Abstrakt

Nanoarchaeum equitans is the only known representative of Archaea phylum Nanoarchaeota and stands out as one of the tiniest known living organism. What is more it has smallest genome, which is only 490.885 base pairs long. It is also one of the most compact genomes. According to predictions about 95% of the DNA encodes proteins or stable RNA. Nanoarchaeum equitans lacks genes for most vital metabolic pathways including lipid, cofactor, amino acid, or nucleotide biosynthesis. However contrary to most known organisms with reduced genome has a full set of enzymes involved in DNA replication, repair and recombination, one of which is a single-stranded DNA binding protein. To date four nanoarchaeal proteins have been identified: tRNA splicing endonuclease, reverse gyrase, neelaredoxin and family B DNA polymerase. Interestingly all of thempossess unusual features. Sequence analysis of Nanoarchaeum equitans SSB protein indicates that it may also fit the trend. We report the identification and characterization of SSB from Nanoarchaeum equitans Kin4-M. NeqSSB is one of the largest known SSB proteins consisting 243 amino acid residues with a calculated molecular mass of 27.78 kDa.It is functional as a monomer with encoding one singlestranded DNA binding domain (OB-fold), which is the smallest and consists of 75 amino acids residues only. Nanoarchaeum SSB is the completely new kind structure of SSB protein. SSBs proteins identified to date are homotetramers or homodimers and theirs OB-fold have about 100 amino acids residues. In fluorescence titrations with poly(dT), NeqSSB protein binds single-stranded DNA with a binding site size about 42 nt per monomer. In addition, a unique feature is the high protein binding capacity of RNA and DNA, both linear and circular. The half-lives of NeqSSB was 5 min at 100°C. These results showed thatNanoarchaeum SSB as the first characterized SSB with ability to binding all kinds of nucleic acids is thermostable SSB protein with unique structure, offering an attractive alternative for other SSB proteins in their applications for molecular biology techniques.

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