Abstract
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 hjc-, hje-, hjm-, and pina single-knockout strains and double-knockout strains of the thermophilic crenarchaeon Sulfolobus acidocaldarius and characterized the mutant phenotypes. Notably, we succeeded in isolating the hjm- and/or pina-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 Δpina was observed at low temperatures (cold sensitivity). When deletion of the HJ resolvase genes was combined, Δpina Δhjc and Δpina Δhje exhibited severe cold sensitivity. Δhjm 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.
Highlights
Publisher’s Note: MDPI stays neutralThermophiles inhabit a hot environment that accelerates the rate of DNA damage [1].It has been proposed that DNA repair in thermophilic archaea has unique properties in order to be robust in extreme environments [2,3]
Most disrupted strains were constructed, except for ∆hjc ∆hje. This exception is compatible with a previous genetic study in “S. islandicus” [19], suggesting that the function of Hjc and Hje is redundant and essential for cellular viability in Sulfolobales
The pina-deleted strain exhibited growth retardation at a lower temperature (55 ◦ C), but not at a higher temperature (Figure 3A–C). This cold-sensitive phenotype was accelerated by additional deletion of the Holliday junction (HJ) resolvases Hjc or Hje (Figure 3C)
Summary
Publisher’s Note: MDPI stays neutralThermophiles inhabit a hot environment that accelerates the rate of DNA damage [1].It has been proposed that DNA repair in thermophilic archaea has unique properties in order to be robust in extreme environments [2,3]. An endonuclease XPF/Hef- and NucS/EndMSdeleted strain exhibits sensitivity to helix-distorting DNA lesions, suggesting that both endonucleases participate in homologous recombination (HR)-mediated stalled-fork DNA repair [3,4,5]. For these reasons, an opinion that this HR-mediated DNA repair serves as the major pathway for the removal of a wide variety of DNA lesions in hyperthermophilic archaea and is unusually efficient and reliable in comparison with other organisms has been argued [3,4,5]. Exploring the HR process in hyperthermophilic archaea is with regard to jurisdictional claims in published maps and institutional affiliations
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
