LAMBDA INTEGRASE CLEAVES DNA IN CIS

In the Int family of site-specific recombinases, DNA cleavage is accom plished by nucleophilic attack on the activated scissile phosphodieste r bond by a specific tyrosine residue, It has been proposed that this tyrosine is contributed by a protomer bound to a site other than the o ne being cleaved ('trans' cleavage). To test this hypothesis, the diff erence in DNA binding specificity between closely related integrases ( Ints) from phages lambda and HK022 was exploited to direct wild type I nts and cleavage- or activation-defective mutants to particular sites on bispecific substrates. Analysis of Int cleavage at individual sites strongly indicates that DNA cleavage is catalyzed by the Int bound to the cleaved site ('cis' cleavage). This conclusion contrasts with tho se from previous experiments with two members of the Int family, FLP a nd lambda Int, that supported the hypothesis of trans cleavage. We sug gest explanations for this difference and discuss the implications of the surprising finding that Int-family recombinases appear capable of both cis and trans mechanisms of DNA cleavage.