Resolution of mismatched overlap Holliday junction intermediates by the tyrosine recombinase IntDOT

Kenneth Ringwald, Sumiko Yoneji, Jeffrey Gardner

Research output: Contribution to journalArticle

Abstract

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 aminoterminal (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 armtype sites and a DNA-bending protein. We constructed synthetic HJs with the armtype 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.

Original languageEnglish (US)
Article numbere00873-16
JournalJournal of bacteriology
Volume199
Issue number10
DOIs
StatePublished - May 1 2017

Keywords

  • Conjugative transposons
  • DNA-protein interactions
  • Holliday junctions
  • Integrase
  • Integrative conjugative elements
  • Recombination
  • Tyrosine recombinases

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

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