Nucleic acid helix-coil transitions mediated by helix-unwinding proteins from calf thymus.
We have studied nucleic acid double helix destabilization mediated by purified calf helix-unwinding proteins, measuring ultraviolet hyperchromicity to detect helix melting. Both calf unwinding protein 1 (UP1) and a high salt eluting protein fraction are found to depress strongly the helix melting temperature (Tm) of the synthetic alternating copolymers poly[d(AT)] and poly[r(AU)], indicating that both DNA and RNA are recognized by these proteins. UP1 also destabilizes natural, GC-containing DNA helices, but to a smaller extent than observed with the above polymers. A simple model is presented to aid in the qualitative interpretation of the data, outlining the expected effect on the helix-coil transition of a protein ligand with differential affinity for the helix or coil form of nucleic acid. The observed helix-destabilizing effect of UP1 is dependent on the protein to nucleic acid ratio in an expected manner. Competition studies demonstrate a low, but appreciable affinity of UP1 for native DNA, opening the possibility that protein-mediated denaturation might be initiated by protein binding to the double helix. "Hairpin" helical regions of denatured DNA are strongly destabilized by UP1. Despite the fact that removal of these hairpin helices might greatly facilitate DNA renaturation, we failed to observe renaturation from the UP1-DNA complex after a switch to helix-stabilizing conditions. Thus, UP1 shows an important difference from its presumed prokaryotic analogue, T4 gene 32-protein. Possible in vivo functions of the calf proteins are discussed in light of these observations.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004247:DNA; D003852:Deoxyribonucleoproteins; D008024:Ligands; D008274:Magnesium; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009698:Nucleoproteins; D009994:Osmolar Concentration; D011119:Polynucleotides; D011485:Protein Binding; D011487:Protein Conformation; D012964:Sodium; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D013950:Thymus Gland / Q000032:analysis*
D003852:Deoxyribonucleoproteins; D008024:Ligands; D009698:Nucleoproteins; D011119:Polynucleotides; D004247:DNA; D012964:Sodium; D008274:Magnesium