Genetic Analysis Of The Structure And Function Of Hjm Helicase And Other Novel DExD/H-box Family Helicases In Sulfolobus Islaudicus

Sulfolobus lives in acid and hot environments and the genomic DNA is prone to damage. However, its genome remains stable and the mutation rate is as same as mesophiles, suggesting that it possesses a very efficient repair system. The protein complexes in archaeal genetic information processing system are simpler than those in eukaryote. Therefore, the studies on the processing of DNA in archaea can provide novel insight into the complicated mechanisms in eukaryote.The replication fork can be stalled by DNA lessions, and the stalled replication fork can be restarted by replication fork regression and repaired by homologous recombination （HR）. Archaeal Hjm （He1308a）, a member of DExD/H-box helicases which represent the largest family of helicases, is believed to participate in homologous recombination repair. The stalled replication fork is processed and reversed to form the important intermediate Holliday junction （HJ）. Hjm may play important roles in annealing and Holliday junction migration. Previous studies showed that domain V of Hjm is a regulatory domain. The deletion of it and the mutation at conserved arginine residues led to change of enzyme activity. However, the in vivo function and the relationship between the domains and functions are not clear.To study the in vivo function of Hjm, the structure and domain organization of Sulfolobus islandicus Hjm was built by homologous modeling using SWISS-MODEL based on Sulfolobus solfataricus Hjm （He1308）. Then the domain knockout plasmids were constructed based on MID （marker insertion and target gene deletion） strategy, and the marker-integration cells were purified. Under counter-selection, the proliferation assay showed that domain V was essential for cell viability. The hjm complementary system was constructed using pSSR plasmid which carries a resistance maker to simvastatin. Chromosomal hjm deletion could be complemented by ectopic expression of hjm under the control of its native promoter but not by arabinose promoter. Point mutants and truncated mutants were constructed by overlap extension PCR. Using the complementary system, we found that the arginine residues within domain V which affected the regulation of the helicase activity were essential for cell viability, and HTH （helix-turn-helix） motif in domain III which participates in the recognization and binding with double strand DNA were essential too. Cellular Hjm levels were kept constant in wild type cells and complementary cells even under ultraviolet （UV） and methyl methanesulfonate （MMS） treatment conditions. These results suggest that Hjm expression and activity are tightly controlled in vivo.DExD/H-box helicases are distributed widely in the three domains of life. They belong to superfamily 2 helicases and participate in RNA and DNA metabolism, such as unwinding, transcription, translation, pre-mRNA processing and mRNA degradation. They are also involved in ribosome biogenesis and nucleocytoplasmic transport. The biochemical properties and structures of some DExD/H-box helicases in archaea have been documented, but many of them have not been characterized and reports on in vivo functional analyses and mechanisms are limited.To investigate the essentialities and in vivo functions of DExD/H-box helicases, we identified 15 DExD/H-box helicases in Sulfolobus islandicus by bioinformatics analysis and attempted gene knockout of 8 genes which have not or barely been reported using marker replacement strategy. Only two deletion mutants △SiRe₀681 and △SiRe₁605 were obtained, suggesting that the two genes were non-essential, and the other six may be essential.Growths of the two deletion strains were monitored using the wild type REY15A as control. In the presence of methylmethane sulfonate （MMS） △SiRe₀681 grew faster than wild type cells. CFU （colony formation unit） assay showed that △SiRe₀681 formed more colonies on plate. Transcription of SiRe₁962 and SiRe₁625 that involved in chromosome segregation were up-regulated by 2.46 and 2.06-fold in △SiRe₀681, respectively. Gene transcription of cell division proteins including CdvB （SiRe₁174）, CdvB2 （SiRe₁200） and CdvB1 （SiRe₁550） were up-regulated by 2.16,4.86 and 5.27-fold, respectively. Flow cytometry analysis showed that △SiRe₀681 had more G2 phase cells which made the strain show more resistance to MMS. Using △SiRe₀681 as recipient cells, SiRe₀681 complementary system was constructed with pSSR plasmid, and REY15A harboring empty pSSR as control. Growth and flow cytometry analyses showed that there was no difference in growth and cell cycle between SiRe₀681 complementary strain and RYE15A under the same culture conditions. The complementation of SiRe₀681 can restore the wild type phenotype. The deletion of SiRe₀681 may affect cell cycle and some non-essential DNA biological processes in G1/S phase were skipped, resulting in more cells passing through and entering into G2 phase.Growth curve analysis showed that △SiRe₁605 was more sensitive to MMS than the wild type cell. In the presence of MMS, many genes of nucleotide metabolism and DNA repair enzymes were found to be down-regulated. For example, transcription levels of DNA polymerase 2, Rad50 and Mrel 1 were down-regulated to 0.22,0.45 and 0.45-fold compared with wild type, respectively. Cell division proteins including CdvA, CdvB, Vps4, CdvB2, CdvBl and CdvB3 were down-regulated to 0.28,0.31,0.33,0.22,0.14 and 0.36-fold, respectively. As an Lhr helicase, SiRe₁605 maybe participates in DNA repair through affecting the transcriptions of DNA repair protein genes.