The interaction between fibronectin and DNA.

Abstract

An examination of the DNA binding domain structure of bovine plasma fibronectin (Fn) was undertaken by a combination of limited proteolytic cleavage and Western blotting. A time course digestion of fibronectin with cathepsin D produced a number of proteolytic fragments possessing DNA binding activity. After two min digestion, two DNA binding fibronectin fragments of Mr approximately 180kd and 120kd were detected. Upon further digestion, a fibronectin fragment of Mr 18kd was detected. Thus it would appear that under physiological ionic strength only a single DNA binding domain exists in the fibronectin molecule. It was also demonstrated that the interaction of fibronectin with DNA is not ionic in nature, as heat denaturation of protein totally abolishes the DNA binding activity. An examination of possible sequence specificity of DNA binding activity of fibronectin was also undertaken by dot blotting the bovine plasma fibronectin and using [32P] labelled lambda FC 40 DNA containing approximately 16 kd of 5' end of the chicken fibronectin gene. Its binding to fibronectin was approximately twice the binding of [32P] labelled wild type lambda, where as binding to control of equimolar concentration of calf thymus histone H 2 A was approximately equal. The use of a smaller subcloned region, a approximately 1.9kb fragment of DNA from the 5' end of chicken fibronectin gene and wild type lambda DNA showed approximately 2 fold increase in histone binding and approximately 7 fold increase in fibronectin binding, indicating preferential fibronectin binding with eukaryotic DNA as compared to prokaryotic DNA. Further investigation of sequence specificity showed that a 0.45kb DNA fragment from the 5' end of chicken fibronectin gene, containing a number of elements characteristic of promoter, demonstrated approximately 2 fold higher level of binding with fibronectin and approximately 3.5 fold less binding activity with equimolar concentration of histone H2A when compared with a 1.4kb fragment of chicken fibronectin gene from the 1st exon. These results suggest fibronectin binding may be preferential to the promoter region of its own gene which could have possibly a regulatory function.