Purification and properties of the Escherichia coli deoxyribonucleic acid-unwinding protein. Effects on deoxyribonucleic acid synthesis in vitro.

The DNA-unwinding protein from Escherichia coli has been purified to homogeneity. It is a single polypeptide of 22,000 daltons; the native molecular weight is 90,000. The effect of the protein on the activity of the three DNA polymerases of E. coli has been studied. The activities of DNA polymerases I and III are significantly reduced, whereas DNA polymerase II activity is enhanced in the presence of unwinding protein. The rate of transcription catalyzed by E. coli RNA polymerase in the presence of the protein is reduced when single-stranded, but not double-stranded, DNAs are employed as templates. Using fd DNA as template in a DNA synthetic reaction that is dependent on both RNA polymerase and DNA polymerase II, the unwinding protein was found to be essential for the synthesis of a DNA product that is equal in size to the template. With the use of crude cell-free extracts of E. coli, it was shown that DNA polymerase II can convert bacteriophage fd DNA to the replicative form. These experiments suggest a possible physiological role for DNA polymerase II and the unwinding protein of E. coli.