PARENT SESSION
1:30 PM to 3:30 PM
Monday, April 22, 2002
Poster Session 12 DNA Damage and Repair I
Room: Nevada Exhibition Center
(P17-166) In vitro activities and complex formation of the human Rad51B and Rad51C DNA repair proteins.
Lio, Yi-Ching*,1, Mazin, Alexander2, Miroshnychenko, Olga1, Kowalczykowski, Stephen2, Chen, David1, 1 Life Sciences Division, Berkeley, CA2 Division of Biological Sciences, Davis, CA
ABSTRACT-
The human Rad51 recombinase is a key player in repairing DNA damages by homologous recombination. Five Rad51 paralogs have currently been identified: Rad51B/Rad51L1, Rad51C/Rad51L2, Rad51D/Rad51L3, XRCC2, and XRCC3. Evidence from the yeast two- and three-hybrid assays as well as the baculovirus system has demonstrated the simultaneous interactions between these human proteins [Schild, D., Lio, Y.-C., Collin, D.W., Tsomondo, T. and Chen, D.J. (2000) J. Biol. Chem. 275(22), 16443-9]. To further characterize the biochemical properties of these proteins, recombinant Rad51, Rad51B-(His)6 and Rad51C proteins were expressed employing the baculovirus system and each was individually purified to homogeneity from Sf9 insect cells. Rad51B and Rad51C proteins were found to bind single- and double-stranded DNA, and preferentially bind 3′-end tailed double-stranded DNA. The ability to bind DNA was elevated with mixed Rad51 and Rad51C, as well as mixed Rad51B and Rad51C, compared to that of individual protein. In addition, both Rad51B and Rad51C display DNA-stimulated ATPase activity in a time-dependent manner. Single-stranded DNA is a better stimulator of the ATPase activity than double-stranded DNA for both proteins. Furthermore, using oligonucleotide substrates, Rad51C was found to display DNA strand transfer activity which is much lower than that of Rad51, while Rad51B does not show any strand transfer activity under examined conditions. The effect of Rad51B and Rad51C on the strand transfer activity of Rad51 will be discussed. Evidence from Ni-NTA pull-down and gel filtration experiments suggests a highly stable Rad51B-Rad51C heterodimer, which interacts weakly with Rad51. By analogy with ScRad55 and ScRad57, our results suggest that Rad51B and Rad51C function through interactions with the human Rad51 recombinase and play a mediating role in the homologous recombinational repair pathway. This work was supported by NIH grant CA 74046, DOD grant DAMD17-99-9170 and California Breast Cancer Research Program grant 7KB-0019.
KEYWORDS: Complex formation, DNA binding activity, ATPase activity, DNA strand transfer activity