The chromosome replication cycle
My lab uses a model cell-free system derived from Xenopus eggs to study how genetic information is faithfully transmitted from one cell generation to the next. We are interested in the following questions:
1. How is DNA replication coordinated with the cell cycle?
DNA replication is restricted to a single round per cell cycle because Cdt1, a key replication initiation protein, is destroyed after the first round of DNA replication. We have discovered that Cdt1 destruction is strictly dependent on the first round of DNA replication, and that it occurs via an unusual mechanism (Arias and Walter, 2005; Arias and Walter, 2006; Jin et al., 2006): Cdt1 and the E3 ubiquitin ligase, Cul4-Ddb1Cdt2, dock onto the replication fork via the replication factor PCNA, leading to Cdt1 ubiquitylation and destruction. Our current goal is to understand how PCNA recruits Cdt1 and its ubiquitin ligase, since this will teach us important lessons about the spatial and temporal regulation of proteolysis.
2. What is the mechanism of the replicative DNA helicase?
Our data indicate that a complex of MCM2-7, Cdc45, and GINS comprises the DNA helicase, which unwinds DNA at the replication fork (Pacek and Walter, 2004; Pacek et al., 2006). We are using biochemical, ultrastructural (EM), and ChIP approaches to determine how it is possible that this helicase can processively unwind up to 200 kb of DNA.
3. How is sister chromosome cohesion established?
Cohesin is a ring-shaped protein complex that holds sister chromatids together, perhaps by forming a molecular “hoola-hoop” that encircles sisters. We discovered that the binding of cohesins to chromosomes is intimately linked to the initiation of DNA replication (Takahashi et al., 2004). We are using this observation to elucidate the mechanism by which cohesion between sisters is established.
4. How are inter-strand DNA cross-links repaired?
Inter-strand DNA cross-links are highly toxic DNA lesions because they block passage of RNA and DNA polymerases. We have found that addition of inter-strand DNA cross-links to Xenopus egg extracts activates the Fanconi anemia (FA) repair pathway, which comprises the "first responder" to these DNA lesions in cells. Interestingly, activation of the FA pathway requires DNA replication, suggesting an intimate interplay between replication and repair. We are taking advantage of this system to understand the molecular mechanism of inter-strand DNA cross-link repair.
Joukov, V., Groen, A.C., Prokhorova, T., Gerson, R., White, E., Rodriguez, A., Walter, J.C., and Livingston, D.M. (2006). The BRCA1/BARD1 Heterodimer Modulates Ran-Dependent Mitotic Spindle Assembly. Cell 127, 539-552. [PDF]
Arias, E.E and Walter J.C. (2007). Strength in numbers: preventing re-replication via multiple mechanisms in eukaryotic cells. Genes&Development 21, 497-518. [PDF]
Jin, J., Arias, E.E., Chen, J., Harper, J.W., and Walter, J.C. (2006). A family of diverse Cul4-Ddb1 interacting proteins includes Cdt2, which is required for S phase destruction of the replication factor Cdt1. Molecular Cell 23, 709-721. [PDF]
Pacek, M., Tutter, A.V., Kubota, Y., Takisawa, H., and Walter, J.C. (2006). Localization of MCM2-7, Cdc45, and GINS to the site of DNA unwinding during eukaryotic DNA replication. Molecular Cell 21, 581-587. [PDF]
Arias, E.E. and Walter, J.C. (2006). PCNA functions as a molecular platform to trigger Cdt1 destruction and prevent re-replication. Nature Cell Biology 8, 84-90. [PDF]
Takahashi, T.S., Wigley, D., and Walter, J.C. (2005). Pumps, paradoxes, and ploughshares: Mechanism of the MCM2-7 helicase. TIBS 30, 437-44. [PDF]
Takahashi, T.S. and Walter, J.C. (2005). Cdc7-Drf1 is a developmentally regulated protein kinase required for the initiation of vertebrate DNA replication. Genes & Development, 19, 2295-2300. [PDF]
Byun, T.S., Pacek, M., Yee, M., Walter, J.C., and Cimprich, K.A. (2005). Functional Uncoupling of MCM Helicase and DNA Polymerase Activities Activates the ATR-Dependent Checkpoint. Genes & Development 19, 1040-52. [PDF]
Arias, E. and Walter, J.C. (2005). Replication-dependent destruction of Cdt1 limits DNA replication to a single round per cell cycle in Xenopus egg extracts. Genes & Development 19, 114. [PDF]
Takahashi, T.S., Yiu, P., Chou, M.F., Gygi, S. and Walter, J.C. (2004). Pre-replication complex-dependent recruitment of Xenopus Scc2 and cohesin to chromatin. Nature Cell Biology 6, 991. [PDF]
Ramachandran, N., Hainsworth, E., Bhullar, B., Eisenstein, Rosen, B.S., Lau, A.Y, Walter, J.C., LaBaer, J. (2004). Self-assembling protein microarrays. Science 305, 86. [PDF]
Pacek, M. and Walter, J.C. (2004). A requirement for MCM7 and Cdc45 in chromosome unwinding during eukaryotic DNA replication. EMBO J. 23, 3667. [PDF]
Prokhorova, T. A., Mowrer, K, Gilbert, C.H. and Walter, J.C. (2003). DNA replication of mitotic chromatin in Xenopus egg extracts. PNAS 100, 13241.[PDF]
Vashee, S., Cvetic, C., Lu, W., Simancek, P., Kelly, T.J., and Walter, J.C. (2003). Sequence-independent DNA binding and replication initiation by the human origin recognition complex. Genes & Development 17, 1894.[PDF]
Edwards, M.C., Tutter, A.V., Cvetic, C., Gilbert, C.H., Prokhorova, T.A. and Walter, J.C. (2002) MCM Complexes Bind to Chromatin in a Distributed Pattern Surrounding the ORC. J Biol. Chem. 277, 33049-57. [PDF]
Wohlschlegel J.A., Dhar S.K., Prokhorova T.A., Dutta, A. and Walter, J.C. (2002) Xenopus Mcm10 Binds to Origins of DNA Replication after Mcm2-7 and Stimulates Origin-Binding of Cdc45. Molecular Cell 9, 233-240.[PDF]
Wohlschlegel, J.A., Dwyer, B.T., Dhar, S.K., Cvetic, C., Walter, J., and Dutta, A. (2000). Inhibition of Eukaryotic DNA Replication by Geminin Binding to Cdt1. Science 290, 2309-2312. [PDF]
Walter, J.C. (2000). Evidence for sequential action of cdc7 and cdk2 protein kinases during initiation of DNA replication in Xenopus egg extracts. J. Biol. Chem. 275, 39773-39778.[PDF]
Walter, J. and Newport, J. (2000). Initiation of eukaryotic DNA replication: origin unwinding and sequential chromatin-association of cdc45, RPA, and DNA polymerase a. Molecular Cell 5, 617-627. [PDF]
Walter, J., Sun, L., and Newport, J.W. (1998). Regulated chromosomal DNA replication in the absence of a nucleus. Molecular Cell 1, 519-529. [PDF]
Walter, J. and Newport, J.W. (1997). Regulation of Replicon size in Xenopus egg extracts. Science 275, 993-995. [PDF]