DNA melting properties of the dityrosine cross-linked dimer of Ribonuclease A

Abstract

Several DNA binding proteins exist in dimeric form when bound with DNA to be able to exhibit various biological processes such as DNA repair, DNA replication and gene expression. Various dimeric forms of Ribonuclease A (RNase A) and other members of the ribonuclease A superfamily are endowed with a multitude of biological activities such as antitumor and antiviral activity. In the present study, we have compared the DNA binding properties between the RNase A monomer and the dityrosine (DT) cross-linked RNase A dimer, and checked the inhibitory effect of DNA on the ribonucleolytic activity of the dimeric protein. An agarose gel based assay shows that like the monomer, the dimer also binds with DNA. The number of nucleotides bound per monomer unit of the dimer is higher than the number of nucleotides that bind with the each monomer. From fluorescence measurements, the association constant (K_a) values for complexation of the monomer and the dimer with ct-DNA are (4.95 ± 0.45) × 10⁴ M⁻¹ and (1.29 ± 0.05) × 10⁶ M⁻¹ respectively. Binding constant (K_b) values for the binding of the monomer and the dimer with ct-DNA were determined using UV–vis spectroscopy and were found to be (4.96 ± 1.67) × 10⁴ M⁻¹ and (4.32 ± 0.31) × 10⁵ M⁻¹ respectively. Circular dichroism studies shows that the dimer possesses significant effect on DNA conformation. The melting profile for the ct-DNA–dimer indicated that the melting temperature (T_m) for the ct-DNA–dimer complex is lower compared to the ct-DNA–monomer complex. The ribonucleolytic activity of the dimer, like the monomer, diminishes upon binding with DNA.