Purification of ribonuclease H as a factor required for initiation ofin vitroColE1 DNA replication

Abstract

Escherichia coli ribonuclease H was purified to near-homogeneity and identified as the only additional factor required for initiation of in vitro Col El DNA replication from the unique origin by RNA polymerase and DNA polymerase I. Both ribonuclease H activity and stimulating activity for ColEl DNA synthesis comigrate with the single protein band in gel electrophoresis. These two activities coincide throughout the process of purification. Some DNA synthesis takes place on covalently closed-circular DNA molecules other than ColEl DNA with the three purified enzymes. This DNA synthesis is suppressed by an Esherlchla coli single-strand DNA binding protein and/or a high concentration of ribonuclease H. Negative superhelicity of template DNA is required for efficient primer formation. No evidence that supports involvement of ribonuclease III in initiation of ColEl DNA replication or its regulation was found.