The National Institute of Diabetes & Digestive & Kidney Diseases

The objective of our research is to gain mechanistic insights into the cellular processes that impact the genomic structure and the heritance of the genomic material. We study mechanisms of reactions that impact the stability of the linear organization of the genome, as well as the three-dimensional dynamics involved in the heritance of bacterial chromosomes.

Genome rearrangement by transpositional recombination.
The genomes of all organisms are under the threat of assault by transposable elements. We study the mechanism of DNA transposition of bacteriophage Mu as a model system of a wide family of DNA rearrangement reactions from bacteria to humans. These reactions are involved in many processes that impact our health, from the spread of drug resistance among pathogenic bacteria to replication of retroviruses such as HIV. The immunoglobulin gene rearrangement reaction in vertebrates also takes place by a closely related mechanism. We use biochemical, molecular biological, and biophysical approaches, including single-molecule techniques for our studies. We have made advances in our understanding of the reaction steps and the chemical mechanisms involved in these reactions. Our recent efforts center around the understanding of the assembly and disassembly dynamics of the macromolecular complexes involved in these reactions by developing and utilizing single-molecule biochemical approaches.

Chromosome segregation and cell division in bacteria.
After DNA replication, two daughter copies of the bacterial chromosome and low copy number plasmids must be segregated into two daughter cells to ensure inheritance. Therefore, systems have evolved to actively partition the replicated copies of the genome to two halves of the cell before cell division takes place. One class of such systems involve three components; a specific DNA sequence on the segregating chromosome that functions as the bacterial equivalent of a “centromere”, and two protein factors, one binds to the “centromere” and the other is an ATPase with ATP-dependent non-specific DNA binding activity. E. coli P1-plasmid and F-plasmid are equipped with such systems. We have reconstituted cell-free reaction systems to study these plasmid DNA partition reactions in a flow cell under a microscope. A variety of mechanistic questions are addressed by combining biophysical and biochemical approaches.

After segregation of replicated chromosome and plasmid copies to two halves of the cell, cell division normally must take place at the mid-cell. Thus, we also study the mechanism of mid-cell localization of the cell division septum in E. coli, which is controlled by a set of Min-proteins, with biophysical techniques making use of a reconstituted cell-free reaction system. Progress in these projects is expected to advance our understanding of a variety of biomolecular transportation and patterning reactions in general, and also to provide a foundation for the possible development of novel anti-bacterial agents.

Savilahti, H., Rice, P. A. and Mizuuchi, K. The Phage Mu Transpososome Core: DNA Requirements for Assembly and Function. EMBO J. 14, 4893-4903, 1995.[ Full Text/Abstract ]

Clubb, R. T., Mizuuchi, M., Huth, J. R., Omichinski, J.G., Savilahti, H., Mizuuchi, K., Clore, G. M. and Gronenborn, A. M. The Wing of the Enhancer-binding Domain of Mu Phage Transposase Is Flexible and Is Essential for Efficient Transposition. Proc. Natl. Acad. Sci. USA 93, 1146-1150, 1996.[ Full Text/Abstract ]

Mizuuchi, K. and Baker, T. A. Chemical Mechanisms for Mobilizing DNA. In Craig, N., Craigie, R., Gellert, M., and Lambowitz, A. (Eds.), Mobile DNA II, ASM Press, Washington DC. pp12-23, 2001.

Tan, X., Mizuuchi, M. and Mizuuchi, K. DNA transposition target immunity: Determinants of the MuB distribution patterns on DNA. Proc. Natl. Acad. Sci. USA 104, 13925-13929, 2007.[ Full Text/Abstract ]

Mizuuchi, M., Rice, P. A.,Wardle, S.J., Haniford, D. B. and Mizuuchi, K. Control of transposase activity within a transpososome by the configuration of the flanking DNA segment of the transposon. Proc. Natl. Acad. Sci. USA 104, 14622-14627, 2007.[ Full Text/Abstract ]

Ivanov, V., Li, M. and Mizuuchi, K. Impact of emission anisotropy on fluorescense spectroscopy and FRET distance measurements. Biophys. J. 97, 922-929, 2009.[ Full Text/Abstract ]

Skoko, D., Li, M., Huang, Y., Mizuuchi, M., Cai, M., Bradley, C. M., Pease, P. J., Xiao, B., Marko, J. F., Craigie, R. and Mizuuchi, K. Barrier-to-autointegration factor (BAF) condenses DNA by looping. Proc. Natl. Acad. Sci. USA 106, 16610-16615, 2009.[ Full Text/Abstract ]

Ivanov, V. and Mizuuchi, K. Multiple modes of interconverting dynamic pattern formation by bacterial cell division proteins. Proc. Natl. Acad. Sci. USA 107, 8071-8078, 2010.[ Full Text/Abstract ]

Han, Y.-W. and Mizuuchi, K. Phage Mu transposition immunity: protein pattern formation along DNA by a diffusion-ratchet mechanism. Mol. Cell, 39, 48-58, 2010.[ Full Text/Abstract ]

Vecchiarelli, A. G., Han, Y-W., Tan, X., Mizuuchi, M., Ghirlando, R., Biertümpfel, C., Funnel, B. E. and Mizuuchi, K. ATP control of dynamic P1 ParA-DNA interactions: a key role for the nucleoid in plasmid partition. Mol. Microbiol. 78, 78-91, 2010.[ Full Text/Abstract ]

Greene EC, Mizuuchi K Visualizing the assembly and disassembly mechanisms of the MuB transposition targeting complex. J Biol Chem (279): 16736-43, 2004. [ Full Text/Abstract ]

Yanagihara K, Mizuuchi K Progressive structural transitions within Mu transpositional complexes. Mol Cell (11): 215-24, 2003. [ Full Text/Abstract ]

Hoskins JR, Yanagihara K, Mizuuchi K, Wickner S ClpAP and ClpXP degrade proteins with tags located in the interior of the primary sequence. Proc Natl Acad Sci U S A (99): 11037-42, 2002. [ Full Text/Abstract ]

Greene EC, Mizuuchi K Direct observation of single MuB polymers: evidence for a DNA-dependent conformational change for generating an active target complex. Mol Cell (9): 1079-89, 2002. [ Full Text/Abstract ]

Greene EC, Mizuuchi K Dynamics of a protein polymer: the assembly and disassembly pathways of the MuB transposition target complex. EMBO J (21): 1477-86, 2002. [ Full Text/Abstract ]

Yanagihara K, Mizuuchi K Mismatch-targeted transposition of Mu: a new strategy to map genetic polymorphism. Proc Natl Acad Sci U S A (99): 11317-21, 2002. [ Full Text/Abstract ]

Greene EC, Mizuuchi K Target immunity during Mu DNA transposition. Transpososome assembly and DNA looping enhance MuA-mediated disassembly of the MuB target complex. Mol Cell (10): 1367-78, 2002. [ Full Text/Abstract ]

Mizuuchi M, Mizuuchi K Conformational isomerization in phage Mu transpososome assembly: effects of the transpositional enhancer and of MuB. EMBO J (20): 6927-35, 2001. [ Full Text/Abstract ]

Zheng R, Ghirlando R, Lee MS, Mizuuchi K, Krause M, Craigie R Barrier-to-autointegration factor (BAF) bridges DNA in a discrete, higher-order nucleoprotein complex. Proc Natl Acad Sci U S A (97): 8997-9002, 2000. [ Full Text/Abstract ]

Kennedy AK, Haniford DB, Mizuuchi K Single active site catalysis of the successive phosphoryl transfer steps by DNA transposases: insights from phosphorothioate stereoselectivity. Cell (101): 295-305, 2000. [ Full Text/Abstract ]

Mizuuchi K, Nobbs TJ, Halford SE, Adzuma K, Qin J A new method for determining the stereochemistry of DNA cleavage reactions: application to the SfiI and HpaII restriction endonucleases and to the MuA transposase. Biochemistry (38): 4640-8, 1999. [ Full Text/Abstract ]

Wei SQ, Mizuuchi K, Craigie R Footprints on the viral DNA ends in moloney murine leukemia virus preintegration complexes reflect a specific association with integrase. Proc Natl Acad Sci U S A (95): 10535-40, 1998. [ Full Text/Abstract ]

Wei SQ, Mizuuchi K, Craigie R A large nucleoprotein assembly at the ends of the viral DNA mediates retroviral DNA integration. EMBO J (16): 7511-20, 1997. [ Full Text/Abstract ]

Mizuuchi K Polynucleotidyl transfer reactions in site-specific DNA recombination. Genes Cells (2): 1-12, 1997. [ Full Text/Abstract ]

Yang W, Mizuuchi K Site-specific recombination in plane view. Structure (5): 1401-6, 1997. [ Full Text/Abstract ]

Clubb RT, Schumacher S, Mizuuchi K, Gronenborn AM, Clore GM Solution structure of the I gamma subdomain of the Mu end DNA-binding domain of phage Mu transposase. J Mol Biol (273): 19-25, 1997. [ Full Text/Abstract ]

Schumacher S, Clubb RT, Cai M, Mizuuchi K, Clore GM, Gronenborn AM Solution structure of the Mu end DNA-binding ibeta subdomain of phage Mu transposase: modular DNA recognition by two tethered domains. EMBO J (16): 7532-41, 1997. [ Full Text/Abstract ]

Rice PA, Yang S, Mizuuchi K, Nash HA Crystal structure of an IHF-DNA complex: a protein-induced DNA U-turn. Cell (87): 1295-306, 1996. [ Full Text/Abstract ]

Savilahti H Mizuuchi K Mu transpositional recombination: donor DNA cleavage and strand transfer in trans by the Mu transposase. Cell (85): 271-80, 1996. [ Full Text/Abstract ]

van Gent DC, Mizuuchi K, Gellert M Similarities between initiation of V(D)J recombination and retroviral integration. Science (271): 1592-4, 1996. [ Full Text/Abstract ]

Mizuuchi M Baker TA Mizuuchi K Assembly of phage Mu transpososomes: cooperative transitions assisted by protein and DNA scaffolds. Cell (83): 375-85, 1995. [ Full Text/Abstract ]

Rice P Mizuuchi K Structure of the bacteriophage Mu transposase core: a common structural motif for DNA transposition and retroviral integration. Cell (82): 209-20, 1995. [ Full Text/Abstract ]