Sequence-specific and non-specific binding of the Rci protein to the asymmetric recombination sites of the R64 shufflon

Specific cleavages within the shufflon-specific recombination site plasmid R64 were detected by primer extension when a DNA fragment carrying the recombination site was incubated with the shufflon-specific Rci recombinase. Rci-dependent cleavages occurred in the form of a 51 protruding 7bp staggered cut, suggesting that DNA cleavage and rejoining in the shufflon system take place at these positions. As a result, shufflon crossover sites were designated as sfx sequences consisting of a central 7bp spacer sequence, and left and right 12bp arms. R64 sfx sequences are unique among various site-specific recombination sites, since only the spacer sequence and the right arm sequence are conserved among various R64 sfxs, whereas the left arm sequence is not conserved and is not related to the right arm sequence. From nuclease protection analyses, Rci protein was shown to bind to entire R64 and artificial sfx sequences, suggesting that one Rci molecule binds to the conserved sfx right arm in a sequence-specific manner and the second to the sfx left arm in a non-specific manner. The sfx left arm sequences as well as the right arm sequences were shown to determine affinity to Rci and subsequently inversion frequency. Asymmetry of the sfx sequence may be the reason why Rci protein acts only on the inverted sfx sequences. (C) 2002 Elsevier Science Ltd. All rights reserved.