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The synaptic protein–DNA complexes, formed by specialized proteins that bridge two or more distant sites on DNA, are critically involved in various genetic processes. However, the molecular mechanism by which the protein searches for these sites and how it brings them together is not well understood. Our previous studies directly visualized search pathways used by SfiI, and we identified two pathways, DNA threading and site-bound transfer pathways, specific to the site-search process for synaptic DNA–protein systems. To investigate the molecular mechanism behind these site-search pathways, we assembled complexes of SfiI with various DNA substrates corresponding to different transient states and measured their stability using a single-molecule fluorescence approach. These assemblies corresponded to specific–specific (synaptic), non-specific–non-specific (non-specific), and specific–non-specific (pre-synaptic) SfiI–DNA states. Unexpectedly, an elevated stability in pre-synaptic complexes assembled with specific and non-specific DNA substrates was found. To explain these surprising observations, a theoretical approach that describes the assembly of these complexes and compares the predictions with the experiment was developed. The theory explains this effect by utilizing entropic arguments, according to which, after the partial dissociation, the non-specific DNA template has multiple possibilities of rebinding, effectively increasing the stability. Such difference in the stabilities of SfiI complexes with specific and non-specific DNA explains the utilization of threading and site-bound transfer pathways in the search process of synaptic protein–DNA complexes discovered in the time-lapse AFM experiments.
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A bipartite thermodynamic-kinetic contribution by an activating mutation to RDF-independent excision by a phage serine integrase
Abstract Streptomyces phage ϕC31 integrase (Int)—a large serine site-specific recombinase—is autonomous for phage integration (attP x attB recombination) but is dependent on the phage coded gp3, a recombination directionality factor (RDF), for prophage excision (attL x attR recombination). A previously described activating mutation, E449K, induces Int to perform attL x attR recombination in the absence of gp3, albeit with lower efficiency. E449K has no adverse effect on the competence of Int for attP x attB recombination. Int(E449K) resembles Int in gp3 mediated stimulation of attL x attR recombination and inhibition of attP x attB recombination. Using single-molecule analyses, we examined the mechanism by which E449K activates Int for gp3-independent attL x attR recombination. The contribution of E449K is both thermodynamic and kinetic. First, the mutation modulates the relative abundance of Int bound attL-attR site complexes, favoring pre-synaptic (PS) complexes over non-productively bound complexes. Roughly half of the synaptic complexes formed from Int(E449K) pre-synaptic complexes are recombination competent. By contrast, Int yields only inactive synapses. Second, E449K accelerates the dissociation of non-productively bound complexes and inactive synaptic complexes formed by Int. The extra opportunities afforded to Int(E499K) in reattempting synapse formation enhances the probability of success at fruitful synapsis.
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- Award ID(s):
- 1949821
- PAR ID:
- 10206682
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 48
- Issue:
- 12
- ISSN:
- 0305-1048
- Page Range / eLocation ID:
- 6413 to 6430
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1093/nar/gkaa401
