Novel thermostable single-stranded DNA-binding protein (SSB) from Deinococcus geothermalis.
To study the biochemical properties of single-stranded DNA-binding (SSB) protein from Deinococcus geothermalis (DgeSSB), we have cloned the ssb gene obtained by PCR and developed an overexpression system. The gene consists of an open reading frame of 900 nucleotides encoding a protein of 300 amino acids with a calculated molecular weight of 32.45 kDa. The amino acid sequence exhibits 43, 44 and 75% identity with Thermus aquaticus, Thermus thermophilus and Deinococcus radiodurans SSBs, respectively. We show that DgeSSB is similar to Thermus/Deinococcus SSB in its biochemical properties. DgeSSB includes two oligonucleotide/oligosaccharide-binding folds per monomer and functions as a homodimer. In fluorescence titrations with poly(dT), DgeSSB bound about 30 nt independent of the salt concentration, and the fluorescence was quenched by about 65%. In a complementation assay in Escherichia coli, DgeSSB took over the in vivo function of EcoSSB. DgeSSB is thermostable with half-lives of 50 min at 70 degrees C and 5 min at 90 degrees C. Hence, DgeSSB offers an attractive alternative for TaqSSB and TthSSB in their applications for molecular biology methods and for analytical purposes.
Filipkowski
Paweł
P
Department of Microbiology, Gdańsk University of Technology, ul. Narutowicza 11/12, 80952 Gdańsk, Poland.
Duraj-Thatte
Anna
A
Kur
Józef
J
D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D034301:Deinococcus / Q000235:genetics*; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D017353:Gene Deletion; D015870:Gene Expression; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009841:Oligonucleotides / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013824:Thermus / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D009844:Oligosaccharides