A highly thermostable, homodimeric single-stranded DNA-binding protein from Deinococcus radiopugnans.

Paweł Filipkowski, Magdalena Koziatek, Józef Kur

Index: Extremophiles 10 , 607-614, (2006)

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Abstract

We report the identification and characterization of the single-stranded DNA-binding protein (SSB) from the mesophile and highly radiation-resistant Deinococcus radiopugnans (DrpSSB). PCR-derived DNA fragment containing the complete structural gene for DrpSSB protein was cloned and expressed in Escherichia coli. The gene consisting of an open reading frame of 900 nucleotides encodes a protein of 300 amino acids with a calculated molecular weight of 32.45 kDa and pI 5.34. The amino acids sequence exhibits 43, 44, 79 and 18% identity with Thermus aquaticus, Thermus thermophilus, Deinococcus radiodurans and E. coli SSBs, respectively. The DrpSSB includes two OB folds per monomer and functions as a homodimer. In fluorescence titrations with poly(dT), DrpSSB bound 24-31 nt depending on the salt concentration, and fluorescence was quenched by about 80%. In a complementation assay in E. coli, DrpSSB took over the in vivo function of EcoSSB. The half-lives of DrpSSB were 120 min at 90 degrees C, 60 min at 95 degrees C and 30 min at 100 degrees C. These results were surprising in the context of half-life of SSB from thermophilic T. aquaticus, which has only 30 s of half-life at 95 degrees C. DrpSSB is the most thermostable SSB-like protein identified to date, offering an attractive alternative for TaqSSB and TthSSB in their applications for molecular biology methods and analytical purposes.