Bacterial-type DNA Holliday junction resolvases in eukaryotic viruses

Abstract

Homologous DNA recombination promotes genetic diversity and the maintenance of genome integrity, yet no enzymes with specificity for the Holliday junction (HJ)-a key DNA recombination intermediate-have been purified and characterized from metazoa or their viruses. Here we identify critical structural elements of RuvC, a bacterial HJ resolvase, in uncharacterized open reading frames from poxviruses and an iridovirus. The putative vaccinia virus resolvase was expressed as a recombinant protein, affinity purified, and shown to specifically bind and cleave a synthetic HJ to yield nicked duplex molecules. Mutation of either of two conserved acidic amino acids abrogated the catalytic activity of the A22R protein without affecting HJ binding. The presence of bacterial-type enzymes in metazoan viruses raises evolutionary questions. Homologous DNA recombination is a ubiquitous process that promotes genetic diversity and the maintenance of genome integrity in prokaryotic and eukaryotic organisms. The central intermediate in the recombination process is a four-way DNA junction usually referred to as a Holliday junction (HJ), consisting of two homologous duplex DNA molecules joined by cross-over strands (1, 2). Although HJ-resolving activities have been found in extracts prepared from a wide variety of organisms, only enzymes from bacteria, bacteriophages, yeast mitochondria, and archaea have been genetically identified and characterized. The latter include Escherichia coli RuvC (3-5), bacteriophage T4 endonuclease VII (6), bacteriophage T7 endonuclease I (7, 8), lambdoid prophage RusA (9), Saccharomyces cerevisiae mitochondrial CCE1 (10), Schizosaccharomyces pombe mitochondrial YDC2 (11), and Pyrococcus furiosus Hjc protein (12). These enzymes can be divided into two functional groups. Members of the first have RuvC as their prototype, include the resolvases from bacteria, mitochondria, and archaea, have high selectivity for HJs, exhibit sequence specificity for cleavage, and are thought to have roles in recombination and DNA repair. Members of the second group, composed of the bacteriophage enzymes, cleave a variety of branched DNAs, exhibit low sequence specificity, and have roles in recombination and the processing of DNA before packaging. Absent from either group, however, is a well-characterized HJ endonuclease from metazoa or their viruses.The poxviruses, of which vaccinia virus is the prototype, are large DNA viruses that replicate in the cytoplasm of infected cells and consequently provide a unique system for studying enzymes involved in RNA and DNA synthesis (13). Although the ability of poxviruses to undergo recombination has been long known (14), this process has not been separated from replication (15), and the viral proteins involved have remained elusive. An enzyme that cleaves HJs may participate in recombination as well as other steps in processing and packaging of poxvirus DNA. The genomes of poxviruses consist of a single linear doublestranded DNA molecule with covalently linked hairpin termini (16, 17) t...

Poxvirus DNA topoisomerase knockout mutant exhibits decreased infectivity associated with reduced early transcription

“…The topoisomerase was the only candidate resolvase (12) before the discovery of the poxvirus HJ endonuclease (13). However, whereas concatemers accumulated when synthesis of the HJ endonuclease was repressed (14), we found no such accumulation in the absence of topoisomerase.…”

“…The vaccinia virus topoisomerase can resolve Holliday junctions (HJs) in vitro, suggesting a role in the resolution of viral DNA concatemers to form unit genomes with hairpin telomeres (9,11,12). Biochemical and genetic studies, however, attribute this function to a recently discovered poxvirus HJ endonuclease with homology to bacterial RuvC (13,14). Roles for poxvirus topoisomerase in DNA replication (15), recombination (16,17), and compaction of the viral genome for packaging have also been considered.…”

Holliday junction-binding peptides inhibit distinct junction-processing enzymes

“…RecG is highly conserved among bacteria but absent from most eukaryotes, where RecQ helicase homologs may fulfill a similar function (19)(20)(21)(22). Enzymes related to RuvC are found in most bacteria, archaea, and some eukaryotic viruses (15,16,23). Although RuvC homologs are not present in eukaryotes, HJ-resolving enzymes from eukaryotes are of great interest (22,(24)(25)(26)(27)(28).…”

Mapping vaccinia virus DNA replication origins at nucleotide level by deep sequencing

“…As expected for a genome with such covalently closed ends, VACV replicative intermediates are headto-head or tail-to-tail concatemers (4,5). The concatemers exist only transiently because they are cleaved by a virus-encoded Holliday junction resolvase into unit length genomes with hairpin ends before incorporation into virus particles (6,7). Because VACV genomes and concatemers resemble the replicative intermediates of the much smaller parvoviruses, the rolling hairpin model of replication originally proposed for the latter family was extended to poxviruses and has become the current scheme for poxvirus genome replication (2).…”