Identification, cloning and expression of p25, an AT-rich DNA-binding protein from the extreme thermophile, Thermus aquaticus YT-1

• Published in: Nucleic acids research Vol. 27; no. 7; pp. 1690 - 1697
• Main Authors: Du, X, Pène, J J
• Format: Journal Article
• Language: English
• Published: England Oxford Publishing Limited (England) 01.04.1999
• Subjects: Amino Acid Sequence; Base Sequence; Cloning, Molecular; DNA, Bacterial - metabolism; DNA-Binding Proteins - genetics; DNA-Binding Proteins - isolation & purification; DNA-Binding Proteins - metabolism; Electrophoresis, Polyacrylamide Gel; Escherichia coli - genetics; Molecular Sequence Data; Protein Binding; Sequence Homology, Amino Acid; Temperature; Thermus - genetics; Thermus aquaticus
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/27.7.1690

Although the G+C content of Thermus aquaticus YT-1 chromosomal DNA is 67.4%, regions with lower G+C content have also been observed. AT-rich DNA-binding proteins may contribute to the thermostability and biological functions of these DNA regions at Thermus growth temperatures. Using double-stranded DNA (dsDNA)-cellulose chromatography, a T.aquaticus YT-1 protein, designated as p25, was identified to bind preferentially to AT-rich DNA. The gene encoding p25 was cloned and sequenced after immunoscreening T.aquaticus YT-1 expression libraries. The deduced primary structure of p25 is 211 amino acids in length with a molecular weight of 23 225 Da. Native p25 was purified and characterized as a homodimer with modification possibly at lysine and arginine residues. Its preferential and temperature-dependent binding to AT-rich DNA was confirmed with mobility-shift DNA-binding assays. The protein was demonstrated to bind preferentially to dsDNA instead of single-stranded DNA. The binding of p25 to dsDNA also improved the thermotolerence of this protein. Overexpression study of fusion p25 suggested that the N-terminus of the protein might form the DNA-binding domain or be closely involved in DNA-binding activity.