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

Josefin Ahlqvist,Slawomir Dabrowski,Salam Al-Karadaghi,Ólafur H Friðjónsson,Arnþór Ævarsson,Maria Håkansson,Karolina Kwiatkowska-Semrau,Anna-Karina Kaczorowska,Eva Nordberg Karlsson,Guðmundur Ó Hreggviðsson,Andrius Jasilionis,Javier A Linares-Pastén,Tadeusz Kaczorowski

doi:10.1107/s2059798321012298

Abstract

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.

Highlights

Holliday junction-resolving enzymes are nucleases that cleave four-way DNA–Holliday junctions (Hjs) into two unconnected DNA duplexes
ORFs within the phage Tth15-6 genome were predicted by the RAST server and suggested the presence of a gene encoding a putative DNA-resolving enzyme/helicase-like enzyme of 155 amino-acid residues extending from coordinate nucleotide 18521 to nucleotide 18988 in the phage Tth15-6 genome
At the same time, the corresponding motifs found in Hj-resolving enzymes from other species are E-PD-EVK or even E-VD-EVK, as in the case of the archeal Hj-resolving enzyme from P. furiosus. Based on this observation and the discussion above, we propose that Hjc_15-6 and several other Hj-resolving enzymes originating from thermophilic phages will have a three-part signature motif corresponding to E-SD-EVK

Summary

Introduction

Holliday junction-resolving enzymes are nucleases that cleave four-way DNA–Holliday junctions (Hjs) into two unconnected DNA duplexes. Hjs are common intermediates during meiotic and mitotic genetic recombination, and Hj-resolving enzymes have been isolated from all types of eukaryotic and prokaryotic cells and their viruses (Wyatt & West, 2014). Hjs were first presented by Robin Holliday in 1964, when he suggested a model for gene conversion during meiosis in fungi (Holliday, 1964), claiming that two homologous chromosomes paired between complementary sequences lead to the formation of a cross-stranded structure that physically links the two component helices. Other models of how and why two dsDNA helices may cross-link have been reported. This type of four-way DNA structure at the point of strand. D78, 212–227 research papers exchange has become known as an Hj, and the enzymes that resolve them are referred to as Hj-resolving enzymes

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