Julin Laboratory, Dept. of Chemistry and Biochemistry, University of Maryland College Park

Enzymology of DNA recombination and repair.

            Living organisms are under constant assault by environmental agents that damage their DNA, including radiation and reactive chemicals. These agents lead to formation of damaged bases and DNA strand breaks which, if unrepaired, can lead to cell death, mutation and cancer in humans. Organisms are not defenseless against this onslaught. The first line of defense is a wide variety of DNA repair and recombination enzymes that are able to undo the damage and restore the DNA to its original undamaged state.

            We are studying two enzymes from bacteria that participate in repair of DNA damage: the RecBCD enzyme from Escherichia coli, and the RecD protein from Deinococcus radiodurans.

E. coli RecBCD enzyme.

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RecBCD is a complex macromolecular machine composed of three protein subunits called the RecB, RecC, and RecD proteins. The enzyme is essential for the repair of double-strand breaks in E. coli and many other bacteria. RecBCD binds to the end of a broken DNA duplex and acts as an ATP-dependent helicase and nuclease to process the DNA for subsequent repair via homologous recombination. RecBCD acts as a DNA helicase, moving rapidly along the DNA and separating the individual strands of the double helix. Meanwhile, the enzyme is also a nuclease which degrades one of the two unwound DNA strands. The activity of the enzyme is subject to an unusual form of regulation when it encounters a specific DNA sequence called Chi (5'-GCTGGTGG). The helicase activity continues to unwind the DNA helix after Chi, but the nuclease switches its specificity and starts to degrade the other unwound strand. Meanwhile, the enzyme loads the RecA protein onto the undegraded single strand. RecA initiates a recombination reaction, in which the broken DNA molecule is re-joined to a homologous chromosome, the critical step in repairing the original double-strand break.             The complete three-dimensional structure of the RecBCD enzyme bound to a short double-stranded DNA molecule (determined in the laboratory of Dr. Dale Wigley, London Research Institute, UK) provided an atomic level view of how the enzyme interacts with a double-stranded DNA molecule. We discovered in previous work that the nuclease activity is localized to a small domain that is part of the RecB subunit. The complete structure of this domain was revealed in the crystal structure. Our current research focuses mainly on the properties of the RecB nuclease domain and how it interacts with the other activities of RecBCD during the complex reaction with double-stranded DNA. We use enzyme kinetics, site-directed mutagenesis, protein chemistry, and other methods in this work.

Deinococcus radiodurans RecD protein.

            D. radiodurans is a fascinating bacterium because of its ability to survive under extreme conditions that are lethal to most other forms of life. The organism, dubbed "Conan the Bacterium", can withstand high levels of ionizing radiation (X-rays and gamma rays), ultraviolet light, and DNA-damaging chemicals. The physiological properties and enzymatic pathways that enable it to survive these conditions are of great interest.

            The most serious consequence of ionizing radiation is the formation of double-strand breaks in DNA. D. radiodurans can survive a much greater number of breaks in its chromosomes than can E. coli and other organisms, suggesting that it has very efficient pathways for repair of these breaks. It was therefore surprising when the complete genome sequence of D. radiodurans revealed that the organism does not have either recB or recC genes, and thus lacks a RecBCD enzyme. However, the genome sequence showed that there is a gene encoding a protein that is very similar to the RecD subunit of RecBCD enzymes. Because there are no RecB and RecC proteins in D. radiodurans, the RecD protein must serve a different function in this organism than it does in E. coli and other bacteria.

            We are studying the biological and enzymatic properties of the D. radiodurans RecD protein. We find that the purified RecD protein is a DNA helicase, and that cells with a mutation in the recD gene are sensitive to irradiation with gamma rays and ultraviolet light, suggesting a role for the protein in DNA repair. We are continuing to carry out genetics experiments with the recD mutant, and biochemical experiments with the purified RecD protein, with the goal of understanding how the protein fits into the overall physiology of the organism.

Selected publications.

Ghatak, A., and Julin, D. A. (2006) "Kinetics of ATP-stimulated nuclease activity of the Escherichia coli RecBCD enzyme." Journal of Molecular Biology 361, 954-968.

Wang, J., and Julin, D. A. (2004) "DNA Helicase Activity of the RecD Protein from Deinococcus radiodurans." Journal of Biological Chemistry 279, 52024-52032.

Kulkarni, A., and Julin, D. A. (2004) "Specific inhibition of the E. coli RecBCD enzyme by Chi sequences in single-stranded oligonucleotides." Nucleic Acids Research 32, 3672-3682.

Wang, J., Chen, R., and Julin, D. A. (2000) "A Single Nuclease Active Site of the Escherichia coli RecBCD Enzyme Catalyzes Single-Stranded DNA Degradation in Both Directions." Journal of Biological Chemistry 275, 507-513.

Zhang, X.-J. J., and Julin, D. A. (1999) "Isolation and Characterization of the C-terminal Nuclease Domain from the RecB Protein of Escherichia coli." Nucleic Acids Research 27, 4200-4207.

Yu, M., Souaya, J., and Julin, D. A. (1998) "The 30-kDa C-terminal domain of the RecB protein is critical for the nuclease activity, but not the helicase activity, of the RecBCD enzyme from Escherichia coli." Proceedings of the National Academy of Sciences USA 95, 981-986.

Yu, M., Souaya, J., and Julin, D. A. (1998) "Identification of the Nuclease Active Site in the Multifunctional RecBCD Enzyme by Creation of a Chimeric Enzyme." Journal of Molecular Biology 283, 797-808.

Chen, H.-W., Randle, D. E., Gabbidon, M., and Julin, D. A. (1998) "Functions of the ATP Hydrolysis Subunits (RecB and RecD) in the Nuclease Reactions Catalyzed by the RecBCD Enzyme from Escherichia coli." Journal of Molecular Biology 278, 89-104.