In vitro site‐specific recombination mediated by the tyrosine recombinase XerA of Thermoplasma acidophilum

• Published in: Genes to cells : devoted to molecular & cellular mechanisms Vol. 22; no. 7; pp. 646 - 661
• Main Authors: Jo, Minji, Murayama, Yasuto, Tsutsui, Yasuhiro, Iwasaki, Hiroshi
• Format: Journal Article
• Language: English
• Published: England Wiley Subscription Services, Inc 01.07.2017
• Subjects: Archaeal Proteins - genetics; Archaeal Proteins - metabolism; Binding Sites; Cell division; Chromosomes; Conserved sequence; DNA, Archaeal - genetics; Genome, Bacterial; Homologous recombination; In Vitro Techniques; Monomers; Mutation; Plasmids; Recombinase; Recombinases - metabolism; Recombination, Genetic; Substrate Specificity; Thermoplasma - enzymology; Thermoplasma - genetics; Thermoplasma - growth & development; Translocase; Tyrosine; Tyrosine - metabolism
• ISSN: 1356-9597; 1365-2443
• DOI: 10.1111/gtc.12503

In organisms with circular chromosomes, such as bacteria and archaea, an odd number of homologous recombination events can generate a chromosome dimer. Such chromosome dimers cannot be segregated unless they are converted to monomers before cell division. In Escherichia coli, dimer‐to‐monomer conversion is mediated by the paralogous XerC and XerD recombinases at a specific dif site in the replication termination region. Dimer resolution requires the highly conserved cell division protein/chromosome translocase FtsK, and this site‐specific chromosome resolution system is present or predicted in most bacteria. However, most archaea have only XerA, a homologue of the bacterial XerC/D proteins, but no homologues of FtsK. In addition, the molecular mechanism of XerA‐mediated chromosome resolution in archaea has been less thoroughly elucidated than those of the corresponding bacterial systems. In this study, we identified two XerA‐binding sites (dif1 and dif2) in the Thermoplasma acidophilum chromosome. In vitro site‐specific recombination assays showed that dif2, but not dif1, serves as a target site for XerA‐mediated chromosome resolution. Mutational analysis indicated that not only the core consensus sequence of dif2, but also its flanking regions play important roles in the recognition and recombination reactions mediated by XerA. XerA resolves a plasmid dimer with the Peak 2 sequence to monomers.