2009 OSU Molecular Life Sciences
Interdisciplinary Graduate Programs Symposium
Poster abstracts
Abstract:
E. coli RecE protein is part of the classical RecET recombination system that has recently been employed in powerful new methods for genetic engineering. RecE binds to free dsDNA ends and processively digests the 5’-ended strand to form 5’-mononucleotides and a 3’-overhang that is a substrate for single strand annealing promoted by RecT. Here, we report the crystal structure of the C-terminal nuclease domain of RecE at 2.8 Å resolution. RecE forms a toroidal tetramer with a central tapered channel that is wide enough to bind dsDNA at one end, but is partially plugged at the other end by the C-terminal segment of the protein. Four narrow tunnels, one within each subunit of the tetramer, lead from the central channel to the four active sites, which lie about 15 Å from the channel. The structure, combined with mutational studies, suggests a mechanism in which dsDNA enters through the open end of the central channel, the 5’-ended strand passes through a tunnel to access one of the four active sites, and the 3’-ended strand passes through the plugged end of the channel at the back of the tetramer.
References:
1. Zhang, J., Xing, X., Herr, A. B., and Bell, C. E. (2009) Structure.
2. Kolodner, R., Hall, S.D., and Luisi-Deluca, C. (1994) Mol. Microbiol. 11, 23-30.
3. Joseph, J.W., and Kolodner, R. (1983a) J. Biol. Chem. 258, 10411-10417.
4. Joseph, J.W., and Kolodner, R. (1983b) J. Biol. Chem. 258, 10418-10424.
5. Chang, H.W., and Julin, D.A. (2001) J. Biol. Chem. 276, 46004-46010.
6. Muyrers, J.P.P., Zhang, Y., Buchholz, F., and Stewart, A.F. (2000) Genes & Devel. 14, 1971-1982.
7. Copeland, N.G., Jenkins, N.A., and Court, D.L. (2001) Nat. Rev. Genet. 2, 769-779.
8. Branton, D., Deamer, D.W., Marziaali, A, Bayley, H., Benner, S.A., Butler, T., Di Ventra, M., Garaj, S., Hibbs, A., and Huang, X. et al. (2008) Nature Biotech. 26, 1146-1153.
9. Tolun, G., and Myers, R.S. (2003) Nucleic Acids Res. 31, e111.
10. Kovall, R., and Mattews, B.W. (1997) Science 277, 1824-1827.
Keywords: double stranded DNA repair, recombination, RecE exonuclease