Abstract
Fluorescent fusion proteins are exceedingly useful for monitoring protein localization in situ or visualizing protein behavior at the single molecule level. Unfortunately, some proteins are rendered inactive by the fusion. To circumvent this problem, we fused a hyperactive RecA protein (RecA803 protein) to monomeric red fluorescent protein (mRFP1) to produce a functional protein (RecA-RFP) that is suitable for in vivo and in vitro analysis. In vivo, the RecA-RFP partially restores UV resistance, conjugational recombination, and SOS induction to recA(-) cells. In vitro, the purified RecA-RFP protein forms a nucleoprotein filament whose k(cat) for single-stranded DNA-dependent ATPase activity is reduced approximately 3-fold relative to wild-type protein, and which is largely inhibited by single-stranded DNA-binding protein. However, RecA protein is also a dATPase; dATP supports RecA-RFP nucleoprotein filament formation in the presence of single-stranded DNA-binding protein. Furthermore, as for the wild-type protein, the activities of RecA-RFP are further enhanced by shifting the pH to 6.2. As a consequence, RecA-RFP is proficient for DNA strand exchange with dATP or at lower pH. Finally, using single molecule visualization, RecA-RFP was seen to assemble into a continuous filament on duplex DNA, and to extend the DNA approximately 1.7-fold. Consistent with its attenuated activities, RecA-RFP nucleates onto double-stranded DNA approximately 3-fold more slowly than the wild-type protein, but still requires approximately 3 monomers to form the rate-limited nucleus needed for filament assembly. Thus, RecA-RFP reveals that its attenuated biological functions correlate with a reduced frequency of nucleoprotein filament nucleation at the single molecule level.
Highlights
The fusion of native proteins to various fluorescent proteins has found widespread use in biology
In Escherichia coli, recombinational repair of double-stranded DNA3 breaks is mediated by the RecBCD pathway, whereas the repair of ssDNA gaps is mediated by the RecF pathway [9]
The assembly of RecA protein onto ssDNA that is complexed with ssDNA binding (SSB) protein is a kinetically slow process, which is catalyzed by so-called mediator or loading proteins [20]
Summary
The fusion of native proteins to various fluorescent proteins has found widespread use in biology. In Escherichia coli, recombinational repair of double-stranded DNA (dsDNA) breaks is mediated by the RecBCD pathway, whereas the repair of ssDNA gaps is mediated by the RecF pathway [9] Both of these recombination pathways require the functions of RecA protein. RecA-RFP Protein an enhanced intrinsic ability to displace SSB from ssDNA [27] One such mutant is the RecA803 protein, in which valine 37 is mutated to methionine [28, 29]. This mutant RecA protein displays a higher intrinsic rate of nucleoprotein filament assembly on ssDNA, which is responsible for its enhanced capacity to displace DNA-bound SSB protein
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