Archaeal MutS5 tightly binds to Holliday junction similarly to eukaryotic MutSγ

<jats:p>Archaeal <jats:styled-content style="fixed-case">DNA</jats:styled-content> recombination mechanism and the related proteins are similar to those in eukaryotes. However, no functional homolog of eukaryotic MutSγ, which recognizes Holliday junction to promote homologous recombination, has been identified in archaea. Hence, the whole molecular mechanism of archaeal homologous recombination has not yet been revealed. In this study, to identify the archaeal functional homolog of MutSγ, we focused on a functionally uncharacterized MutS homolog, MutS5, from a hyperthermophilic archaeon <jats:italic>Pyrococcus horikoshii</jats:italic> (phMutS5). Archaeal MutS5 has a Walker <jats:styled-content style="fixed-case">ATP</jats:styled-content>ase motif‐containing amino acid sequence that shows similarity to the <jats:styled-content style="fixed-case">ATP</jats:styled-content>ase domain of MutSγ. It is known that the <jats:styled-content style="fixed-case">ATP</jats:styled-content>ase domain of MutS homologs is also a dimerization domain. Chemical cross‐linking revealed that purified phMutS5 has an ability to dimerize in solution. phMutS5 bound to Holliday junction with a higher affinity than to other branched and linear <jats:styled-content style="fixed-case">DNA</jats:styled-content>s, which resembles the <jats:styled-content style="fixed-case">DNA</jats:styled-content>‐binding specificities of MutSγ and bacterial MutS2, a Holliday junction‐resolving MutS homolog. However, phMutS5 has no nuclease activity against branched <jats:styled-content style="fixed-case">DNA</jats:styled-content> unlike MutS2. The <jats:styled-content style="fixed-case">ATP</jats:styled-content>ase activity of phMutS5 was significantly stimulated by the presence of Holliday junction similarly to MutSγ. Furthermore, site‐directed mutagenesis revealed that the <jats:styled-content style="fixed-case">ATP</jats:styled-content>ase activity is dependent on the Walker <jats:styled-content style="fixed-case">ATP</jats:styled-content>ase motif of the protein. These results suggest that archaeal MutS5 should stabilize the Holliday junction and play a role in homologous recombination, which is analogous to the function of eukaryotic MutSγ.</jats:p>