Characterization Of RadA Homologues From The Hyperthermophilic Archaeon Sulfolobus Tokodaii

Homologous recombination is a well conserved DNA repair mechanism that prevails in all living organisms.It is essential for repair of DNA breaks,the preservation of genomic integrity,restart of stalled replication forks and eukaryotic meiosis.Less has been known about homologus recombination in Archaea.However, many archaeal proteins are homologues to their eukaryotic counterparts in homologus recombination.For example,archaea possess homologues of the eukaryotic DNA double-strand break processing proteins Mrell and Rad50 and the single-stranded DNA(ssDNA)-binding protein RPA.The archaeal RadA shares～40%amino acid identity with the eukaryotic Rad51,but only～20%identity with bacterial RecA.As homologues of Rad51 exist in Eukaryota,homologues of RadA also exist in Euryarchaeota.The RecA/Rad51/RadA proteins in three domains of life are recognized as recombinases that play a crucial role in Holliday junction formation by strand exchange reaction.Many proteins in bacteria and eukaryotes have been characterized to facilitate the formation of nucleoprotein filament of RecA/Rad51 or to facilitate strand exchange promoted by RecA/Rad51.Such proteins are RecBCD,RecFOR complexes in bacteria and Rad52,Rad54,Rad55 and Rad57 in yeast.Rad55,Rad57 in yeast and Rad51B,Rad51C,Rad51D,XRCC2 and XRCC3 in vertebrate are Rad51 homologues.Genetic analysis has confirmed their participation in homologous recombination and Rad55/Rad57 complex has been characterized to stabilize the nucleoprotein filament of Rad51.While there is RadB in Euryarchaeota,which is the homologue of RadA,no report about homologues of RadA in Crenarchaeota has been published.Through sequence alignment analysis,four RadA homologues have been found in Sulfolobus tokodaii Str.7.Phylogenetic analysis based on the entire amino acid sequences indicated that they exhibited more homology to Rad55 in yeast,so the four homologues were designated as stRad55A(ST0579),stRad55B(ST0838),stRad55C (ST1830)and stRad55D(ST2522)respectively.RT-PCR analysis revealed that transcription of S.tokodaii radA,rad55A and rad55B could be induced by UV irradiation,suggesting that these proteins may be involved in DNA repair.We expressed these proteins and S.tokodaii ssDNA-binding protein SSB in E.coli.RadA, stRad55A,stRad55C and SSB were expressed in soluble forms,but stRad55B and stRad55D were expressed in insoluble forms.Gel filtration of RadA,stRad55A, stRad55C and SSB revealed that RadA,stRad55A and stRad55C exist as oligomers while SSB as monomer in solution.We further characterized the biochemical properties of stRad55A,stRad55B and stRad55C.It is found that(1)stRad55A has biochemical properties much more similar to Rad52:it preferred binding to ssDNA and had ssDNA-dependent ATPase activity;it could release the suppression of excessive SSB on the strand exchange catalyzed by RadA;it could facilitate RadA nucleoprotein formation on SSB-coated ssDNA and interact directly with both RadA and SSB.However,there are also different properties between the two proteins:stRad55A inhibited strand exchange promoted by RadA when there was no SSB in reaction and no DNA annealing activity was found for stRad55A.(2)stRad55B could express in soluble form when coexpressed with RadA in E.coli and it formed complex with RadA.It preferred binding to ssDNA and had very weak DNA-independent ATPase activity.It suppressed strand exchange catalyzed by RadA in reaction without SSB.The characterized properties of stRad55B have similarity to that of RadB.(3)Like stRad55A,stRad55C preferred binding to ssDNA and had ssDNA-dependent ATPase activity.It interacted physically with RadA and Hjc.stRad55C suppressed strand exchange catalyzed by RadA in reaction without SSB,but could promote the Holliday junction resolving activity of Hjc.This is the first report of biochemical properties of RadA homologues in Crenarchaeota.Although it is not enough to elucidate the physiological function of these proteins in the cells,our work has proved the involvement of these proteins in DNA repair.More biochemical characterization and genetic analysis are needed to elucidate the mechanism of the involvement of these proteins in DNA repair.