A bipartite thermodynamic-kinetic contribution by an activating mutation to RDF-independent excision by a phage serine integrase.
Journal
Nucleic acids research
ISSN: 1362-4962
Titre abrégé: Nucleic Acids Res
Pays: England
ID NLM: 0411011
Informations de publication
Date de publication:
09 07 2020
09 07 2020
Historique:
accepted:
08
05
2020
revised:
02
05
2020
received:
23
01
2020
pubmed:
2
6
2020
medline:
15
9
2020
entrez:
2
6
2020
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 32479633
pii: 5849906
doi: 10.1093/nar/gkaa401
pmc: PMC7337939
doi:
Substances chimiques
DNA-Binding Proteins
0
Viral Proteins
0
Integrases
EC 2.7.7.-
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
6413-6430Subventions
Organisme : NIGMS NIH HHS
ID : P20 GM104420
Pays : United States
Informations de copyright
© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.
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