Genetic Analysis Of Holliday Junction Resolvases Hje And Hjc In Sulfolobus Islandicus

Homologous Recombination (HR) plays important roles in DNA double-strand breaks repair and the repair of stalled replication forks. A key step in HR is the resolution of Holliday Junctions (HJs). Now, two HJ resolving enzymes (HJ resolvase) Hje and Hjc which have28%sequence identity have been identified from S. islandicus. They resolve HJs in a manner analogous to the E. coli HJ resolvase RuvC, which introduces symmetric nicks into the substrate. Here, we used the genetic system developed recently in Sulfolobus to study the in vivo functions of hje and hjc.Firstly, in Δhje background, we constructed strains in which the endogenous hje allele was replaced with those coding Hje, Hje-C-His, HjeE52AK54A or HjeE52A/K54A-C-His Cells harboring Hje-C-His or HjeE52A/K54A-C-His displayed sensitivity to HU as the same as Ahje, while Δhje/Hje and Δhje/HjeE52A/K54A showed HU sensitivity between those of SisE233S and Ahje, indicating that the His tag can affect the in vivo function of Hje. And the sensitivity of Δhje/HjeE52A/K54A-C-His is not caused by the loss of its nuclease activity, but may be due to disturbing of an unidentified function of Hje in which the C terminal of Hje is involved. To further analyze the in vivo roles of Hje, we constructed overexpression strains which harboring pSeSD-Hje-C-His and pSeSD-Hje. The strain overexpressing Hje showed negative effect on growth, while cells containing pSeSD-Hje-C-His showed more severe growth defect. Next, we analyzed the effect of expression Hje (native promoter), Hjc-C-His or Xpf-C-His. We found that the sensitivity of Δhje/pSeSD-Hjc-C-His to hydoxyurea (HU) was the same as Sis/pSeSD, which indicating that overexpression Hjc-C-His could rescue the HU sensitivity of Ahje. But to our surprise, Δhje/pSeSD-NP Hje was more sensitive to HU than Δhje/pSeSD. These results implied that the level or the activity of Hje needs to be strictly controlled, as reported in yeast, where the level or the activity of Mus81-Emel/Mms4or Yen1is controlled by phosphorylation. We found that Hje-C-His purified from S. islandicus and E. coli migrated differently in SDS-PAGE gel. The former was as a band of18kDa, while the latter was16kDa, the predicted molecular size, supporting that Hje in the cells is posttranlationally modified.Lastly, we investigated the relationship of structure-specificc endonucleases Hje, Hjc, and Xpf in S.islandicus. We attempted to delete xpf in Δhje or Δhjc, and delete hjc in Δhje using an unmarked deletion method. We have obtained Ahje/Axpf and Δhjc/Δxpf mutants as were confirmed by PCR. However, deletion of both hje and hjc was not possible, because double deletion led to synthetic lethality. These results suggest that Hje and Hjc may function in independent pathways in the cleavage of recombination intermediates, while Xpf is not involved in HR but in other processes like nucleotide excision repair.