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

Abstract

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.

Archaeal tyrosine recombinases

“… 2013 ; Jo et al . 2017 ). Moreover, archaeal PaXerA and TaXerA proteins display the canonical structure of tyrosine recombinases comprising two domains surrounding the DNA substrate in a C-shape conformation (Serre et al .…”

“… 2013 ; Jo et al . 2017 ). The two suicidal Thermococcales integrases IntpTN3 from plasmid pTN3 and IntpT26-2 from plasmid pT26-2, were also shown to catalyze site-specific recombination on circular substrates in vitro .…”

“… 2017 ; Jo et al . 2017 ). Linear substrates are not their natural substrates but are useful to characterize some aspects of the integrase activity such as the strand cleavage site (Serre et al .…”

“…The archaeal recombinases IntpT26-2, IntSSV1 and TaXerA also presented non-specific DNA relaxation activities (Letzelter, Duguet and Serre 2004 ; Jo et al . 2017 ; Badel et al . 2020 ).…”

“… 2002 , 2013 ; Jo et al . 2017 ). ( B) General organization of a tRNA indicating the location of the T, A and D loops and the three preferred integration locations for bacterial integrases (I, II and III) (Williams 2002 ).…”