The Carboxy-Terminal αN Helix of the Archaeal XerA Tyrosine Recombinase Is a Molecular Switch to Control Site-Specific Recombination

Abstract

Tyrosine recombinases are conserved in the three kingdoms of life. Here we present the first crystal structure of a full-length archaeal tyrosine recombinase, XerA from Pyrococcus abyssi, at 3.0 Å resolution. In the absence of DNA substrate XerA crystallizes as a dimer where each monomer displays a tertiary structure similar to that of DNA-bound Tyr-recombinases. Active sites are assembled in the absence of dif except for the catalytic Tyr, which is extruded and located equidistant from each active site within the dimer. Using XerA active site mutants we demonstrate that XerA follows the classical cis-cleavage reaction, suggesting rearrangements of the C-terminal domain upon DNA binding.Surprisingly, XerA C-terminal αN helices dock in cis in a groove that, in bacterial tyrosine recombinases, accommodates in trans αN helices of neighbour monomers in the Holliday junction intermediates. Deletion of the XerA C-terminal αN helix does not impair cleavage of suicide substrates but prevents recombination catalysis. We propose that the enzymatic cycle of XerA involves the switch of the αN helix from cis to trans packing, leading to (i) repositioning of the catalytic Tyr in the active site in cis and (ii) dimer stabilisation via αN contacts in trans between monomers.

Structural snapshots of Xer recombination reveal activation by synaptic complex remodeling and DNA bending

“…A simple rotation of the N-terminal domain pivoting at the interdomain linker is sufficient to transition from the ‘closed’ XerD conformation to the ‘open’ DNA-bound XerH conformation (Figure 2—figure supplement 3), consistent with previous proposals that Xer recombinases may undergo a major conformational change upon DNA binding (Jo et al, 2016; Serre et al, 2013; Subramanya et al, 1997). …”

“…However, in the absence of direct structural data the exact architecture of Xer-DNA complexes has remained unknown. Previous DNA-free crystal structures of Xer recombinases (Jo et al, 2016; Serre et al, 2013; Subramanya et al, 1997) showed a domain arrangement that is incompatible with DNA binding. Another puzzling aspect of the mechanism concerned activation by FtsK.…”

Site-Specific Recombination by SSV2 Integrase: Substrate Requirement and Domain Functions

“…The ability of Int SSV2 to catalyze both integration and excision by itself and the characteristics of the site of recombination for the enzyme suggest that SSV-type integrases function in a simple type of site-specific recombination. Recently, a similar observation was made for XerA, a tyrosine recombinase involved in chromosome resolution, from Pyrococcus abyssi (24,25). The protein was shown to be able to recombine dif sites in vitro in the absence of any protein partners.…”

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

“…If the free 5′‐OH groups of the uncleaved strands can attack the covalent complex intermolecularly, as previously observed with P. abyssi XerA (Serre et al . ), the reaction should generate a recombinant product containing the top (63‐nt) and bottom (65‐nt) strands.…”

“…The crystal structure of the full‐length XerA recombinase from P. abyssi has been determined at 3.0 Å resolution (Serre et al . ). Very recently, Jo et al .…”