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Curr Biol. 1995 Nov 1;5(11):1312-21.

Suicide substrates reveal properties of the homology-dependent steps during integrative recombination of bacteriophage lambda.

Author information

1
Laboratory of Molecular Biology, National Institute of Mental Health, Bethesda, Maryland 20892-4034, USA. burgin@csn.org

Abstract

BACKGROUND:

A fundamental feature of bacteriophage lambda site-specific recombination is the strict requirement for a region of sequence identity between recombining DNA duplexes. It has been difficult to understand how the recombination machinery identifies and responds to nonhomologies as subtle as a single base-pair substitution, because the reaction intermediates are transient and there are likely to be several different homology-dependent steps. In order to understand better how the recombination machinery compares parental sequences, we have used the recently developed 'suicide substances'--DNA containing 5'-bridging phosphorothioate linkages--to monitor the timing of homology-sensing relative to the strand cleavage reactions.

RESULTS:

The cleavage reactions for the two different strands of attB, the bacterial recombination locus for lambda integration, show very different degrees of dependence on homology with the partner locus, attP. Strand cleavage at the B binding site for Int recombinase is insensitive to homology. In contrast, cleavage at the B' binding site strongly depends on homology in the three base pairs adjacent to the B site. Strand cleavage at the B site is apparently required for the readout of this homology but, surprisingly, joining of the cleaved B site to a partner is not.

CONCLUSIONS:

Our finding that cleavage at the B site is insensitive to homology shows that effective synapsis between partners does not depend on sequence matching. Cleavage at the B' site provides the earliest positive signal for a homology-dependent switch in the lambda recombination machinery. Because this switch can occur in the absence of strand joining, the results argue against models that invoke strand ligation as the critical element of homology-sensing. Alternative mechanisms are presented that involve varieties of non-covalent strand swapping. A synthesis of the present results and other recent experiments highlights the importance of the disannealing of complementary strands and their reannealing to new partners, a process traditionally described as branch migration. The reversibility of branch migration and its bias away from mismatched combinations are proposed to be the major mechanisms of homology-sensing during lambda integration.

PMID:
8574589
[Indexed for MEDLINE]
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