Mouw, K.W., Steiner, A.M., Ghirlando, R., Li, N.-S., Rowland, S.-J., Boocock, M.R., Stark, W.M. , Piccirilli, J.A. and Rice, P.A. (2010) Sin resolvase catalytic activity and oligomerization state are tightly coupled. Journal of Molecular Biology, 404(1), pp. 16-33. (doi: 10.1016/j.jmb.2010.08.057)
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Abstract
Serine recombinases promote specific DNA rearrangements by a cut-and-paste mechanism that involves cleavage of all four DNA strands at two sites recognized by the enzyme. Dissecting the order and timing of these cleavage events and the steps leading up to them is difficult because the cleavage reaction is readily reversible. Here, we describe assays using activated Sin mutants and a DNA substrate with a 3′-bridging phosphorothiolate modification that renders Sin-mediated DNA cleavage irreversible. We find that activating Sin mutations promote DNA cleavage rather than simply stabilize the cleavage product. Cleavage events at the scissile phosphates on complementary strands of the duplex are tightly coupled, and the overall DNA cleavage rate is strongly dependent on Sin concentration. When combined with analytical ultracentrifugation data, these results suggest that Sin catalytic activity and oligomerization state are tightly linked, and that activating mutations promote formation of a cleavage-competent oligomeric state that is normally formed only transiently within the full synaptic complex.
Item Type: | Articles |
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Status: | Published |
Refereed: | Yes |
Glasgow Author(s) Enlighten ID: | Rowland, Dr Sally-Jane and Boocock, Dr Martin and Stark, Professor Marshall |
Authors: | Mouw, K.W., Steiner, A.M., Ghirlando, R., Li, N.-S., Rowland, S.-J., Boocock, M.R., Stark, W.M., Piccirilli, J.A., and Rice, P.A. |
Subjects: | Q Science > QR Microbiology |
College/School: | College of Medical Veterinary and Life Sciences > School of Molecular Biosciences |
Journal Name: | Journal of Molecular Biology |
ISSN: | 0022-2836 |
Published Online: | 22 September 2010 |
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