Sequences in attB that affect the ability of C integrase to synapse and to activate DNA cleavage

作者：Milind Gupta, Rob Till2 and Margaret C. M. Smith    作者单位：1Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD and 2Institute of Genetics, Queens Medical Centre, University of Nottingham, Nottingham NG7 2UH, UKTo whom correspondence should be addressed. Tel: 01224 555739; Fax: 01224 555844; Email: Maggie.smith@abdn.ac.uk

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【摘要】  Phage integrases are required for recombination of the phage genome with the host chromosome either to establish or exit from the lysogenic state. C31 integrase is a member of the serine recombinase family of site-specific recombinases. In the absence of any accessory factors integrase is unidirectional, catalysing the integration reaction between the phage and host attachment sites, attP x attB to generate the hybrid sites, attL and attR. The basis for this directionality is due to selective synapsis of attP and attB sites. Here we show that mutations in attB can block the integration reaction at different stages. Mutations at positions distal to the crossover site inhibit recombination by destabilizing the synapse with attP without significantly affecting DNA-binding affinity. These data are consistent with the proposal that integrase adopts a specific conformation on binding to attB that permits synapsis with attP. Other attB mutants with changes close to the crossover site are able to form a stable synapse but cleavage of the substrates is prevented. These mutants indicate that there is a post-synaptic DNA recognition event that results in activation of DNA cleavage.

【关键词】  sequences integrase activate cleavage

INTRODUCTION

C31 integrase and several of its relatives are being widely used for precise engineering of complex genomes (1¨C8) and are emerging as promising new tools for gene therapy (9¨C15). In addition to being highly portable C31 integrase is, unlike other recombinases used for genome manipulation such as Cre and Flp, unidirectional (7,16,17). In nature phage integrases are required for recombination of the phage genome with the host chromosome either to establish or exit from the lysogenic state. For integration the host-encoded attB site undergoes a conservative and reciprocal recombination with the phage attP site to form the hybrid product sites, attL and attR. During induction into the lytic cycle, the phage genome excises and this reaction normally requires integrase and an accessory protein Xis (18,19). Phage-encoded integrases can belong to the tyrosine or the serine recombinase families (20). Both families of proteins act by binding to their cognate substrates and bringing the DNAs together in a synapse. Recombination is initiated by cleaving DNA strands, which undergo strand exchange to form recombinant products and these are then released (21). While the mechanism of phage integrase, a tyrosine recombinase, is well understood (18,22,23), the mechanism of action of integrases such as C31 integrase that belong to the serine recombinase family, is less clear.

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