λ integrase cleaves DNA in cis

S. E. Nunes-Düby, R. S. Tirumalai, L. Dorgai, E. Yagil, R. A. Weisberg, Arthur Landy*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

In the Int family of site-specific recombinases, DNA cleavage is accomplished by nucleophilic attack on the activated scissile phosphodiester 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 one being cleaved ('trans' cleavage). To test this hypothesis, the difference in DNA binding specificity between closely related integrases (Ints) from phages λ and HK022 was exploited to direct wild type Ints 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 those from previous experiments with two members of the Int family, FLP and λInt, that supported the hypothesis of trans cleavage. We suggest 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.

Original languageEnglish
Pages (from-to)4421-4430
Number of pages10
JournalEMBO Journal
Volume13
Issue number18
DOIs
StatePublished - 1994

Keywords

  • DNA cleavage
  • HK022 integrase
  • Holiday junctions
  • Site-specific recombination
  • λ integrase

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