Direct Evaluation of a Mechanism for Activation of the RecA Nucleoprotein Filament
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- 作者：Alberto I. Roca and Scott F. Singleton
- 刊名：Journal of the American Chemical Society
- 出版年：2003
- 出版时间：December 17, 2003
- 年：2003
- 卷：125
- 期：50
- 页码：15366 - 15375
- 全文大小：216K
- 年卷期：v.125,no.50(December 17, 2003)
- ISSN：1520-5126
文摘
The RecA protein of Escherichia coli controls the SOS response for DNA damage toleranceand plays a crucial role in recombinational DNA repair. The formation of a RecA·ATP·ssDNA complexinitiates all RecA activities, and yet this process is not understood at the molecular level. An analysis ofRecA·DNA interactions was performed using both a mutant RecA protein containing a tryptophan (Trp)reporter and oligodeoxyribonucleotides (ODNs) containing a fluorescent guanine analogue, 6-methylisoxanthopterin (6MI). Experiments using fluorescent ODNs allowed structurally distinct nucleoprotein filaments,formed in the absence and presence of ATP
fchars/gamma.gif" BORDER=0 >S (a slowly hydrolyzed analogue of ATP), to be differentiateddirectly. Stopped-flow spectrofluorometry, combined with presteady-state kinetic analyses, revealedunexpected differences in the rates of RecA·ODN and RecA·ATPfchars/gamma.gif" BORDER=0 >S·ODN complex assembly. This is thefirst demonstration that such intrinsically fluorescent synthetic DNAs can be used to characterize definitivelythe real-time assembly and activation of RecA·ssDNA complexes. Surprisingly, the ssDNA binding eventis almost 50-fold slower in the presence of the activating ATPfchars/gamma.gif" BORDER=0 >S cofactor. Furthermore, a combination oftime-dependent emission changes from 6MI and Trp allowed the first direct chemical test of whether aninactive filament can isomerize to the active state. The results revealed that, unlike the hexameric motorproteins, the inactive RecA filament cannot directly convert to the active state upon ATPfchars/gamma.gif" BORDER=0 >S binding. Theseresults have implications for understanding how a coincidence of functions-an ATP-communicated signal-like activity and an ATP-driven motorlike activity-are resolved within a single protein molecule.
fchars/gamma.gif" BORDER=0 >S (a slowly hydrolyzed analogue of ATP), to be differentiateddirectly. Stopped-flow spectrofluorometry, combined with presteady-state kinetic analyses, revealedunexpected differences in the rates of RecA·ODN and RecA·ATPfchars/gamma.gif" BORDER=0 >S·ODN complex assembly. This is thefirst demonstration that such intrinsically fluorescent synthetic DNAs can be used to characterize definitivelythe real-time assembly and activation of RecA·ssDNA complexes. Surprisingly, the ssDNA binding eventis almost 50-fold slower in the presence of the activating ATPfchars/gamma.gif" BORDER=0 >S cofactor. Furthermore, a combination oftime-dependent emission changes from 6MI and Trp allowed the first direct chemical test of whether aninactive filament can isomerize to the active state. The results revealed that, unlike the hexameric motorproteins, the inactive RecA filament cannot directly convert to the active state upon ATPfchars/gamma.gif" BORDER=0 >S binding. Theseresults have implications for understanding how a coincidence of functions-an ATP-communicated signal-like activity and an ATP-driven motorlike activity-are resolved within a single protein molecule.