Purification, characterization and molecular cloning of TGP1, a novel G-DNA binding protein from tetrahymena thermophila

Abstract

G-DNA, a polymorphic family of four-stranded DNA structures, has been proposed to play roles in a variety of biological processes including telomere function, meiotic recombination and gene regulation. Here we report the purification and cloning of TGP1, a G-DNA specific binding protein from Tetrahymena thermophila. TGP1 was purified by three-column chromatographies, including a G-DNA affinity column. Two major proteins (~ 80 and ~ 40 kDa) were present in the most highly purified column fraction. Renaturation experiments showed that the ~ 80 kDa protein contains TGP1 activity. Biochemical characterization showed that TGP1 is a G-DNA specific binding protein with a preference for parallel G-DNAs. The TGP1/DNA complex has a dissociation constant (K(d)) of ~ 2.2 x 10-8 M and TGP1 can form supershift in gel mobility shift assays. The cDNA coding TGP1 was cloned and sequenced based upon an internal peptide sequence obtained from the ~ 80 kDa protein. Sequence analyses showed that TGP1 is a basic protein with a pi of 10.58, and contains two extensively hydrophilic and basic domains. Homology searches revealed that TGP1 is a novel protein sharing weak similarities with a number of proteins.