Scott Bailey
HHMI Post Doctoral Fellow
Center for Structural Biology
Department
of Molecular Biophysics & Biochemistry
Yale University
266 Whitney Avenue, 413 Bass Center
New Haven, CT 06520-8114
Phone:
(203) 432-5626
FAX: (203) 432-3282
Email: bailey{at}csb.yale.edu
X-ray Crystallographic Studies of the Prokaryotic Primosome
Summary:
DNA replication is a fundamental process in all living organisms. Bacterial, DNA replication is carried out by a multienzyme assembly, the replisome, that moves together with the replication fork and simultaneously replicates the leading and the lagging strands [1]. The replisome is typically viewed as being composed of three functional subassemblies: (1) the leading and lagging strand polymerases, which catalyze the template-directed 5' to 3' synthesis of DNA; (2) accessory proteins that confer exceptional processivity on the DNA polymerase; and (3) the primosome, composed of helicase, primase and in some systems an accessory protein.
The helicase functions to unwind the duplex DNA in the 5’ to 3’ direction with concomitant ATP hydrolysis, providing a single-stranded template for the primase and polymerase [2]. The primase primes DNA synthesis by producing short RNA primers, from which the polymerase can elongate the daughter strands [3]. While previous structures have provided valuable insights into the replicative helicase and primase, a lack of structural information on their complexes with DNA substrate leaves several fundamental questions about their mechanism unanswered. The primary questions of how the helicase unwinds duplex DNA, and how the primase initiates primer synthesis, remain. In addition, as the helicase and primase only function optimally in complex with each other, a detailed understanding of the mechanism of primosome function can only come with structural information on the entire primosome complex: that is helicase, primase, and forked-DNA substrate. The aim of the project is to gain insight into these questions through an X-ray crystallographic analysis of the primosome and its components.
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References: 1. Kornberg, A. and Baker, T. (1992). DNA replication, W.H. Freeman and Company, NY. 2. LeBowitz, J.H. and McMacken, R. (1986). J. Biol. Chem. 261, 4738-4748. 3. Frick, D. N. and Richardson, C. C. (2001). Annu. Rev. Biochem. 70, 39-80 |