Integration and excision by the large serine recombinase phiRv1 integrase.
The Mycobacterium tuberculosis prophage-like element phiRv1 encodes a site-specific recombination system utilizing an integrase of the serine recombinase family. Recombination occurs between a putative attP site and the host chromosome, but is unusual in that the attB site lies within a redundant repetitive element (REP13E12) of which there are seven copies in the M. tuberculosis genome; four of these elements contain attB sites suitable for phiRv1 integration in vivo. Although the mechanism of directional control of large serine integrases is poorly understood, a recombination directionality factor (RDF) has been identified that is required for phiRv1 integrase-mediated excisive recombination in vivo. Here we describe defined in vitro recombination reactions for both phiRv1 integrase-mediated integration and excision and show that the phiRv1 RDF is not only required for excision but inhibits integrative recombination; neither reaction requires DNA supercoiling, host factors, or high-energy cofactors. Integration, excision and excise-mediated inhibition of integration require simple substrates sites, indicating that the control of directionality does not involve the manipulation of higher-order protein-DNA architectures as described for the tyrosine integrases.
Bibb
Lori A
LA
Pittsburgh Bacteriophage Institute and Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Hancox
Maria I
MI
Hatfull
Graham F
GF
D001287:Attachment Sites, Microbiological / Q000235:genetics / Q000502:physiology; D001483:Base Sequence; D004269:DNA, Bacterial / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D019426:Integrases / Q000502:physiology*; D008969:Molecular Sequence Data; D009160:Mycobacteriophages / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D009169:Mycobacterium tuberculosis / Q000821:virology*; D039002:Prophages / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D011995:Recombination, Genetic; D013379:Substrate Specificity; D016662:Virus Integration
D004269:DNA, Bacterial; D004279:DNA, Viral; D019426:Integrases