- D.S. (Ph.D.), Osaka University, 1972
- M.S., Osaka University, 1969
- B.S., Osaka University, 1967
The objective of our research is to gain insights into the mechanics of 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 3-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 mechanisms of DNA transposition of the bacteriophage Mu as a model system for 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 involves three components: a specific DNA sequence on the segregating chromosome that functions as the bacterial equivalent of a “centromere,” a protein factor that binds to the “centromere,” and a second protein factor—an ATPase with ATP-dependent nonspecific 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. Based on the experimental results we obtained by combining biophysical and biochemical approaches, we have uncovered a previously unappreciated biological cargo transport and positioning mechanism that involves a variation of the reaction-diffusion principle, which we call diffusion-ratchet mechanism.
After segregation of replicated chromosome and plasmid copies to two halves of the cell, cell division normally must take place at the mid-cell. We 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 tAfter segregation of replicated chromosome and plasmid copies to two halves of the cell, cell division normally must take place at the mid-cell. We 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 observed by fluorescent microscopy. The position of cell division septum formation is determined by the location where a bacterial tubulin-homologue, FtsZ polymerizes on the inner membrane, and FtsZ polymerization is inhibited by membrane-localized MinC protein. MinC is brought to membrane by binding to MinD, an ATP-dependent membrane binding protein. Together with MinE, a stimulator of MinD ATPase, Min proteins self-organizes forming standing wave-type oscillation on the membrane inside a cell, generating time-averaged concentration minimum of MinD and MinC at mid-cell where cell division takes place. We have successfully recapitulated the standing-wave-type oscillatory dynamic pattern self-organization by MinD/E proteins in a cell-free reaction. Based on our findings, we now have a detailed mechanistic model of the system, which will be subjected to further experimental tests and refinements. Progress in these projects is expected to advance our general understanding of a variety of biomolecular transportation and patterning reactions and also to provide a foundation for the possible development of novel antibacterial agents.
Applying our Research
Progress in these projects will advance our understanding of the movement of a variety of molecular assemblies produced inside bacterial cells and could provide a basis for developing new drugs to combat disease-carrying bacteria.
- Cell-free study of F plasmid partition provides evidence for cargo transport by a diffusion-ratchet mechanism.
- Vecchiarelli AG, Hwang LC, Mizuuchi K.
- Proc Natl Acad Sci U S A (2013 Apr 9) 110:E1390-7. Abstract/Full Text
- ParA-mediated plasmid partition driven by protein pattern self-organization.
- Hwang LC, Vecchiarelli AG, Han YW, Mizuuchi M, Harada Y, Funnell BE, Mizuuchi K.
- EMBO J (2013 May 2) 32:1238-49. Abstract/Full Text
Research in Plain Language
The molecule called deoxyribonucleic acid (DNA) encodes the biological instructions that make each species and individuals unique. DNA-- and with it the instructions needed to develop, survive and reproduce-- is the molecular entity that constitutes genome of an organism that is passed from adult to their offspring during reproduction.
Our lab is interested in how certain genetic elements (segments of DNA) cause changes to the arrangement of genomic DNA—a reaction with many health consequences. Genetic elements called transposons, researchers have learned, are able to change positions within a genome or among separate genomes, in effect, by cutting and pasting or copying and pasting themselves from place to place. Drugs that once were effective become ineffective as a result of rearranged DNA in disease-carrying bacteria. We study these rearrangements in the virus Mu, a model system for a wide range of DNA rearrangement reactions that occur from bacteria to humans.
Using biochemical, molecular biological, and biophysical approaches, our lab is learning new detail about the chemical reactions that drive DNA rearrangements. Single-molecule approaches we are developing are helping us answer many questions about how the molecular machines involved in this process work.
In other projects, we focus on two aspects of how a parent bacterial cell divides into two daughter cells and distributes its genetic blueprint. Our experiments using E. coli bacteria are revealing new detail about how the cell organizes its genetic blueprint, its entire genome, and position two replicated copies of the genome inside a parental cell before cell division so that each daughter cell inherits a full complement of the genome. Our second interest is the bacterial cell’s ability to precisely position the membrane that separates the two daughter cells at the middle of the parental cell. Progress in these projects will advance our understanding of the movement of a variety of molecular assemblies produced inside bacterial cells and could provide a basis for developing new drugs to combat disease-carrying bacteria.