Genetic Study of Four Candidate Holliday Junction Processing Proteins in the Thermophilic Crenarchaeon .

Homologous recombination (HR) is thought to be important for the repair of stalled replication forks in hyperthermophilic archaea. Previous biochemical studies identified two branch migration helicases (Hjm and PINA) and two Holliday junction (HJ) resolvases (Hjc and Hje) as HJ-processing proteins; however, due to the lack of genetic evidence, it is still unclear whether these proteins are actually involved in HR in vivo and how their functional relation is associated with the process. To address the above questions, we constructed -, -, -, and single-knockout strains and double-knockout strains of the thermophilic crenarchaeon and characterized the mutant phenotypes. Notably, we succeeded in isolating the - and/or -deleted strains, suggesting that the functions of Hjm and PINA are not essential for cellular growth in this archaeon, as they were previously thought to be essential. Growth retardation in Δ was observed at low temperatures (cold sensitivity). When deletion of the HJ resolvase genes was combined, Δ Δ and Δ Δ exhibited severe cold sensitivity. Δ exhibited severe sensitivity to interstrand crosslinkers, suggesting that Hjm is involved in repairing stalled replication forks, as previously demonstrated in euryarchaea. Our findings suggest that the function of PINA and HJ resolvases is functionally related at lower temperatures to support robust cellular growth, and Hjm is important for the repair of stalled replication forks in vivo.