期刊
NUCLEIC ACIDS RESEARCH
卷 43, 期 12, 页码 6134-6143出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkv521
关键词
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资金
- Biotechnology and Biosciences Research Council [BB/E0022200, B18146, BB/K003356/1]
- Wellcome Trust [086413]
- Biotechnology and Biological Sciences Research Council [BB/E022200/1, BB/K003356/1] Funding Source: researchfish
- BBSRC [BB/K003356/1, BB/E022200/1] Funding Source: UKRI
To analyse the mechanism and kinetics of DNA strand cleavages catalysed by the serine recombinase Tn3 resolvase, we made modified recombination sites with a single-strand nick in one of the two DNA strands. Resolvase acting on these sites cleaves the intact strand very rapidly, giving an abnormal half-site product which accumulates. We propose that these reactions mimic second-strand cleavage of an unmodified site. Cleavage occurs in a synapse of two sites, held together by a resolvase tetramer; cleavage at one site stimulates cleavage at the partner site. After cleavage of a nicked-site substrate, the half-site that is not covalently linked to a resolvase subunit dissociates rapidly from the synapse, destabilizing the entire complex. The covalent resolvase-DNA linkages in the natural reaction intermediate thus perform an essential DNA-tethering function. Chemical modifications of a nicked-site substrate at the positions of the scissile phosphodiesters result in abolition or inhibition of resolvase-mediated cleavage and effects on resolvase binding and synapsis, providing insight into the serine recombinase catalytic mechanism and how resolvase interacts with the substrate DNA.
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