研究领域
(1) The Mechanism of Homologous Recombination
The long-term objective of this research program is to study the mechanism of human homologous recombination. Currently, we focus on the chromosome remodeling activity of human Rad54 (hRad54) in the human Rad51 (hRad51) mediated homologous recombination, and the interactions of p53 with both human Rad51 and human Rad54 that have been postulated to modulate homologous recombination in response to DNA damage. In addition, we explore possible roles of human Rad51 in replication fork restart. How does human Rad54 protein remodel chromosome during recombination? The hRad54 protein serves as an accessory factor in hRad51 mediated homologous recombination. However, exactly how hRad54 interacts with hRad51 to stimulate homologous recombination is not well understood. We are attempting to address the following questions. (I) Is hRad54 an active component of the nucleoprotein filament formed by hRad51 on ssDNA? (II) If the role of hRad54 in vivo were to induce chromatin remodeling to facilitate homologous recombination, does the remodeling involve repositioning (sliding) of nucleosomes or does it involve conformational changes of nucleosomes? (III) Can hRad54 promote histone octamer transfer? Can hRad54 assist hRad51-ssDNA to promote strand exchange when the target duplex DNA is part of a nucleosomal array? We also ask, can hRad51 catalyze replication fork regression? DNA recombination is considered to be integral to DNA replication and cell survival. Recombination allows replication to successfully bypass the roadblocks created by DNA damage or the collapse of a replication fork. Using synthetic replication forks, we are testing: (I) Can hRad51 catalyze replication fork regression that has been demonstrated in prokaryotic system? (II) Is there any role for hRad54 in this process? Finally, we ask does p53 modulate homologous recombination through its interactions with human Rad51 and human Rad54? p53 suppresses genome instability, particularly in response to DNA damage. p53 has been implicated in homologous recombination, and recently has been shown to interact with hRad51 and hRad54 in vivo. We are testing the hypothesis that p53 contributes to genome stability by transcription-independent modulation of homologous recombination, and a stalled replication fork. Specifically, we try to address: (I) Can p53 inhibit fork regression promoted by hRad51 thus blocking the restart of replication? (II) Can p53 inhibit hRad51 promoted strand exchange? (III) Does hRad54 play any role in the above processes?
(2) Topological problems of kinetoplast DNA replication
Kinetoplast DNA (kDNA) is the mitochondrial (mt) DNA of trypanosomatids and related protozoan parasites. The structure of kDNA is unique in nature, consisting of a network of thousands of topologically interlocked DNA circles, which resembles the chain mail of medieval armor. There are two types of DNA circles found in various kDNA networks, typically a few dozen maxicircles (~ 20-40 kb) and several thousand minicircles about 0.5-3 kb in length. The complex topology makes the replication of the entire network a unique and interesting problem in biology. Studies of the last two decades have revealed that kDNA network replication includes releasing of each minicircle from the network, replication of the minicircles, segregation of the replicated daughter minicircles, and finally reattaching them back to the network. After each of the maxi- and minicircles has been replicated, the network is then divided into two progeny networks. All these topological transformations of kDNA network are catalyzed by DNA topoisomerase(s). In theory, although the topoisomerase can make any topological conversions, there have to be mechanisms that provide for specificity and for directionality of these conversions. What factors direct the unlinking and reattachment of minicircles from the kDNA network during the replication? We attempt to understand the mechanisms that regulating the directionality of these topological conversions. The goal of this project is to investigate above question directly by using a Crithidia fasciculata mitochondrial topoisomerase II (mt Topo II) that we have recently purified. Specifically, we are trying to address the following questions: (I). Does the mt Topo II shift the steady state fraction of catenanes formed in vitro by plasmid DNAs away from thermodynamic equilibrium? What is the direction of the shift? (II). Does the intrinsically bent segments of the minicircles affect the topoisomerization reaction catalyzed by mt Topo II? (III). Do the single-stranded gaps and nicks found in the newly synthesized minicircles affect the topoisomerization reaction catalyzed by mt Topo II? (IV). Can mt Topo II catalyze the reconstruction of the two-dimensional kDNA network with the purified maxicircles and monomer minicircles as the substrates?
(3) Large-Scale Genomic Monitoring of Ecosystems Using a DNA-based Memory
Collaborating with computer scientist, bioengineer, and environmental scientist, we are developing and implementing a new methodology for a genome-enabled, ecological monitoring system.
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J. Chen and D. H. Wood. “A New DNA Separation Technique with a Low Error Rate” DIMACS Series in Discrete Mathematics and Theoretical Computer Science. 48, 47-56. (1999).
J. Chen, E. Antipov, B. Lemieux, W. Cedeno, and D. H. Wood. “ DNA Computing Implementing Genetic Algorithms” in L. Landweber, E. Winfree, R. Lipton, and S. Freeland (eds.) Proceedings of DIMACS Workshop on Evolution as Computation, pp 39-49 (Piscataway, NJ) (1999).
D. H. Wood, E. Antipov, B. Lemieux, W. Cedeno, and J. Chen. “A DNA Implementation of the Max 1s Problem ” in W. Banzhaf, A. Eiben, M. Garzon, V. Honavar, M. Jakiela, and R. E. Smith (eds.) GECCO-99: Proceedings of the Genetic and Evolutionary Computation Conference pp1835-1842 (Morgan Kaufman, San Francisco) (1999).
D. H. Wood, and J. Chen. “ Toward Large Populations for Royal Road Problems via DNA” in P. Angeline, Z. Michalewicz, M Schoenauer, X. Yao, and A. Zalzaza (eds.) 1999 Congress on Evolutionary Computation. (IEEE Computer Society Press, New York) (1999).
J. Chen, E. Antipov, B. Lemieux, W. Cedeno, and D. H. Wood. “In vitro Selection for a Max 1s DNA Genetic Algorithm” D. K. Gifford & E. Winfree (eds.) in press DNA based Computer V (1999).
D. H. Wood. J. Chen, E. Antipov, B. Lemieux, W. Cedeno “ A Design for DNA Computation of OneMax Problem”. Special issue on Biomolecular Approaches to Soft Computing. Soft Computing, to appear (2000).
N. C. Seeman, F. Liu, C. Mao, X. Yang, J. Chen and E. Winfree. “ Two Dimensions and Two states in DNA Nanotechnology”. J. Biomolecular Structure & Dynamics. 11, 253-262. (2000).
J-T. Oh, Y. Cajal, E. M. Skowronska, S. Belkin, J. Chen, T. K. Van Dyk, M. Sasser, M. K. Jain. “ Cationic Peptide Antimicrobials Induce Selective Transcription of micF and osmY in Escherichia coli” Biochimica et Biophysica Acta. 1463, 43-54. (2000).
A. Liechty, J. Chen, and M. K. Jain. “Origin of Antibacterial Stasis by Polymyxin B in Escherichia coli”. Biochimica et Biophysica Acta. 1463, 55-64. (2000).
J. Chen and D. H. Wood. “Computation with Biomolecules”. Proc. Nat. Acad. Sci. USA. 97, 1328-1330. (2000).
N. C. Seeman, C. Mao, F. Liu, R. Sha, X. Yang, L. Wenzler, X. Li, Z. Shen, H. Yan, P. Sa-Ardyen, Y. Pinto, J. Qi, B. Liu, H. Qiu, S. Du, H. Wang, W. Sun, Y. Wang, T-J Fu, Y. Zhang, J. E. Mueller and J. Chen. “Nicks, Nodes, and New Mortifs for DNA Nanotechnology. In L. Pavesi and E. Buzaneva (eds.), Fromtiers of Nano-Optoelectronic Systems, 177-197. (2000) Kluwer Academic Publishers.
E. Goode, D. H. Wood, and J. Chen. “DNA Implementation of a Royal Road Fitness Evolution” in G. Rozenberg & H. J. Hoogeboom (eds.), DNA Based Computers VI accepted (2000).
L. Cai, U. Marquardt, Z. Zhang, M. J. Taisey and J. Chen “Influences of DNA Topology on Synaptic Complex Formation Promoted by RecA Protein of E. coli” Nucleic Acids Research 29, 1389-1398 (2001).
Z. Zhang, D. Yoon, J. R. LaPorte, and J. Chen. “Appropriate Initiation of Strand Exchange Reaction Promoted by RecA Protein Requires ATP Hydrolysis” J. Mol. Biol. 309, 29-43 (2001).
J. Lukes, J. C. Hines, C. J. Evans, N. K. Avliyakulov, V. P. Prabhu, J. Chen and D. S. Ray “Disruption of the Crithidia fasciculata KAP1 gene results in structural rearrangement of the kinetoplast disc” Molecular & Biochemical Parasitology 117, 179-186. (2001).
V. P. Prabhu, A. M. Simons, H. Iwasaki, D. Gai, D. T. Simmons, and J. Chen. “The Effect of p53 on the Rate of Spontaneous Branch Migration and Resolution of the Holliday Junction by RuvC” J. Mol. Biol. 316, 1023-1032 (2002).
R. Deaton, J. Chen, H. Bi, M. Garzon, H. Rubin, and D. H. Wood “A PCR-based Protocol for In Vitro Selection of Non-crosshybridzing Oligonucleotides” in M. Hagiya & A. Ohuchi (eds.), DNA Computing VIII pp. 196-204. (2002).
R. Deaton, J. Chen, H. Bi, and J. A. Rose “ASoftware Tool for Generating Non-crosshybridizing Libraries of DNA Oligonucleotides” in M. Hagiya & A. Ohuchi (eds.), DNA Computing VIII pp. 252-261. (2002).
R. Deaton, J-W, Kim, and J. Chen. “Design and test of noncrosshybridizing oligonucleotide building blocks for DNA computers and nanostructures” Appl. Phys. Lett., 82, 1305-1307 (2003).
D. Yoon, Y. Wang, K. Stapleford, L. Wiesmüller, and J. Chen.” P53 Inhibits Strand Exchange and Replication Fork Regression Promoted by Human Rad51”. J. Mol. Biol. 336, 639–654 (2004).
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