Genetic Analysis Of A Novel P-loop NTPase In Sulfolobus Islandicus

Holliday Junction is the important intermediate of homologous recombination repair pathway which can be processed by special enzymes.One kind of the enzymes is Holliday Junction resolvase.HJ resolvase Hjc(Holliday junction cleavage)is conserved in archaea.In previous studies,we identified an unknown protein which may interact with Hjc in Sulfolobus islandicus.Bioinformatic and biochemical analysis showed that it has a PIN domain(Pili T N-terminal)at the amino end of the protein,a P-loop NTPase domain in the middle,and a lysine-rich carboxyl terminal.Because the porotein possesses an ATPase activity,we named it PINA(for PIN domain ATPase).In order to further reveal the mechanism of Hjc and understand the physiological function of SPINA,we performed genetic analysis of SisPINA.Firstly,we analysed the necessity of SisPINA and its key active residues by genetic complement assay.The genotype and phenotype were analysed if the complementary strains were obtained.The results showed that SisPINA is necessary for maintaining cell growth and the PIN domain,the P-loop NTPase domain and the C-terminal are all essential.In addition,anlinine mutants at the Lys261 and Glu323 of the Walker A and Walker B motif,respectively,could not complement gene function of the wild type.Next,we tried to obtain strains with SisPINA being expressed at lower or higher levels by modifying SisPINA promoter,and analyzed their growth and sensitivity to UV radiation and HU.Strain Sis/pK-A(38/2)SisPlNA-T where SisPINA was confirmed to be expressed at lower level,did not exhibited higher sensitivity to UV and HU.We assummed that SisPINA protein may not be primarily involved in the repair of lesions caused by UV or HU.Further experiments showed that if the level of SisPINA was kept at a certain higher level in the cell,the normal cell growth were affected,implying that the expression level of SisPINA needs to be strictly regulated in vivo.Finally,we constructed strains in which the wild type or ATPase and DNA binding deficient mutants were overexpressed and the corresponding phenotypes were analyzed.Overpression of some mutant SisPINA proteins which lost ATPase activity or DNA binding activity had an inhibitory effect on the growth of the strains.The stains exhibited slow growth,but the cell morphology was unchanged.