Crystallography And Biochemical Characterization Of A Novel P-loop NTPase From Sulfolobus Islandicus

Archaea are one of the most ancient life forms on Earth.Their genetic information transmission mechanism resembles eukaryotic countpart,and could serve as a simple model for the study on nucleic acid metabolism in eukaryotes.Most archaea live in harsh conditions,where extreme environmental factors threaten their genome stability and cause DNA damages.Homologous recombination is one of the most efficient pathways for double-strand break repair.Holliday Junctions is an intermediate in homologous recombination.The last step in homologous DNA recombination is the resolvation of Holliday Junctions?HJ?by HJ resolving enzymes or other enzymes.HJ Resolvase are present in three domains of life.The main HJ-resolving enzyme in bacteria is RuvC,while those in eukaryote are Mus81-Mms4/Emel and Gen1/Yenl.The archaeal resolvases?in most Sulfolobus spescies?are Hjc and Hje.However,at the present,we have not found any motor proteins catalyzing branch migration in archaea.ASCE?the additional strand catalytic E division?is one of the major divisions of P-loop NTPases.Bio-information analysis showed that a group of novel P-loop NTPase is highly conserved and specific in archaea.The encoding gene in Sulfolobobus islandicus Rey15A is SiRe₁432,which is essential and located adjacent to hjc.This ATPase contains an additional PIN domain?Pili N-terminal domain?at the N-terminal,so we named it as PINA?for PIN-domain ATPase?.Previous research showed that PINA is an ATPase and Holliday junction helicase which prefers divalent calcium as the co-factor.It can specifically bind to Holliday junction by its C-terminal lysine-rich motif.Pull-down and yeast two-hybrid analysis revealed that SisPINA has interaction with Hjc in vitro.However,the function of SisPINA in vivo is still unknown.In this study,we aim to explain the functional role of SisPINA in DNA recombination in S.islandicus by using X-ray diffraction method and biochemical analysis.In this thesis,we first expressed SisPINA and its truncate mutant SisPINAE323K?1-473?in E.coli.Through heat-treatment,heparin HP affinity chromatography and gel filtration,we obtained purified proteins.The crystals of SisPINA grew under the following conditions:1.8 M MgSO4,0.1M CH3COONa·3H2O.We got better crystals after optimization.Meanwhile,the crystals of SisPINAE323K?1-473?mutant grown on two conditions:1%w/v Trypton,0.05 M HEPES pH 7.0 and 20%PEG 3350 ? 0.2 M CH3COONa · 3H20,20%PEG 3350,pH 8.0.At last we got better crystals under conditions of 1%w/v Trypton,0.05 M HEPES pH7.0.But the obtained crystals did not have sufficiently high resolution for structural analysis.Furthermore,by multiple sequence alignment of SisPINA and its homologous proteins in archaea,we found that the P-loop domain and its C-terminus were highly conserved.Eight conserved residues were mutated into alanine.These site-directed mutants were expressed and purified.The ATPase activity was analyzed.Among them,R286,R315 and R358 were critical for the ATPase activity.The alanine mutants lost ATPase activity..At last,we investigated if SisPINA affects the resolvase activity of Hjc by in vitro biochemical assay.We found that SisPINA stimulates the resolvase activity of Hjc.At the same time,in order to know which properties of SisPINA was important to its stimulation effect,we chose two mutants of SisPINA:ATPase activity deficient mutant SisPINAK261A and DNA binding activity deficient mutant SisPINAK498A/K500A/K502A for the analysis.We found that SisPINAK261A was still able to stimulate the resolvase activity of Hjc,however SisPINAK498A/K500A/K502A lost its stimulating effects.Therefore,its DNA binding activity is responsible for the stimulation of the resolvase activity of Hjc..