Study On The Co-expression Of The DSB Repair-related Operon HerA-mre11-rad50-nurA Proteins From Sulfolobus Islandicus And Crystallography Of The Helicase HerA

A number of archaea live in extreme environments,so their genomic DNA is prone to damage.The DNA double-strand breaks(DSBs)are one of the most serious DNA lesions,which can lead to death of the organism.Homologous recombination repair is a relatively precise pathway for repair of DNA DSBs.In bacteria,homologous recombination repair mainly includes RecBCD,RecFOR and SbcC-SbcD pathways.In eukaryotes,homologous recombination repair is mediated by Mrel1-Rad50 and Nbsl(mammalian cells)or Xrs2(Saccharomyces cerevisiae),but the detailed mechanism is not very clear.Archaea,as the third domain of life,has a genomic structure similar to that of bacteria,while its DNA metabolic mechanism resembles that in eukaryotes.Archaea living in harsh conditions exhibit comparable spontaneous mutantion rates with those of bacteria and eukaryotes,indicating that they harbor efficient DNA repair systems.Therefore,study on the high efficient DNA repair mechanisms of archaea in extreme environments would help reveal the molecular mechanisms of DNA repair in eukaryotes and play a guiding role in the prevention of a variety of human diseases.Homologous recombination repair proteins in Eukaryotes,Mrell and Rad50,also exist in archaea,but no homologs of Xrs2 or Nbs1 has been found in archaea.It seems that archaea harbor a Mrel1-Rad50-mediated pathway,not identical with that in eukaryotic cells.Mrel1 is a nuclease which has 3'-5' ssDNA exonuclease and endonuclease activity,while Rad50 is ABC(ATP-binding cassette)type ATPase.MR complexes can degradate double-stranded DNA to form a 3'-overhang structure.In thermophilic archaeal genomes,her A,nurA,mrell and rad50 are in the same operon.Sulfolobus tokodaii NurA is a nuclease which has 5'-3' ssDNA and dsDNA exonuclease activity and single-stranded circular DNA endonuclease activity.It has been reported that StoNurA and StoSSB have physical and functional interaction.StoHerA is a helicase which can unwind a variety of DNA substrates.Study on the proteins from Pyrococcus furiosus show that NurA interacts with HerA to form a complex,which participates in homologous recombination repair of DNA DSBs together with MR complex.However,the biochemical characterization did not reveal the mechanisms of protein interaction and broken DNA ends resection.Here,we tried to directly clone the large fragment of MRHN gene cluster from S.islandicus Rey15A genome into an Escherichia coli expression vector by Red/ET recombineering.The proteins were expressed and purified together to preliminarily study the complex formation of these proteins.Among the four proteins,the crystal structures of Mrel1 and Rad50 have been reported by Hopfner in 2001 and 2000,respectively.Mrel1 and Rad50 form a stable tetramer heterologous(Mrel1)2(Rad5 0)2,which can process the ends of DNA DSBs to form a 3'-overhang.The crystal structure of NurA was reported in 2012,showeing that NurA forms a dimer,and contains a hydrophobic interface that can interact with HerA.In this study,we tried to obtain the crystal structure of S.islandicus HerA.Firstly,we overexpressed SisHerA in S.islandicus to get sufficient protein for screening crystals.The obtained crystals were further optimized in a buffer containing 4%Tacsimate pH 7.4,13.5%PEG3350.However,the resolution of the HerA crystal is 17 A,which was high enough for structural analysis.Then we tried to overexpress SisHerA and its mutant proteins in E.coli,but the obtained crystals did not have a higher resolution for structural analysis.In addition,the physical and functional interactions between StoHerA and StoMrel1 were previously found via in vitro pull-down experiments in our laboratory.We also tried to confirm this result.Wild type StoHerA,its mutant proteins,and truncation mutant proteins of Mrel1 were expressed in,and purified from E.coli,but we failed to get the wild type Mrel 1 protein.