Crystal structure and initial characterization of a novel archaeal-like Holliday junction-resolving enzyme from Thermus thermophilus phage Tth15-6

Acta Crystallogr D Struct Biol. 2022 Feb 1;78(Pt 2):212-227. doi: 10.1107/S2059798321012298. Epub 2022 Jan 24.


This study describes the production, characterization and structure determination of a novel Holliday junction-resolving enzyme. The enzyme, termed Hjc_15-6, is encoded in the genome of phage Tth15-6, which infects Thermus thermophilus. Hjc_15-6 was heterologously produced in Escherichia coli and high yields of soluble and biologically active recombinant enzyme were obtained in both complex and defined media. Amino-acid sequence and structure comparison suggested that the enzyme belongs to a group of enzymes classified as archaeal Holliday junction-resolving enzymes, which are typically divalent metal ion-binding dimers that are able to cleave X-shaped dsDNA-Holliday junctions (Hjs). The crystal structure of Hjc_15-6 was determined to 2.5 Å resolution using the selenomethionine single-wavelength anomalous dispersion method. To our knowledge, this is the first crystal structure of an Hj-resolving enzyme originating from a bacteriophage that can be classified as an archaeal type of Hj-resolving enzyme. As such, it represents a new fold for Hj-resolving enzymes from phages. Characterization of the structure of Hjc_15-6 suggests that it may form a dimer, or even a homodimer of dimers, and activity studies show endonuclease activity towards Hjs. Furthermore, based on sequence analysis it is proposed that Hjc_15-6 has a three-part catalytic motif corresponding to E-SD-EVK, and this motif may be common among other Hj-resolving enzymes originating from thermophilic bacteriophages.

Keywords: archaeal-like Holliday junction-resolving enzymes; signature motifs; structure–function relationship; thermophilic bacteriophages.

MeSH terms

  • Archaea / genetics
  • Archaea / metabolism
  • Bacteriophages* / genetics
  • Bacteriophages* / metabolism
  • DNA, Cruciform*
  • Holliday Junction Resolvases / chemistry
  • Holliday Junction Resolvases / genetics
  • Holliday Junction Resolvases / metabolism
  • Thermus thermophilus


  • DNA, Cruciform
  • Holliday Junction Resolvases