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J. Biol. Chem., Vol. 276, Issue 33, 31092-31098, August 17, 2001
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and
From the Department of Molecular and Medical Genetics, University
of Toronto, Toronto, Ontario M5S 1A8, Canada
The Cre recombinase of bacteriophage P1
cleaves its target site, loxP, in a defined order.
Recombination is initiated on one pair of strands to form a Holliday
intermediate, which is then resolved by cleavage and exchange
of the other pair of strands to yield recombinant products. To
investigate the influence of the loxP sequence on the
directionality of resolution, we constructed synthetic Holliday (
)
structures containing either wild-type or mutant lox sites.
We found that Cre preferentially resolved the synthetic wild-type
structures on a particular pair of strands. The bias in the
direction of resolution was dictated by the asymmetric loxP
sequence since the resolution bias was abolished with symmetric lox sites. Systematic substitutions of the loxP
site revealed that the bases immediately 5' to the scissile
phosphodiester bonds were primarily responsible for the directionality
of resolution. Interchanging these base pairs was sufficient to reverse
the resolution bias. The Cre-lox co-crystal structures show
that Lys86 makes a base-specific contact with guanine
immediately 5' to one of the scissile phosphates. Substituting
Lys86 with alanine resulted in a reduction of the
resolution bias, indicating that this amino acid is important for
establishing the bias in resolution.
Supported by scholarships from the Natural Sciences and
Engineering Research Council of Canada and from the Medical Research Council of Canada.
§
To whom correspondence should be addressed. Tel.: 416-978-6061;
Fax: 416-978-6885; E-mail: p.sadowski@utoronto.ca.
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