Study On The Biochemical Property In Vivo Function And Crystal Structure Of The AP Endonucleases And Phosphorylation Modification Of The Holliday Junction Resolvase Hje From Sulfolobus Islandicus

Base damages and modifications are recognized and repaired by base excision repair?BER?pathway.Typical BER starts by a specialized DNA glycosidase which can recognize and remove the damaged base resulting in an apurinic/apyrimidinic site?AP site?.AP endonuclease cleaves the first phosphodiester bond 5' to the AP site,producing an accessible 3'-OH and 5'-dRP for the subsequent nick processing,new strand synthesis and ligation by nuclease,DNA polymerase and ligase,respectively.AP endonucleases are divided into two families,Exo? and Endo?,of which the difference is that besides AP endonuclease activity,Exo? proteins always have 3'-5'exonuclease activity.Homologues of these two families from Eukaryote?human Ape1,Ape2,yeast Apnl and Apn2?and Bacteria?E.coli EcoExo? and EcoEndo??have been widely studied both biochemically and functionally.In Archaea,EndoIV family proteins are conserved among all the archael domain,while Exo? family proteins can only be found in some species.Although several AP endonucleases from Archaeoglobus fulgidus,Methanobacterium thermo auto trophicum and Pyrobaculum aerophilum have been characterized,the studies mainly concerned about their in vitro properties.So far,in vivo functional analysis of archaeal AP endonucleases has not been reported.Sulfolobus islandicus is one of the few archaea that have both Exo? and EndoIV homologues.The genetic manipulation system has also been well constructed.In this study,we firstly focused on the biochemical properties and in vivo functions of SisExo? and SisEndoIV We cloned and expressed SisExo? and SisEndo?.Then we tested their AP endonuclease activity using a synthetic oligonucleotide containing an AP site.Both proteins have obvious AP endonuclease activity but not any exonuclease activity.The optimal pH is 9.5 and the best cation is Mn2+ for both species.The Michaelis constants of SisExo? and SisEndoIV are similar with each other but the Kcat of SisEndoIV is about 28 times that of SisExo?,indicating a higher catalytic efficiency of SisEndoIV Unlike other known archaeal AP endonucleases,SisExo? and SisEndo? can not recognize modified bases such as dI,dU and 8-oxo-dG.To further investigate the role of SisExo? and SisEndoIV in S.islandicus,we performed genetic analysis.We successfully obtained the single and double knockout strains of SiRe₀100?code for SisExo??and SiRe₂666?code for SisEndoIV?.While ?SiRe₀100 exhibits no obvious phenotype,?SiRe₂666 is more sensitive to the alkylating agent MMS,implying that SiRe₂666 plays a more significant role in base excision repair pathway in vivo.What's more,over-expression of SisEndoIV under arabinose induction can also makes the strain more sensitive to MMS,and even a relatively lower over-expression by sucrose can not complement the phenotype of ?SiRe₂666.This result indicates that as the main functional AP endonuclease,the protein level of Sis EndoIV needs be strictly regulated during BER.Interestingly,we also found that over-expression of SisExoI? under sucrose induction can partly complement ?SiRe₂666.We suspect that the restriction of the in vivo role of SisExoIII is mainly the poor expression level or enzymatic activity.Taken together,we conclude that SisEndoIV is the major functional AP endonuclease in Suloflobus islandicus,while SisExo? can only be served as a background.To gain more insights into the catalytic mechanism of SisExo?,we solved the crystal structure of SisExo??Y105A?.The overall structure of SisExo??Y105A?resembles other known Exo? family proteins,but differences can be found mainly in the organization of key residues within the catalytic center and the flexibility of DNA binding loops.That may be the reason why SisExo? has lower AP endonuclease and poor exonulcease activity.We also characterized the properties of analine mutants of residues formed the disulfide bond,Cys142 and Cys215.Bio-informational alignment revealed that this intramolecular linkage is only conserved among crenarchaeal Exo?AP endonuclease proteins.The formation of this disulfide bond increases the protein thermostability under high tempreture,according to the amino acid mutation analysis.Holliday junction resolvase is the structure specific nuclease that can recognize and process Holliday junction?HJ?,an intermediate product during homologous recombination repair.By symmetrically cleavage the double strand of HJ,HJ resolvase midiates the "resolution" pathway in which the subsequent DNA ligase can seal the resultant terminals directly.So far many HJ resolvases have been identified including bacterial RuvC,and eukaryotic Mus81-Emel/Mms4,Slxl-Slx4 and Gen1/Yen1.In eukaryote cells,the activity,interaction and subcellular localization of HJ resolvase have been found to be regulated through phosphorylation modification to maintain the efficiency and accuracy of homologous recombination repair.In Archaea,Hjc,the well known HJ resolvase,is conserved in nearly all the species,but another resolvase Hje has been identified with higher catalytic activity in a few of Crenarchaea.Our previous studied have reavealed that Hje is the major HJ resolase in Sulfolobus islandicus,but little is known about whether it can be functionally regulated by phosphorylation as in Eukaryote.In this thesis,we investigated the phosphorylation of Hje.We found that Hje can be phosphorylated by SiRe₂056 and the modification sites include Ser3,Ser127 and Thr134.Further experiments showed that phosphorylated Hje has a slightly lower HJ binding activity compared with non-modified protein.To study the in vivo function of Hje phosphorylation,we constructed the in situ complementation strains harboring mutations at Ger127 or/and Thr137 site.When we mutate these two sites to alanine,the strain shows higher sensitivity to hydroxyurea?HU?.On the other hand,mutation of Ser127 and Thr134?especially for Ser127?to glutamate to mimic phosphorylation caused higher resistence to HU.Considering that the Ser127 and Thr134 are located at the carboxylic terminal which is far away from the active center of Hje,we suspect that the phosphorylation on these two sites may modulate the interactions between Hje and other HR factors.Further investigation in Hje phosphorylation in Sulfolobus islandicus will deepen our understanding of the regulation mechanism of HR and help the studies of DNArepair proteins in other archaea species.