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Dr. Megan J. Davey

BSc, York; PhD, Toronto; Postdoc, Rockefeller

Assistant Professor

Dept. of Biochemistry

MSB 358

519-661-2111 x81414

mdavey5@uwo.ca

Positions Available for Graduate and Summer Students

Mechanisms of DNA Replication

My laboratory studies mechanisms of protein function though investigation of the molecular machinery that drives DNA replication. In particular, we investigate the assembly of replication forks at replication origins. Our approach is primarily in vitro using pure proteins. Since we work with the model organism Saccharomyces cerevisiae, the potential exists to expand our studies into genetics and cell biology.

DNA replication is an essential cellular process that helps to maintain the genome.  Failure to completely and accurately duplicate the genome at the appropriate time is catastrophic to the cell, leading to altered cellular states or even cell death.  Thus, it is not surprising that proteins involved in DNA replication have been implicated in cancer and aging as well as other conditions that affect human health.

Our studies focus on the initiation of DNA replication, which is the assembly of replication forks at origins. This process is a highly regulated and coordinated process, and involves the assembly of over forty different polypeptides into structures that are at least 2 MDa in size (about the size of a ribosome) almost simultaneously at approximately 400 origins in the yeast genome (thousands in humans).

Our current studies center on the replicative helicase, which unwinds DNA ahead of DNA polymerase at the replication fork.  The association of replicative helicases with origins is dependent on a helicase loader, which uses the energy of ATP binding to drive the loading of helicase onto DNA. In eukaryotic cells, the replicative helicase is thought to be comprised of minichromosome maintenance proteins (Mcm) 2 through 7.  These six proteins are homologous to each other and form a ring shaped complex (Mcm2-7) that is required for replication initiation and elongation in yeast cells. It's assembly onto origins is dependent on a number of proteins, including Cdc6. Cdc6 is of particular interest because it shares homology to helicase loaders in bacterial cells.

Currently, there are three different projects in my laboratory that examine helicase loading, helicase activation and mechanisms of DNA unwinding by MCM proteins.

Helicase Loading: A key step in the initiation of DNA replication is the loading of the replicative helicase onto origins. One of our aims in these studies is to develop an in vitro assay for helicase loading using pure proteins.  These assays will then be used to probe the mechanisms of helicase loading, such as how does the helicase load? What is the identity of the helicase loader?  The best candidate for the helicase loader is Cdc6, which shares sequence similarity with helicase loaders from bacterial cells. In addition to directly testing this protein's ability to function as the helicase loader in loading assays, we will also examine Cdc6 for other helicase loader activities, such as ATPase, DNA binding and interaction with Mcm2-7. These studies examine the essential DNA replication proteins, Mcm2-7 and Cdc6, for their roles in replication initiation, specifically as the replicative helicase and helicase loader, respectively.

Helicase Activation:  Loading of Mcm2-7 onto DNA does not immediately result in DNA unwinding. There are additional event that must occur. These events include the action of specific protein kinases and association of other proteins with origins.  These observations are consistent with the observation that even though all six MCM proteins are required for DNA replication in vivo, Mcm2-7 does not unwind DNA in vitro. Only a complex of Mcm(4/6/7)2 is able to unwind DNA.  Indeed, Mcm2 and Mcm3/5 inhibit DNA unwinding by Mcm(4/6/7)2. Thus, Mcm2-7 must be activated to unwind DNA, either by structural rearrangement so that Mcm(4/6/7)2 can unwind DNA or by activation of the entire complex to unwind DNA. The aims of this project are to determine the requirements for activation of Mcm2-7 to unwind DNA.  To do so, we will investigate the activity of Mcm2-7 in response to two likely activators of Mcm2-7: the Cdc7/Dbf4 kinase complex and Cdc45, an essential DNA replication protein that interacts with Mcm2-7 and origins just prior to DNA unwinding in vivo.

Mechanism of DNA unwinding by MCM proteins: Replicative helicases in other organisms are comprised of six copies of the same subunit rather than six different subunits as Mcm2-7 is.  One possible explanation for the "extra" subunits is that each subunit has a different role to perform in DNA unwinding.  DNA unwinding is coupled to ATP binding and hydrolysis, therefore we will mutate that ATP binding sites of the MCM subunits to investigate the roles of each subunit in DNA unwinding. With the Mcm4, 6 and 7 subunits we can investigate their roles in DNA unwinding directly.  With Mcm2, 3 and 5 we will determine whether their ATP sites are necessary for the inhibition of DNA unwinding.

Selected References 

1. Ma X, Stead BE, Rezvanpour A, Davey MJ. (2010) The effects of oligomerization on Saccharomyces cerevisiae Mcm4/6/7 function. BMC Biochem. 11(1): 37. [PubMed]
2. Davey MJ. (2010) Towards defining a role for DDK in replication fork stabilization and/or recovery. Cell Cycle. Jun 15;9(12). [PubMed]
3. Stead BE, Sorbara CD, Brandl CJ, Davey MJ (2009) ATP Binding and Hydrolysis by Mcm2 Regulate DNA Binding by Mcm Complexes. J Mol Biol. 391(2): 301-313. [PubMed]
4. Muecke M, Samuels M, Davey M, Jeruzalmi D (2008) Preparation of multimilligram quantities of large, linear DNA molecules for structural studies. Structure 16(6): 837-841. [PubMed]
5. Alan J. Tackett, David J. Dilworth, Megan J. Davey, Michael O’Donnell, John D. Aitchison, Michael P. Rout, Brian T. Chait (2005) Proteomic and genomic characterization of a boundary chromatin complexes. J. Cell Biol. 169: 35-47. [PubMed]
6. Daniel L. Kaplan, Megan J. Davey and Mike O'Donnell (2003) Mcm4/6/7 unwinds DNA by steric exclusion and can actively translocate along a duplex. J. Biol. Chem.278: 49171-49182. [PubMed] 
7. Megan J. Davey and Mike O'Donnell (2003) Replicative helicase loaders: Ring Makers and Ring Breakers Current Biology 13: R594-R596. [PubMed]
8. Megan J. Davey, Chiara Indiani and Mike O'Donnell (2003) Reconstitution of the Mcm2-7p heterohexamer, subunit arrangement and ATP site architecture. J. Biol. Chem. 278: 4491-4499. [PubMed] 
9. Megan J. Davey, David Jeruzalmi, John Kuriyan, and Mike O'Donnell (2002) Motors and Switches: AAA+ machines within the replisome. Nature Rev. Molec. Cell Bio., 3, 826 -835. [PubMed]
10. Megan J. Davey and Mike O'Donnell (2000) Mechanisms of DNA replication. Current Opinion Chem. Bio. 4:581-586. [PubMed]