Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Genomics:GTL Program Projects
J. Craig Venter Institute
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Development of a Deinococcus radiodurans Homologous Recombination System
Sanjay Vashee*, Ray-Yuan Chuang* (RChuang@venterinstitute.org), Christian Barnes, Hamilton O. Smith, and J. Craig Venter
J. Craig Venter Institute, Rockville, MD
A major goal of our Institute is to rationally design synthetic microorganisms that are capable of carrying out the required functions. One of the requirements for this effort entails the packaging of the designed pathways into a cohesive genome. Our approach to this problem is to develop an efficient in vitro homologous recombination system based upon Deinococcus radiodurans (Dr). This bacterium was selected because it has the remarkable ability to survive 15,000 Gy of ionizing radiation. In contrast, doses below 10 Gy are lethal to almost all other organisms. Although hundreds of double-strand breaks are created, Dr is able to accurately restore its genome without evidence of mutation within a few hours after exposure, suggesting that the bacterium has a very efficient repair mechanism. The major repair pathway is thought to be homologous recombination, mainly because Dr strains containing mutations in recA, the bacterial recombinase, are sensitive to ionizing radiation.
Since the mechanism of homologous recombination is not yet well understood in Dr, we have undertaken two general approaches to study this phenomenon. First, we are establishing an endogenous extract that contains homologous recombination activity. This extract can then be fractionated to isolate and purify all proteins that perform homologous recombination. We are also utilizing information from the sequenced genome. For example, homologues of E. coli homologous recombination proteins, such as recD and ruvA, are present in Dr. Thus, another approach is to assemble the homologous recombination activity by purifying and characterizing the analogous recombinant proteins. However, not all genes that play a major role in homologous recombination have been identified by annotation.
We have over-expressed and purified D. radiodurans homologues of repair proteins, including RecA, SSB, RecD, RuvB, and RuvC. We showed that the properties of DrSsb DNA binding and strand-exchange properties are very similar to that of E. coli SSB. In addition, using antibodies we have raised, we have determined that the amount of both DrSsb and RecA protein increases in the cell when exposed to a DNA damaging agent, whereas the level of DrRecD protein remains the same.
* Presenting author
