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Molecular and Cellular Biology, November 2007, p. 7594-7602, Vol. 27, No. 21
0270-7306/07/$08.00+0     doi:10.1128/MCB.00997-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Structural Changes in Mcm5 Protein Bypass Cdc7-Dbf4 Function and Reduce Replication Origin Efficiency in Saccharomyces cerevisiae{triangledown}

Margaret L. Hoang,1,{dagger} Ronald P. Leon,2 Luis Pessoa-Brandao,2 Sonia Hunt,1,{ddagger} M. K. Raghuraman,1 Walton L. Fangman,1 Bonita J. Brewer,1 and Robert A. Sclafani2*

Department of Genome Sciences, University of Washington, Seattle, Washington,1 Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado2

Received 6 June 2007/ Returned for modification 30 June 2007/ Accepted 15 August 2007

Eukaryotic chromosomal replication is a complicated process with many origins firing at different efficiencies and times during S phase. Prereplication complexes are assembled on all origins in G1 phase, and yet only a subset of complexes is activated during S phase by DDK (for Dbf4-dependent kinase) (Cdc7-Dbf4). The yeast mcm5-bob1 (P83L) mutation bypasses DDK but results in reduced intrinsic firing efficiency at 11 endogenous origins and at origins located on minichromosomes. Origin efficiency may result from Mcm5 protein assuming an altered conformation, as predicted from the atomic structure of an archaeal MCM (for minichromosome maintenance) homologue. Similarly, an intragenic mutation in a residue predicted to interact with P83L suppresses the mcm5-bob1 bypass phenotype. We propose DDK phosphorylation of the MCM complex normally results in a single, highly active conformation of Mcm5, whereas the mcm5-bob1 mutation produces a number of conformations, only one of which is permissive for origin activation. Random adoption of these alternate states by the mcm5-bob1 protein can explain both how origin firing occurs independently of DDK and why origin efficiency is reduced. Because similar mutations in mcm2 and mcm4 cannot bypass DDK, Mcm5 protein may be a unique Mcm protein that is the final target of DDK regulation.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Genetics, MS 8101, PO Box 6511, University of Colorado School of Medicine, Aurora, CO 80045. Phone: (303) 724-3271. Fax: (303) 724-3215. E-mail: robert.sclafani{at}uchsc.edu

{triangledown} Published ahead of print on 27 August 2007.

{dagger} Present address: Department of Embryology, Carnegie Institution, Baltimore, MD 21218.

{ddagger} Present address: Chemical and Biological Sciences Unit, FBI Laboratory, Quantico, VA 22135.

Molecular and Cellular Biology, November 2007, p. 7594-7602, Vol. 27, No. 21
0270-7306/07/$08.00+0     doi:10.1128/MCB.00997-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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