Identification and properties of the Deinococcus grandis and Deinococcus proteolyticus single-stranded DNA binding proteins (SSB).
To study the biochemical properties of SSB's from Deinococcus grandis (DgrSSB) and Deinococcus proteolyticus (DprSSB), we have cloned the ssb genes obtained by PCR and have developed Escherichia coli overexpression systems. The genes consist of an open reading frame of 891 (DgrSSB) and 876 (DprSSB) nucleotides encoding proteins of 296 and 291 amino acids with a calculated molecular mass of 32.29 and 31.33 kDa, respectively. The amino-acid sequence of DgrSSB exhibits 45%, 44% and 82% identity and the amino-acid sequence of DprSSB reveals 43%, 43% and 69% identity with Thermus aquaticus (TaqSSB), Thermus thermophilus (TthSSB) and Deinococcus radiodurans SSBs, respectively. We show that DgrSSB and DprSSB are similar to Thermus/Deinococcus SSBs in their biochemical properties. They are functional as homodimers, with each monomer encoding two single-stranded DNA binding domains (OB-folds). In fluorescence titrations with poly(dT), both proteins bind single-stranded DNA with a binding site size of about 33 nt per homodimer. In a complementation assay in E. coli, DgrSSB and DprSSB took over the in vivo function of EcoSSB. Thermostability with half-lives of about 1 min at 65-67.5 degrees C make DgrSSB and DprSSB similar to the known SSB of Deinococcus radiodurans (DraSSB).
Filipkowski
Paweł
P
Gdańsk University of Technology, Chemical Faculty, Department of Microbiology, Gdańsk, Poland.
Kur
Józef
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D034301:Deinococcus / Q000378:metabolism*; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins