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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.
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
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