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Segall, Anca

Professor

Center for Microbial Sciences

CMB



asegall@sunstroke.sdsu.edu


Anca Mara Segall, Ph. D.

The mechanism of site-specific recombination; structure/function analysis of recombination proteins


Site-specific recombination is a mechanism used to rearrange DNA through relatively short sequences that may share less than 20 bases of homology. This mechanism is used by viruses which insert their chromosomes into the chromosomes of their cellular hosts. Cells use this mechanism to control gene expression when adapting to environmental challenges and to separate replication intermediates of their chromosomes. Site-specific recombinases are specific for unique target sequences; as a model system for recombination in general, these enzymes are much more amenable to study than enzymes involved in homologous recombination.

We study the site-specific recombination of the bacterial virus lambda. The virus inserts its genome into and excises its genome out of the E. coli chromosome in a tightly regulated fashion, in response to environmental conditions. The site-specific recombination reaction it uses was the first to be reconstituted in vitro; the proteins which carry out the reaction have been purified, and the DNA sequences necessary have been characterized. We are using this recombination system as a model to study basic mechanistic questions about recombination. The system is easily apporached using molecular, biochemical and genetic tools, and the advances that have been made so far allow us to ask extremely sophisticated questions about how the proteins involved actually carry out the recombination reaction.

One question we are asking concerns the efficiency of the recombination event. In order for the virus to recombine with the chromosome, the recombinase must start recombination by nicking DNA and then it must very efficiently re-seal the ends of the DNA in a new arrangement. Although we know the residues that are directly involved in cutting DNA, we don't know which part of the recombinase is necessary for resealing the cuts. To answer this, we have isolated mutants of the recombinase protein which cut DNA but cannot ligate the ends. We are mapping these mutants to determine the amino acid changes in the protein, and characterizing the mutant proteins biochemically.

Another question we are investigating is what kind of movement(s) do the proteins make in order to complete recombination? First, using protein-protein crosslinking, we determined that the recombinase most likely works as a tetramer. This means that there must be at least two surfaces of the protein that are involved in contacts with other recombinase monomers. Second, we are genetically mapping the regions involved in protein-protein contacts : we have found one cluster of amino acids involved in contacts and have hints of a second cluster. To determine how much the recombinase monomers must move with respect to each other during the reaction, we are >fixing< the tetramer prior to adding DNA substrates. The DNA substrates can still wrap around the tetramer, but this tethered tetramer can no longer carry out recombination. Thus, the proteins must move with respect to each other during recombination.

A last example of the kinds of questions we ask is how much do the DNA substrates move during recombiantion? We are approaching this using a microscopy technique called Atomic Force Microscopy. This method will allow us to visualize the position of the DNA molecules with respect to the recombination proteins, and their conformation (how bent or straight they are). By comparing the DNA-protein complexes before and after recombination has taken place, we should be able to see how much the DNA molecules during the reaction.


The questions we are addressing are important for all sorts of other reactions. For example, the HIV virus must similarly find and attack target sites in human chromosomes in order to efficiently insert the DNA copy of the virus genome. If the HIV integrase enzyme quits mid-way, the replication cycle of the virus will be interrupted (a hopeful target for anti-viral drugs). However, at the moment it is not possible to answer these kinds of questions for HIV because its recombination reaction has not been completely reconstituted in vitro. Thus, our insights into the mechanism of lambda's integrase protein have wide-ranging implications.

More About the Lab


Bibliography:

Cassell,G., Moision, R., Rabani, E. and A. Segall, 1999 The geometry of a synaptic intermediate in a pathway of bacteriphage lambda site-specific recombination. Nucl. Acids Res. 27: 1145-1151. Download PDF
Reproduced with permission from NAR Online http://www.oup.co.uk/nar

Segall, AM 1998 Analysis of higher order intermediates and synapsis in the bent-L pathway of bacteriophage lambda site-specific recombination. J. Biol. Chem. 273, 24258-24265. Download PDF

Anca Segall and Howard Nash, 1996. Architectural flexibility in lambda site-specific recombination: Three alternate conformations channel the attL site into three alternate pathways. Genes to Cells, May issue.

Anca Segall, Steve Goodman and Howard Nash, 1994. Architectural elements in nucleoprotein complexes: Interchangeability of specific and nonspecific DNA binding proteins. EMBO J. 13: 4536-4548.

Lynn Miesel, Anca Segall and John Roth, 1994. Construction of chromosomal rearrangements in Salmonella by P22 transduction: Inversions of nonpermissive intervals are not lethal. Genetics 137: 919-932.

Anca Segall and John Roth, 1994. Approaches to half-tetrad analysis in bacteria: Recombination between repeated, inverse-order chromosomal sequences. Genetics 136: 27-39.

Anca Segall and Howard Nash, 1993. Synaptic intermediates in bacteriophage lambda site-specific recombination: Integrase can align pairs of attachment sites. EMBO J. 12: 4567-4576.

Anca Segall and John Roth, 1989. Recombination between homologies in direct and inverse orientation in the chromosome of Salmonella. Genetics 122:737-747.

Anca Segall, Michael J. Mahan, and John Roth, 1988. Rearrangement of the bacterial chromosome: Forbidden inversions. Science 241: 1314-1318.

Ph.D. students

Troy Bankhead, Geoffrey Cassell, Lea Jessop

Master's students

Gina Allicotti, Marcy Klemm, Tien Le, Margaret Lee, Victor Seguritan

Undergraduates

Jeff Boldt, Tim Harmon, Luz Ramos, Scott Robinson

Anca Segall, Ph.D.

San Diego State University
Dept. of Biology
5500 Campanile Dr.
San Diego, CA 92182-4614

office (619) 594-4490
lab (619) 594-4638
fax (619) 594-5676

Note: Entrez Medline entries for a particular Author name may correspond with multiple authors with the same initials. Also, the list is limited to entries stored in the Entrez Medline Database and may not accurately reflect the true number of publications