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Homology-dependent interactions determine the order of strand exchange by IntDOT recombinase.

ABSTRACT: The Bacteroides conjugative transposon CTnDOT encodes an integrase, IntDOT, which is a member of the tyrosine recombinase family. Other members of this group share a strict requirement for sequence identity within the region of strand exchange, called the overlap region. Tyrosine recombinases catalyze recombination by making an initial cleavage, strand exchange and ligation, followed by strand swapping isomerization requiring sequence identity in the overlap region, followed by the second cleavage, strand exchange and ligation. IntDOT is of particular interest because it has been shown to utilize a three-step mechanism: a sequence identity-dependent initial strand exchange that requires two base pairs of complementary DNA at the site of cleavage; a sequence identity-independent strand swapping isomerization, followed by a sequence identity-independent cleavage, strand exchange and ligation. In addition to the sequence identity requirement in the overlap region, Lambda Int interactions with arm-type sites dictate the order of strand exchange regardless of the orientation of the overlap region. Although IntDOT has an arm-binding domain, we show here that the location of sequence identity within the overlap region dictates where the initial cleavage takes place and that IntDOT can recombine substrates containing mismatches in the overlap region so long as a single base of sequence identity exists at the site of initial cleavage.

SUBMITTER: Laprise J

PROVIDER: S-EPMC2817482 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Homology-dependent interactions determine the order of strand exchange by IntDOT recombinase.

Laprise Jennifer J Yoneji Sumiko S Gardner Jeffrey F JF

Nucleic acids research 20091201 3

The Bacteroides conjugative transposon CTnDOT encodes an integrase, IntDOT, which is a member of the tyrosine recombinase family. Other members of this group share a strict requirement for sequence identity within the region of strand exchange, called the overlap region. Tyrosine recombinases catalyze recombination by making an initial cleavage, strand exchange and ligation, followed by strand swapping isomerization requiring sequence identity in the overlap region, followed by the second cleava ...[more]

PMID: 19952068

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Project description:CTnDOT is an integrated conjugative element found in Bacteroides species. CTnDOT contains and transfers antibiotic resistance genes. The element integrates into and excises from the host chromosome via a Holliday junction (HJ) intermediate as part of a site-specific recombination mechanism. The CTnDOT integrase, IntDOT, is a tyrosine recombinase with core-binding, catalytic, and amino-terminal (N) domains. Unlike well-studied tyrosine recombinases, such as lambda integrase (Int), IntDOT is able to resolve Holliday junctions containing heterology (mismatched bases) between the sites of strand exchange. All known natural isolates of CTnDOT contain mismatches in the overlap region between the sites of strand exchange. Previous work showed that IntDOT was unable to resolve synthetic Holliday junctions containing mismatched bases to products in the absence of the arm-type sites and a DNA-bending protein. We constructed synthetic HJs with the arm-type sites and tested them with the Bacteroides host factor (BHFa). We found that the addition of BHFa stimulated resolution of HJ intermediates with mismatched overlap regions to products. In addition, the L1 site is required for directionality of the reaction, particularly when the HJ contains mismatches. BHFa is required for product formation when the overlap region contains mismatches, and it stimulates resolution to products when the overlap region is identical. Without this DNA bending, the N domain of IntDOT is likely unable to bind the L1 arm-type site. These findings suggest that BHFa bends DNA into the necessary conformation for the higher-order complexes, including the L1 site, that are required for product formation.IMPORTANCE CTnDOT is a mobile element that carries antibiotic resistance genes and moves by site-selective recombination and subsequent conjugation. The recombination reaction is catalyzed by an integrase IntDOT that is a member of the tyrosine recombinase family. The reaction proceeds through ordered strand exchanges that generate a Holliday junction (HJ) intermediate. Unlike other tyrosine recombinases, IntDOT can resolve HJs containing mismatched bases in the overlap region in vivo, as is the case under natural conditions. However, HJ intermediates including only IntDOT core-type sites cannot be resolved to products if the HJ intermediate contains mismatched bases. We added arm-type sites in cis and in trans to the HJ intermediates and the protein BHFa to study the requirements for higher-order nucleoprotein complexes.

Dataset Information

Homology-dependent interactions determine the order of strand exchange by IntDOT recombinase.

Publications

Homology-dependent interactions determine the order of strand exchange by IntDOT recombinase.

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