Structure-Based Genetic Analysis of Hel308a in the Hyperthermophilic Archaeon Sulfolobus islandicus.

In archaea, the HEL308 homolog Hel308a (or Hjm) is implicated in stalled replication fork repair. The biochemical properties and structures of Hjm homologs are well documented, but in vivo mechanistic information is limited. Herein, a structure-based functional analysis of Hjm was performed in the genetically tractable hyperthermophilic archaeon, Sulfolobus islandicus. Results showed that domain V and residues within it, which affect Hjm activity and regulation, are essential and that the domain V-truncated mutants and site-directed mutants within domain V cannot complement hjm chromosomal deletion. Chromosomal hjm deletion can be complemented by ectopic expression of hjm under the control of its native promoter but not an artificial arabinose promoter. Cellular Hjm levels are kept constant under ultraviolet (UV) and methyl methanesulfonate (MMS) treatment conditions in a strain carrying a plasmid to induce Hjm overexpression. These results suggest that Hjm expression and activity are tightly controlled, probably at the translational level.

Knockout and functional analysis of two DExD/H-box family helicase genes in Sulfolobus islandicus REY15A.

DExD/H-box helicases represent the largest family of helicases. They belong to superfamily 2 helicases and participate in nucleotide metabolism, ribosome biogenesis, and nucleocytoplasmic transport. The biochemical properties and structures of some DExD/H-box helicases in the archaea have been documented, but many of them have not been characterized; and reports on in vivo functional analyses are limited. In this study, we attempted gene knockout of 8 putative DExD/H-box helicases in Sulfolobus islandicus REY15A and obtained two deletion mutants, SiRe_0681 and SiRe_1605. We determined that ΔSiRe_0681 grew faster than wild type cells in the presence of methyl methanesulfonate (MMS). Flow cytometry analysis showed that this strain had fewer G1/S phase cells than the wild type, and the genes coding for cell division proteins were up-regulated. The stain ΔSiRe_1605 was more sensitive to MMS than the wild type cell, and many nucleotide metabolism and DNA repair enzymes were found to be down-regulated. Intriguingly, deletion of either gene led to silencing simultaneously of over 80 genes located at a specific region. This study provides a novel insight into the in vivo functions of predicted DExD/H-box family helicases in the archaea.