Phosphorylation of the Archaeal Holliday Junction Resolvase Hjc Inhibits Its Catalytic Activity and Facilitates DNA Repair in Sulfolobus islandicus REY15A

Abstract

Protein phosphorylation is one of the main protein post-translational modifications and regulates DNA repair in eukaryotes. Archaeal genomes encode eukaryotic-like DNA repair proteins and protein kinases (ePKs), and several proteins involved in homologous recombination repair (HRR) including Hjc, a conserved Holliday junction (HJ) resolvase in Archaea, undergo phosphorylation, indicating that phosphorylation plays important roles in HRR. Herein, we performed phosphorylation analysis of Hjc by various ePKs from Sulfolobus islandicus . It was shown that SiRe_0171, SiRe_2030, and SiRe_2056, were able to phosphorylate Hjc in vitro . These ePKs phosphorylated Hjc at different Ser/Thr residues: SiRe_0171 on S34, SiRe_2030 on both S9 and T138, and SiRe_2056 on T138. The HJ cleavage activity of the phosphorylation-mimic mutants was analyzed and the results showed that the cleavage activity of S34E was completely lost and that of S9E had greatly reduced. S. islandicus strain expressing S34E in replacement of the wild type Hjc was resistant to higher doses of DNA damaging agents. Furthermore, SiRe_0171 deletion mutant exhibited higher sensitivity to DNA damaging agents, suggesting that Hjc phosphorylation by SiRe_0171 enhanced the DNA repair capability. Our results revealed that HJ resolvase is regulated by protein phosphorylation, reminiscent of the regulation of eukaryotic HJ resolvases GEN1 and Yen1.

Archaeal DNA Repair Mechanisms

“…The resultant newly formed junctions can have significant impacts on genomic integrity and Holliday junction resolution is likely an important point of regulation for HR-DSBR. The cleavage activity of Hjc is repressed by phosphorylation in Sulfolobus islandicus, which is consistent with bacterial and eukaryotic resolvases [59,60]; cells are more resistant to high doses of DNA damaging agents when the phosphomimetic version of Hjc is expressed [61].…”

Phosphoproteomic Analysis Reveals Rio1-Related Protein Phosphorylation Changes in Response to UV Irradiation in Sulfolobus islandicus REY15A

“…Assay of the sensitivity of strains to UV irradiation on plates was performed as described previously ( Huang et al, 2019 ). For UV-treatment of cells in liquid medium, a 30 mL aliquot at an OD 600 ∼ 0.2–0.3 was transferred to a petri dish (9 cm in diameter).…”

“…Although archaeal Rios was suggested to have a conserved role in ribosome biogenesis ( Knuppel et al, 2018 ), their function has not been established thus far. Our previous study revealed that Rio1 phosphorylated the Holliday junction resolvase Hjc in S. islandicus and regulated its nuclease activity ( Huang et al, 2019 ). However, it remains unclear what other role(s) archaeal Rio1 plays in DNA damage-induced regulatory networks.…”

Haloferax volcanii —a model archaeon for studying DNA replication and repair

“…In S. islandicus , Hjc is regulated by phosphorylation, which acts to inhibit its catalytic activity [ 433 ]. Interestingly, Hjc phosphomimetic mutants of S. islandicus exhibit increased resistance to DNA damaging agents, suggesting that phosphorylation acts to redirect repair to avoid HR that is dependent on Hjc [ 433 ].…”

“…In S. islandicus , Hjc is regulated by phosphorylation, which acts to inhibit its catalytic activity [ 433 ]. Interestingly, Hjc phosphomimetic mutants of S. islandicus exhibit increased resistance to DNA damaging agents, suggesting that phosphorylation acts to redirect repair to avoid HR that is dependent on Hjc [ 433 ]. While the serine residues targeted for phosphorylation in S. islandicus Hjc are not conserved in other archaeal species, modification of Hjc and other HR proteins by phosphorylation in S. acidocaldarius is well documented [ 434 ] .…”