Studies of the molecular mechanisms of DNA replication

Analysis of single molecules by transmission electron microscopy (EM) provides a powerful means of studying the mechanics of DNA replication. Input from EM is as important to understanding large scale structural questions as obtaining Xray structures is to showing how polymerases catalyze DNA synthesis. While a detailed understanding of the mechanics of replication has been built up through a combination of biochemical and structural approaches, many key questions remain unanswered. Two in particular are how the architecture of the DNA strands at the fork facilitates the coupling of leading and lagging strand replication, and how origins are activated in the cell. The recent purification and characterization of the proteins which catalyze replication in a variety of prokaryotic and eukaryotic systems makes it possible to reconstruct the key steps in vitro using defined DNA templates and proteins. This allows us to study intermediates in these reactions at the single molecule level using EM. Our approach is to examine the structure of a statistically large number of replicating molecules and then to combine this information with data about the average population derived from biochemical assays. No single system will provide all of the structural answers, and it will be critical to compare results across a number of systems to determine which features are fundamental to all. We have established a highly interactive program with laboratories expert in specific replication systems including the laboratories of Johannes Walter (Harvard), Tom Broker and Louise Chow (Univ. of Alabama), Antoine van Oijen (Netherlands), Charles Richardson (Harvard) and Sandra Weller (Univ. of Conn.).

Specific areas being investigated:

1. Architecture of origin complex in yeast. Using several high resolution EM techniques, the structure of the yeast Origin Recognition Complex (ORC) is being examined both alone and bound to yeast ARS DNA elements.  Opening of the origin by the action of the mcm2-7 helicase is being examined by EM.

2. Origin opening and activation in HPV. The nature of eukaryotic origin recognition and opening is being probed using the human papilloma virus as a model. The ability of the viral E1 protein to unwind the origin in a reaction facilitated by E2 and host chaperone proteins is being examined using a combination of EM and biochemical tools. The role of host DNA polymerase alpha and cellular cyclin E and cdk2 kinase will be probed. ()Experiments will be initiated using cell extracts and SV40 and HPV origin based plasmids to obtain basic information about the folding of the DNA strands at a moving eukaryotic replication fork.

3. Architecture of the moving replication fork in the T7 and T4 systems. This long standing effort to understand the basic looping of the lagging strand at a moving replication fork will continue to be probed using the simple and well characterized T7 and T4 replication systems. We use EM to focus on the question of how Okazaki fragment size is controlled and the role of the structural ‘spools’ created by the binding of single stranded DNA on the lagging strand by the T7 SSB. Using the more complex T4 replication system we will continue our efforts to establish the generality of the looping of the lagging strand as a means of coupling leading and lagging strand synthesis. The nature of the loop and proteins required for its formation will be examined. Recent work has combined EM with the single molecule methods pioneered by Antoine van Oijen making this dual approach particularly powerful.

Selected References: (see also recent papers, 2009-2011 under publications).

Selected References: (see also recent papers, 2009-2011 under publications).

  • Kyusung Park, Zeger Debyser, Stanley Tabor, Charles C. Richardson, and Jack D. Griffith. Formation of a DNA Loop at the Replication Fork Generated by Bacteriophage T7 Replication Proteins. J. Biol. Chem. 273: 5260-5270. 1998.
  • Joonsee Lee, Paul Chastain II, Takahiro Kusakabe, Jack D. Griffith, and Charles C. Richardson. Coordinated Leading and lagging strand DNA synthesis on a mini-circular template. Molecular Cell. 1: 1001-1010. 1998.
  • Daniel G. Lee, Alexander M. Makhov, Richard D. Klemm, Jack D.Griffith, and Stephen P. Bell. Regulation of ORC conformation and ATPase Activity: differential effects of single-stranded and double-stranded DNA binding. EMBO J. 19: 1-9, 2000.
  • Biing Yuan Lin, Alexander M. Makhov, Jack D. Griffith, Thomas R. Broker and Louise T. Chow. Chaperone Proteins Abrogate the Inhibition of the Human Papillomavirus E1 Replicative Helicase by the HPV E2 Protein. Molecular Cell. Biol. 22: 6592-6604. 2002.
  • Paul D. Chastain I, Alexander M. Makhov, Nancy G. Nossal, and Jack D. Griffith. Analysis of the Okazaki Fragment Distributions along Single Long DNAs Replicated by the Bacteriophage T4 Proteins. Molecular Cell. 6: 803-814, 2000.
  • Paul D. Chastain, Alexander M. Makhov, Nancy G. Nossal, and Jack Griffith. Architecture of the replication complex and DNA loops at the fork generated by the bacteriophage T4 proteins. J. Biol. Chem. 278:21276-21285, 2003.
  • Nancy G. Nossal, Alexander M. Makhov, Paul D. Chastain, II, Charles E. Jones, and Jack D. Griffith. Architecture of the bacteriophage T4 replication complex revealed with nanoscale biopointers. J. Biol. Chem. 282: 1098-1108, 2007.
  • Concerted loading of Mcm2-7 double hexamers around DNA during DNA replication origin licensing. Remus D, Beuron F, Tolun G, Griffith JD, Morris EP, Diffley JF. Cell. 2009 Nov 13;139(4):719-30.  PMID: 19896182
  • E. coli DNA replication in the absence of free β clamps. Tanner NA, Tolun G, Loparo JJ, Jergic S, Griffith JD, Dixon NE, van Oijen AM.  EMBO J.  2011 30(9):1830-40. PMID: 21441898