) at replication origins and a component of the replisome at replication forks [2, 3] . Mcm10 mediates the transition of the pre-RC to the replisome [4, 5] by interacting with the origin recognition complex (ORC), Cdt1, the Mcm2-Mcm7 helicase  and DNA polymerase a  . It is believed to be part of the replicating fork. In contrast, Mcm1's role in DNA replication has been elusive. Mcm1 has been shown to be a global transcription factor and its role in DNA replication was viewed as indirect. However, recent studies from our lab suggest that Mcm1 may play a direct role in regulating origin usage by binding replication origins. Furthermore, Mcm1 interacts physically and genetically with Mcm10 and subunits of the Mcm2-Mcm7 helicase suggesting that it may regulate origin usage by cooperating with components of the pre-RC. To elucidate the functions of each of these MCM proteins, we would like to focus our efforts in the context of the following questions.
1. What is the mechanism that regulates origin usage in eukaryotes?
The rate of DNA replication is dependent on origin usage. This correlation has been observed in prokaryotes and during development in metazoans. Recently, our laboratory demonstrated that Mcm1 plays a direct role in the initiation of DNA synthesis at replication origins. We showed that Mcm1 binds to all replication origins tested  but the affinity of Mcm1 for origins varies  . When Mcm1 activity is limiting in the cell, the affinity of replication origins for Mcm1 correlates with the efficiency of their usage  . Intracellular Mcm1 activity has been shown to be responsive to stress, cell growth and nutrient availability  . We will test the hypothesis that genomic replication origins conform to a hierarchy of usage based on their affinity for Mcm1. According to this hypothesis, under adverse conditions when Mcm1 activity is limiting, only selective replication origins are activated. Thus, Mcm1 may regulate the length of S phase, frequency of cell division and the rate of cell proliferation by regulating the number of origins activated and the relevant array of genes expressed. We will investigate the mechanism by which Mcm1 regulates origin usage by examining the effect of Mcm1 binding on the recruitment and activation of the pre-RC.
2. What is the structure of the hexameric MCM2-7 helicase?
The MCM2-7 helicase consists of six highly conserved but nonidentical subunits. Although intact hexameric complexes can be readily isolated from a number of eukaryotes, none demonstrates helicase activity  . The best evidence that the MCM complex indeed possesses helicase activity comes from studies of the Methanobacterium thermoauthotrophicum MCM helicase. There, a single progenitor MCM protein self assembles into a hexameric ring structure that has robust 3’-5’ helicase activity  . In eukaryotes, when three of the subunits are removed from the hexameric MCM complex, weak helicase activity can be generated from the remaining three subunits (Mcm4, Mcm6 and Mcm7) in the form of a double trimer  (Figure 1). One explanation is that three of the subunits act as regulators of the three catalytic subunits and that removal of the regulatory subunits is an obligatory step in the activation of the eukaryotic MCM helicase. Another explanation is that posttranslation modifications of the regulatory subunits are necessary to activate the hexameric MCM2-7 complex. Removal of these regulatory subunits bypasses that requirement in the test tube. One way to dissect the function of the MCM2-7 helicase is to understand its structure. To date, crystal structure of a hexamer of the N-terminal portion of the archael MCM helicase which does not contain the helicase catalytic domain has been solved  . Solving the crystal structure of the full-length archael monmoeric or hexameric MCM protein would be the next obvious step. Dr. Quan Hao’s group is making an effort towards these goals. Our laboratory will make use of genetic suppressor analysis to identify the nearest neighbors of the MCM2-7 complex in S. cerevisiae. Mutations in the conserved regions that destroy interactions between the subunits will be generated and suppressors will be used to identify the nearest neighbor.
3. What is the process of assembly of the pre-RC at replication origins?
The MCM2-7 proteins are known to assemble at replication origins at G1 phase in an ORC, Cdc6 and Cdt1 dependent manner  (Figure 2). It is also believed to be topologically linked to DNA at the beginning of S phase in the capacity of a replicative helicase. However, physical interactions between the MCM2-7 complex and ORC, Cdc6 or Cdt1 have not been verified. Furthermore, neither subunits nor complexes of the Mcm2-Mcm7 proteins bind DNA. To elucidate the process of assembly of the MCM2-7 complex, we will use UV laser mediated protein-DNA crosslinking to investigate the exact time of contact between the Mcm2-Mcm7 complex and genomic origin DNA in yeast. This work will be carried out in collaboration with Dr. Watt Webb’s group using their ultrafast, tunable laser system that is used to photoinitiate covalent crosslinks between protein and DNA rapidly and with a high effective yield. This work is funded by a NSF collaborative research grant (NSF 0242328).
4. What is the role of Mcm10 in transcriptional silencing and heterochromatin assembly?
The abundance of the MCM proteins suggests that they have functions beyond DNA replication perhaps in transcription and chromatin structure or organization  . Indeed, we have shown that Mcm7 acts as a transcription factor to modulate its own expression and the expression of other early cell cycle genes  . Recently, we showed that Mcm10, as well as most members of the Mcm2-Mcm7 family affects transcription in yet another manner, they are required for the proper silencing of the silent mating type loci and telomeric genes (I. Liachko and N. Douglas, unpublished results). Study of Mcm10 in Drosophila showed that it interacts with the heterochromatin protein 1 (Hp1)  . Similarly, study in S. cerevisiae showed that it interacts with Sir2 and Sir3, two proteins involved in the maintenance of silencing ( N. Douglas , unpublished data). Tethering and genetic analyses indicate that Mcm10 is involved in the maintenance of silencing. We will investigate the role of Mcm10 in heterochromatin assembly and chromatin organization in two different models, S. cerevisiae and Drosophila. For these studies, we have generated a panel of reagents and mutants including null, transgenic and separation of function mutants. Each model offers different and complementary advantages. Approaches taken in the Drosophila study include immunofluoresnce microscopy, fluorescence resonance energy transfer (FRET) analysis, position effect variegation (PEV) analysis and two-hybrid analysis. Approaches taken in the yeast study includes chromatin immunoprecipitation, microarray and suppressor analysis.
1. Tye, B.K., MCM proteins in DNA replication. Annu Rev Biochem, 1999. 68: p. 649-686.
2. Ishimi, Y., A DNA helicase activity is associated with an MCM4, -6 and -7 protein complex. J. Biol Chem, 1997. 272: p. 24508-24513.
3. Lee, J.K. and Hurwitz, J., Isolation and characterization of various complexes of the minichromosome maintenance proteins of Schizosaccharomyces pombe. J Biol Chem, 2000. 275: p. 18871-18878.
4. Homesley, L., Lei, M., Kawasaki, Y., Sawyer, S., Christensen, T., and Tye, B.K., Mcm10 and the MCM2-7 complex interact to initiate DNA synthesis and to release replication factors from origins. Genes Dev, 2000. 14: p. 913-926.
5. Sawyer, S.L., Cheng, I.H., Chai, W., and Tye, B.K., Mcm10 and Cdc45 Cooperate in Origin Activation in Saccharomyces cerevisiae. J Mol Biol, 2004. 340: p. 195-202.
6. Christensen, T.W. and Tye, B.K.,Drosophila Mcm10 Interacts with Members of the Pre-Replication Complex and is Required for Proper Chromosome Condensation. Mol Biol Cell, 2003. 14: p. 2206-2215.
7. Fien, K., Cho, Y.S., Lee, J.K., Raychaudhuri, S., Tappin, I., and Hurwitz, J., Primer utilization by DNA polymerase alpha -primase is influenced by its interaction with Mcm10p. 2004.
8. Chang, V.K., Fitch, M.J., Donato, J.J., Christensen, T.W., Merchant, A.M., and Tye, B.-K., Mcm1 binds replication origins. J Biol Chem, 2003. 278: p. 6093-6100.
9. Chang, V.K., Donato, J.J., Chan, C.S.M., and Tye, B.K., Mcm1 promotes replication initiation by binding specific elements at replication origins. Mol Cell Biol, 2004. 24: p. 6514-6524.
10. Chen, Y. and Tye, B.K., The yeast MCM1 protein is regulated posttranscriptionally by the flux of glycolysis. Mol Cell Biol, 1995. 15: p. 4631-4639.
11. Tye, B.K. and Sawyer, S., The hexameric eukaryotic MCM helicase: building symmetry from nonidentical parts. J Biol Chem, 2000. 275: p. 34833-34836.
12. Kelman, Z., Lee, J.-K., and Hurwitz, J., The single minichromosome maintenance protein of Methanobacterium thermoautotrophicum DH contains DNA helicase activity. Proc. Natl. Acad. Sci. USA, 1999. 96: p. 14783-14788.
13. Fletcher, R.J., Bishop, B.E., Leon, R.P., Sclafani, R.A., Ogata, C.M., and Chen, X.S., The structure and function of MCM from Archaeal M. Thermoautotrophicum. Nat Struct Biol, 2003. 10: p. 160-167.
14. Lei, M. and Tye, B.K.,Initiating DNA synthesis: from recruiting to activating the MCM complex. J Cell Sci, 2001. 114: p. 1447-1454.
15. Fitch, M.J., Donato, J.J., and Tye, B.K., Mcm7, a subunit of the presumptive MCM helicase, modulates its own expression in conjunction with Mcm1. J Biol Chem, 2003. 278: p. 25408-25416.
Fig. 1. A, sequence conservation among S. cerevisiae MCM proteins. Black bars represent regions conserved between S. cerevisiae MCMs and the single MCM protein of Methanobacterium thermoautotrophicum, and colored bars represent regions conserved between yeast and mammalian MCMs of the same class. The largest conserved domain contains a nucleotide binding motif. B, a model for the assembly of an active hexameric MCM helicase via two different pathways. Both pathways involve a conformational change from an unspecified structure to a planar hexameric ring structure during the transition from an inactive complex to an active helicase. In vivo activation of the MCM helicase requires chemical modifications of the double trimer, including phosphorylation of Mcm2 by Cdc7-Dbf4. Conformational changes resulting from chemical modifications give rise to an active ring-shaped helicase containing all six MCM proteins. In vitro assembly of an active MCM helicase requires the removal of Mcm2, Mcm3, and Mcm5 from the inactive complex. Dimerization and fusion of two Mcm4, -6, and -7 trimers give rise to a hexameric ring structure that is catalytically active.
Fig. 2. Initiating DNA synthesis, from recruitment to activation of the MCM complex – a schematic representation. (a) Assembly of the pre-RC begins during the G1 phase when Cdc6 and Cdt1 are recruited to replication origins where ORC and Mcm10 bind. (b) Cdc6 and Cdt1 facilitate the loading of the MCM complex. Anchoring of the MCM complex is mediated through interaction between individual subunits of the MCM complex and multimers of the Mcm10 protein. Cdc6 is removed from origins once the MCM complex is recruited. The Cdc7-Dbf4 kinase is also recruited to the origin during G1 phase. (c) Phosphorylation of the MCM complex by Cdc7-Dbf4 occurs during S phase and is controlled locally at individual origins. Phosphorylation of the MCM complex is coupled to a conformation change of the complex that results in the melting of origin DNA. ORC and Mcm10 are hidden from view. (d) This conformational change is believed to convert the inactive MCM complex into the enzymatically active helicase whose ring shaped structure becomes topologically linked to DNA (see Figure 1). Recruitment of Cdc45 requires both the phosphorylation of the MCM complex and the activity of CDKs. Disassociation of the MCM helicase by Cdc45 from the Mcm10 anchor initiates the melting of DNA and recruits RPA, DNA polymerase ?, and primase to the origins for the initiation of DNA synthesis. (e) The melting of the dsDNA induces a conformational change in ORC before replication origins assume a post-replication chromatin state. Like the ORC, Mcm10 is believed to remain bound to origins throughout the cell cycle.
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Liu, Z., Frantz, D., Gilbert, W. and Tye, B.K. (1993) Identification and characterization of a nuclease activity specific for G4 tetrastranded DNA. Proc. Natl. Acad. Sci. 90, 3157-3161.
Yan, H., Merchant, A.M., and Tye, B.K. (1993) Cell cycle-regulated nuclear localization of MCM2 and MCM3 which are required for the initiation of DNA synthesis at chromosomal replication origin in yeast. Genes & Develop. 7, 2149-2160.
Chen, Y. and Tye, B. (1995) The yeast MCM1 protein is regulated post-transcriptionally by the flux of glycolysis. Mol. Cell. Biol. 15, 4631-4639.
Lei, M., Kawasaki, Y. and Tye, B. (1996) MCM proteins, their physical interactions and dosage effects on DNA replication in yeast. Mol. Cell Biol. 16, 5081-5090.
Merchant, A.M., Kawasaki, Y., Chen, Y., Lei, M. and Tye, B.K. (1997) A lesion in the DNA replication initiation factor Mcm10 induces pausing of elongation forks through chromosomal replication origins in S. cerevisiae. Mol. Cell. Biol. 17: 3261-3271.
Young, M. R. and Tye, B.K. (1997) Mcm2 and Mcm3 are constitutive nuclear proteins that exhibit distinct isoforms and bind chromatin during specific cell cycle stages of S. cerevisiae. Mol. Biol. Cell. 8:1587-1601.
Young, M.R., Suzuki, K., Yan, H., Gibson, S. and Tye, B. K. (1997) Nuclear accumulation of S. cerevisiae Mcm3 is dependent on its nuclear localization sequence. Genes Cells. 2: 631-643.
Lei, M., Kawasaki, Y., Young, M., Kihara, K., Sugino, A. and Tye, B. K. (1997) Mcm2 is a target of regulation by Cdc7-Dbf4 during the initiation of DNA synthesis in S. cerevisiae. Genes Dev. 11: 3365-3374.
Wu, C., Weiss, K., Yang, C., Harris, M., Tye, B.-K., Newlon, C., Simpson, R., Haber, J. (1998) Mcm1 regulates the recombination enhancer controlling donor preference in Saccharomyces mating-type switching. Genes & Dev. 12: 1726-1737.
Homesley, L., Lei, M., Kawasaki, Y., Sawyer, S., Christensen, T. and Tye, B.K. (2000) Mcm10 and the MCM2-7 complex interact to initiate DNA synthesis and to release replication factors from origins. Genes Dev. 14: 913-926.
Tye, B.-K. (1994) The MCM2-3-5 protein family, are they replication licensing factors? Trends in Cell Biology. 4, 160-166.
Tye, B.K. (1998) Minichromosome maintenance as a genetic assay for defects in DNA replication. Methods, A Companion to Methods Enzymol. Ed. Paul Fisher. Academic Press. New York.
Tye, B.K. (1999) MCM proteins in DNA replication. Ann. Rev. Biochem. Vol. 68: 649-686 Editor C.C. Richardson Publisher,.Ann Rev. Inc. Palo Alto, California
Tye, B.K. (2000) Insights into DNA replication from the third domain of life. Proc. Natl. Acad. Sci. 97: 2399-2401.
Tye, B.K. and Sawyer, S.L. (2000) The Hexameric Eukaryotic MCM Helicase: Building Symmetry from Nonidentical Parts. J Biol Chem. 275: 34833-34836.