Research Projects

 

I am interested in the application of topology, geometry nd computational methods to understand biological processes. 

DNA topology

I am interested in studying natural processes which affect the topology of DNA. Most of my work has focused on the study of enzymes, such as site-specific recombinases and DNA topoisomerases, although I have also worked extensively on the problem of DNA condensation in phage capsids.


A. Tangle Model for site-specific recombination

The tangle model is a mathematical method due to De Witt Sumners and Claus Ernst [Sumners et al. Quart. Rev. Biophysics 28, 3 (1995), 253 - 313], which uses knot theory and low-dimensional topology to understand the mechanisms of binding and strand-exchange by site-specific recombinases. A web description of the tangle model can be found here.



    TangleSolve. While at Berkeley, I worked with an undergraduate research assistants, Yuki Saka, Wenjing Zheng and Stefanus Jasin on a computer implementation of the tangle model. The resulting software, TangleSolve, is available for use on the web or for download. A description of TangleSolve can be found in [Saka and Vazquez, 2002; Zheng et al. 2007], and on the TangleSolve website.


I have worked on several site-specific recombination systems:
    Gin and mutant Gin, Vazquez and Sumners, 2004

Gin is a site-specific recombination system of bacteriphage Mu. Bacteriophage Mu infects a large family of bacteria, including several strains of Escherichia coli. Gin's role in the phage's development is to invert a segment of DNA, called the G-segment, thus extending the host range of the phage. The DNA knots and/or links produced by site-specific recombination on circular DNA substrates were characterized in Nicholas Cozzarelli's lab in Berkeley, mainly by Roland Kanaar (Gin) and Nancy Crisona (mutant Gin).


    XerC/XerD at psi, Vazquez, Colloms and Sumners, 2005

       XerC and XerD are two site-specific recombinases of E. coli which act cooperatively to resolve chromosomal dimers formed by Homologous Recombination, thus allowing proper segregation at cell division. XerC/XerD act at dif sites in the E. coli chromosome, but also at other sites such as psi and cer in naturally occurring plasmids. When acting at psi sites the Xer system produces 4-crossing torus links. The experiments were done in David Sherratt's lab (University of Oxford) by Jonathan Bath and Sean Colloms.


    FtsK-XerC/D at dif, Grainge et al. 2007

    To act at dif sites, XerC and Xer D are recruited by the powerful translocase FtsK and co-localize at the septum to perform a simple recombination event turning a dimeric chromosomes into two monomers which can properly segregate at cell division. We have recently reported that this system is able to unlink replication catenanes in vivo when topoIV is inhibited. I have analyzed the mathematical pathway of unknotting. The experiments were done by Ian Grainge and Migena Bregu in Sherratt's lab in Oxford, and I am collaborating with Koya Shimokawa (Saitama University, Japan) on the mathematical analysis.


    TnpI-IRS, Zheng et al. 2007


Bacillus thuringiensis is a bacterium that produces specific toxins that are lethal to a variety of
insect species, but inoffensive to most other organisms Therefore, B. thuringiensis and its toxin
crystals are used in organic farming to protect crops from harmful moths and butterflies.
Transposons are segments of DNA that can move to different regions (transposition) within the
genome of a single cell. Tn4430 is a transposon from from B. thuringiensis. During this process, co-integrate intermediates between the donor and target replicons are generated. Tn4430 encodes the TnpI protein, a member of the tyrosine site-specific recombinase family that catalyzes the site-specific recombination reaction used to resolve these co-integrate intermediates. In-vitro recombination reactions on circular DNA substrates with two directed repeats of the full IRS sites yield products with specific topology: all products are two-node links (Hopf Links). The DNA knots and/or links produced by TnpI recombination were characterized by Christine Galloy in Bernard Hallet's lab (Universite Catholique de Louvain).


B. Difference Topology: extending the Tangle Model

Jointly with John Luecke and Isabel Darcy, I have analyzed data from novel difference topology experiments to unveil the structure of the Mu transpososome. The technique was developed in Rasika Harshey's and Makkuni Jayaram's lab, and the experiments were done by Shailja Pathania.


The purpose in difference topology experiments is to shed light on the topological structure of a stable protein/DNA complex (such as the Mu transpososome) by using a known site-specific recombinase to create DNA knots or links when given the complex as a substrate. The resulting knots/links contain the information needed to understand the whole complex. Analyzing difference topology experiments requires extending the tangle model, especially since the complex to be analyzed may be better modeled by n-string tangles, where n is greater than 2.



    The Mu transpososome

Transposable elements, also called mobile elements, are fragments of DNA able to move
along a genome by a process called transposition. Mobile elements play an important role
in the shaping of a genome, and they can impact the health of an organism by introducing genetic mutations. Of special interest is that transposition is mechanistically very similar to the way certain retroviruses, including HIV, integrate into their host genome.
Bacteriophage Mu is a system widely used in transposition studies due to the high
efficiency of Mu transposase. The MuA protein performs the first steps required to transpose the Mu genome from its starting location to a new DNA location. MuA binds to specific DNA sequences which we refer to as attL and attR sites (named after Left and Right attaching regions). A third DNA sequence called the enhancer (E) is also required to assemble the Mu transpososome. The Mu transpososome is a very stable complex consisting of 3 segments of double- stranded DNA captured in a protein complex. In our paper we are interested in studying the topological structure of the DNA within the Mu transpososome.   
   Look at the preprint: Darcy et al, submitted   


C. Type-2 Topoisomerases


I am very interested in understanding the mechanism of action of type-2 topoisomerases. I am currently modelling their DNA unknotting reaction. This project was inspired by our work on DNA packing in bacteriophages (Arsuaga et al. 2002) while working with Javier Arsuaga and Joaquim Roca (molecular biologist). The theoretical part took off as an undergraduate project involving several Berkeley undergraduates: first Barath Raghavan (now a CS graduate student at UCSD). Since then several students have worked on the project: Diana Nguyen (UCLA Medical School), Miki Suga and Xia (Carol) Hua (MIT), Nathan Shayefar (UCB), Itamar landau (UCB), Janella Slaga (SFSU), Juliet Portillo (SFSU), Ben Dalziel (SFSU), Nicholas Normandin (SFSU), Andrew Herrmann (SFSU), Reuben Brascher (SFSU), Jeff halperin (SFSU). We use knot theory and computer simulations to model strand-passage on polygonal chains (the DNA) in space. You can see a description of the early stages of the project in [Hua et al, 2007; Hua and Vazquez, 2007]. More papers are in preparation.


This project is funded by NIH- MBRS SCORE grant S06 GM052588


D. DNA packing in bacteriophage capsids

Repeating experiments of Liu and Wang (1981) we showed that DNA extracted from bacteriophage P4 capsids is highly knotted. 95% of the observed DNA is knotted, and most of the knots are very complicated. Using experimental techniques, mathematical arguments and computer simulations we have shown that the probability of a chain being knotted i confined volumes is very high. We also showed that the DNA packing in bacteriophages is ruled by a chirality bias. In collaboration with Joaquim Roca, Javier Arsuaga and Yuanan DIao we are investigating the nature of this bias. See [Trigueros et al. 2001, Arsuaga et al, 2002, Arsuaga et al. 2002 and Arsuaga et al. 2005] for more information.

   

              


Radiation Research:

 

I was a postdoc at UC Berkeley from 2000 to 2005, and I currently have a visiting scholar position in the mathematics department at UC Berkeley. There, I have been part of the Mathematical Radiobiology group led by Professor Rainer Sachs. Our group typically consists of one senior faculty member, two junior faculty, one graduate student and several undergraduate assistants. We develop mathematical and biophysical models of DNA repair in human cells by analyzing the aberrations induced on them by low-dose ionizing radiation. We use Monte-Carlo simulation to give a quantitative analysis of various models for chromosomal aberration production. This analysis is compared to experimental data given by mFISH (multiplex fluorescence in situ hybridization). Most of our results have been published; the relevant papers can be found in the list of publications.

 

 


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