Analytical ultracentrifugation studies of translin: analysis of protein-DNA interactions using a single-stranded fluorogenic oligonucleotide.
Translin is a recently identified nucleic acid binding protein that appears to be involved in the recognition of conserved sequences found at many chromosomal breakpoints. Previous reports indicate that, based on gel filtration analysis and electron microscopy of protein-DNA complexes, translin forms an octameric structure that binds the DNA. In this study, we further examine the possibility of self-association of translin and its interactions with DNA by analytical ultracentrifugation. Sedimentation velocity analysis of translin indicates that the predominant species sediments with a sedimentation coefficient of 8.5 S and has a frictional ratio, f/f(omicron), of 1.35; these data are consistent with the presence of an octamer with an ellipsoidal configuration; a small amount of a component with significantly higher mass is also present. Equilibrium sedimentation studies of translin at three different protein concentrations also indicate that the predominant species present is an octamer with a minor fraction of aggregated species. Neither monomer nor dimer was detected. Sedimentation equilibrium studies of translin with an FITC-labeled single-stranded oligonucleotide were performed to examine the interaction. A novel analysis method has been developed to analyze protein-nucleic acid interactions based on global fitting of scans of 280 and 490 nm to appropriate mathematical models. Utilizing this method, it was determined that the DNA binding species of translin is an octamer binding a single-stranded oligonucleotide with a DeltaG degrees value of -9.49 +/- 0.12 kcal/mol, corresponding to a dissociation constant, K(d), of 84 +/- 17 nM. On the basis of this evidence and electron microscopy, it is envisioned that translin forms an annular structure of eight subunits, hydrodynamically an oblate ellipsoid, which binds DNA at chromosomal breakpoints.
Lee
S P
SP
Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC 20007, USA.
Fuior
E
E
Lewis
M S
MS
Han
M K
MK
D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D008956:Models, Chemical; D009841:Oligonucleotides / Q000378:metabolism*; D014461:Ultracentrifugation / Q000379:methods
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D009841:Oligonucleotides; C095563:TSN protein, human; D016650:Fluorescein-5-isothiocyanate