Functional Analysis Of DExD/H-box Helicases SiRe₀681 And SiRe₁605 In Sulfolobus Islandicus

DExD/H box proteins are DNA or RNA helicase that use NTPs to bind and remodel the DNA and RNA.DExD/H box proteins are classified into supperfamily ?,and they are very conserved in all eukaryotic cells and in many bacteria and archaea.DEAD/H box helicases are characterized by the presence of an very conserved Asp-Glu-Ala-Asp?DEAD?motif which is also called Walker B motif,and it is the binding site of ATP.DEAD/H box helicases take part in lots of cellular processes and play very important roles in metabolism of nucleic acids.For example,DEAD/H-box helicases participate in double strand unwinding,pre-mRNA process,transcription,translation,mRNA degradation,DNA repair,ribosome biosynthesis and so on.Sulfolobus islandicus is a hyperthermophilic archaea which has a wide distribution in the northern hemisphere.DExD/H box helicases play a very important role in helping S.islandicus living in extreme environment,but the study of DExD/H box helicases is little.Previous studies of our lab have successed to knockout two DExD/H box helicases genes SiRe₀681 and SiRe₁605,and analyzed their functions in vivo.But the phenotypic analysis is not enough,and the biochemical properties are not tested.In this project,we want to do further studies on SiRe₀681 and SiRe₁605.We overexpressed the proteins,tested the growth curve,and measured the growth rate of the two deletion mutants at relative low temperature.Meanwhile,we analyzed the changes of gene expression quantities at transcriptomic level.To explore the functions of SiRe₀681 and SiRe₁605 in vivo,we expressed and purified SiRe₀681 and SiRe₁605 in E.coli,and tested the helicase activity of them.Overexpression of SiRe₀681 and SiRe₁605 in vivo did not have any influence on the growth of the strains.But the growth rates of ?SiRe₀681 and ?SiRe₁605 were slower than REY15A at relative low tempreture?60??.The result indicates that the DExD/H box helicase are important to S.islandicus when it suffer from cold pressure.Previous results found that ?SiRe₀681 grew faster than wild type strain.Flow cytometry analysis showed that deletion of SiRe₀681 might accelerate cell progress and some DN A biological processes probably skipped in G1/S phase.But at low temperature,?SiRe₀681 grew slower than the wild type strain.It suggests that the DNA processes regulated by SiRe₀681 benefit the cells living in low temperature.SiRe₁605 is important to the cells when they suffer from cold stress and damages,since deletion of SiRe₁605 lead to cells growing slower at 60 ?,and also cause the cells more prone to aggregate when treated with MMS.Transcriptome analysis showed that almost all genes related to cell division in?SiRe₀681were up-regulated.And the reverse gyrase were significant up-regulated in ?SiRe₀681,but obvious down-regulated in ?SiRe₁605.Reverse gyrase is important to the hyperthermophilics living in high temperature,so the changes of reverse gyrase in ?SiRe₀681 and ?SiRe₁605 may be related to these two mutants growing faster or slower when treated with MMS,respectively.In the presence of MMS,many genes of nucleotide metabolism and DNA repair enzymes were found to be down-regulated in ?SiRe₁605.It indicates that SiRe₁605 probably take part in DNA repair pathway,and deletion of SiRe₁605 cause the cells prone to damage so that the cells would grew slower to give more time to repair it.According to analysis of the whole transcriptomic data and KEGG pathway,we found that ?SiRe₁605 caused far more influence on metabolic pathway and gene expression level,compared with ?SiRe₀681.It means that SiRe₁605 may regulate lots of proteins expression and metabolic pathways.Deletion of SiRe₀681 and SiRe₁605 leads to the expression level of many genes at 0 in a special region.This phenomenon may be related to the regulation of DExD/H-box helicases in the gene silencing mechanism.We expressed and purified SiRe₀681 with Ni-NTA after heat treatment.But SiRe₁605 is difficult to purified,because it is prefered to form precipitation and several proteins might be simultaneously expressed from the middle sequnence of gene.We then improved the expression and purification conditions and finally got the relatively purified protein.Next,we used three DNA substrates to test the unwind ability of SiRe₁605 and SiRe₀681,but did not found obvious DNA helicase activity.Because of SiRe₁605 and SiRe₀681 were predicted to be RNA helicase,and the homologous protein of SiRe₀681 in Thermococcus kodakarensis only have the activity to unwind the stem-loop RNA,so we need do further study to test the RNA helicase activity of them.