Nicholas R. Cozzarelli
Our recent work has been on the partitioning of chromosomes and the unlinking of DNA during replication. We have used prokaryotic and eukaryotic systems to study the requisite DNA-protein interactions in vitro and the physiological roles in vivo. Traditional structural, genetic, and enzymological techniques are complemented with the use of DNA microarrays and single molecule biophysical measures.
The two strands of DNA must be continuously unlinked during replication. The topoisomerases that accomplish this might instead be expected to entangle and knot chromosomes because of the huge DNA concentration in vivo. We have identified several factors that solve this problem and contribute to the orderly unlinking of DNA. A major contributor to chromosome partitioning is the condensation of daughter DNA upon itself soon after replication. We showed that DNA condensation is due primarily to supercoiling that is introduced by topoisomerases and maintained by Structural Maintenance of Chromosome proteins, often called condensins. We are studying condensins of frogs, worms, yeast, and bacteria in an attempt to understand how these proteins cause the orderly long-range folding of DNA.
Another factor promoting chromosome partitioning is that the type -2 topoisomerases of all organisms do not just speed up the approach to topological equilibrium, but actually change the equilibrium position. They actively remove all DNA entanglements. This requires that topoisomerases sense the global conformation of DNA even though they interact with DNA only locally. We showed that topoisomerases achieve this because they are like Maxwell's demon and by positioning themselves at sharp bends in DNA carry out net disentanglement of DNA.
All the enzymes that play critical roles in DNA unlinking and chromosome segregation, helicases, topoisomerases, and condensins, are motor proteins. They use the energy of ATP hydrolysis to move large pieces of DNA over long distances. We have used single molecule methods to study their action. We can easily detect a single catalytic event by a lone topoisomerase and are extending these studies to helicases and condensins. Single molecule measures of topoisomerases indicate that traditional bulk enzymatic assays underestimate the activity of topoisomerases by orders of magnitude. This is because dead enzymes and pauses contribute to bulk rates and because the DNA substrate is in a relatively inactive form in solution.
An equal partner to the topoisomerases in chromosome segregation is the helicases. We are exploring how helicases convert the energy of ATP hydrolysis into unwinding DNA. We have shown that the helicases encoded by SV40 and bovine papilloma virus encircle their substrate DNA and have developed a detailed model of their action.
We have used DNA microarrays to measure simultaneously the replication and transcription of all E. coli genes. We have proven that the E. coli chromosome is, indeed, folded into topological domains. We are now mapping the domain barriers. Our most recent work in this area looks at the timing and localization of replication forks in bacteria and yeast. We found that neighboring replication forks often fire at the same time and are investigating whether this is an expression of clustering in the cell in foci.
Organization of the domains of the SV40 large T-antigen hexamer. [M.S. Van Loock, A. Alexandrov, D. Yu, N.R. Cozzarelli, and E.H. Egelman (2002) Curr. Biol. 12, 472-476]
C. elegans condensin promotes mitotic chromosome architecture, centromere organization, and sister chromatid segregation during mitosis and meiosis II. [K.A. Hagstrom, V.F. Holmes, N.R. Cozzarelli and B. Meyer (2002) Genes Dev. 16, 729-742]
Positive torsional strain causes the formation of a four-way junction at replication forks. [L. Postow, C. Ullsperger, R. W. Keller, C. Bustamante, A. V. Vologodskii, and N. R. Cozzarelli (2001) J. Biol. Chem. 276, 2790-2796]
Mechanism of toplogy simplification by type II DNA topoisomerases. [A. V. Vologodskii, W. Zhang, V. V. Rybenkov, A. A. Podtelezhnikov, D. Subramanian, J. D. Griffith, and N. R. Cozzarelli (2001) Proc. Natl. Acad. Sci. USA 98, 3045-3049]
Topological challenges to DNA replication: Conformations at the fork. [L. Postow, N.J. Crisona, B.J. Peter, C.D. Hardy, and N.R. Cozzarelli (2001) Proc. Natl. Acad. Sci. USA 98, 8219-8226]
Closing the ring: Links between SMC proteins and chromosome partitioning, condensation, and supercoiling. [V. F. Holmes and N. R. Cozzarelli (2000) Proc. Natl. Acad. Sci. USA 97, 1322-1324]
Preferential relaxation of positively supercoiled DNA by Escherichia coli topoisomerase IV in single-molecule and ensemble measurements. [N. J. Crisona, T. R. Strick, D. Bensimon, V. Croquette, and N. R. Cozzarelli. (2000). Genes Dev 14(22):2881-2892]
Analysis of topoisomerase function in bacterial replication fork movement: Use of DNA microarrays. [A.B. Khodursky, B.J. Peter, M.B. Schmid. J. DeRisi, D. Botstein, P.O. Brown, and N.R. Cozzarelli (2000) Proc. Natl. Acad. Sci. USA, 97, 9419-9424]
Last Updated 2003-09-02