Single-molecule Analysis of ϕC31 Integrase-mediated Site-specific Recombination by Tethered Particle Motion

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2016 Dec 18

PMID 27986956

Citations 9

Authors

Hsiu-Fang Fan

Tao-Shih Hsieh

Chien-Hui Ma

Makkuni Jayaram

Affiliations

Soon will be listed here.

Abstract

Serine and tyrosine site-specific recombinases (SRs and YRs, respectively) provide templates for understanding the chemical mechanisms and conformational dynamics of strand cleavage/exchange between DNA partners. Current evidence suggests a rather intriguing mechanism for serine recombination, in which one half of the cleaved synaptic complex undergoes a 180° rotation relative to the other. The 'small' and 'large' SRs contain a compact amino-terminal catalytic domain, but differ conspicuously in their carboxyl-terminal domains. So far, only one serine recombinase has been analyzed using single substrate molecules. We now utilized single-molecule tethered particle motion (TPM) to follow step-by-step recombination catalyzed by a large SR, phage ϕC31 integrase. The integrase promotes unidirectional DNA exchange between attB and attP sites to integrate the phage genome into the host chromosome. The recombination directionality factor (RDF; ϕC31 gp3) activates the excision reaction (attL × attR). From integrase-induced changes in TPM in the presence or absence of gp3, we delineated the individual steps of recombination and their kinetic features. The gp3 protein appears to regulate recombination directionality by selectively promoting or excluding active conformations of the synapse formed by specific att site partners. Our results support a 'gated rotation' of the synaptic complex between DNA cleavage and joining.

Citing Articles

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Sales T, de Oliveira M, Florentino L, Lima R, Rech E Front Bioeng Biotechnol. 2025; 13:1478413.

PMID: 40066361 PMC: 11891168. DOI: 10.3389/fbioe.2025.1478413.

Direct observation of subunit rotation during DNA strand exchange by serine recombinases.

Cadden G, Schloetel J, McKenzie G, Boocock M, Magennis S, Stark W Nat Commun. 2024; 15(1):10407.

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Quantitation of DNA Binding Affinity Using Tethered Particle Motion.

Henneman B, Erkelens A, Heinsman J, Battjes J, Dame R Methods Mol Biol. 2024; 2819:497-518.

PMID: 39028521 DOI: 10.1007/978-1-0716-3930-6_23.

Protocol for the establishment of a serine integrase-based platform for functional validation of genetic switch controllers in eukaryotic cells.

de Oliveira M, Florentino L, Sales T, Lima R, Barros L, Limia C PLoS One. 2024; 19(5):e0303999.

PMID: 38781126 PMC: 11115199. DOI: 10.1371/journal.pone.0303999.

The influence of coiled-coil motif of serine recombinase toward the directionality regulation.

Chen Y, Su B, Van Duyne G, Fogg P, Fan H Biophys J. 2023; 122(24):4656-4669.

PMID: 37974397 PMC: 10754689. DOI: 10.1016/j.bpj.2023.11.009.

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