Purification and properties of the uvrA protein from Escherichia coli

Abstract

The uvrA+ gene product from E. coli was purified to apparent homogeneity; the assay measured its ability to restore repair endonuclease activity in extracts from uvrA mutated cells. The uvrA protein is a 115,000 molecular weight DNA-binding protein having higher affinity for single-stranded than double-stranded DNA. It does not introduce single-strand breaks or alkali-labile bonds in native or UV-irradiated DNA, but it catalyzes hydrolysis of ATP to ADP and P(i). The ATPase activity is not DNA dependent and has a K(m) of 0.23 mM, which corresponds to the K(m) for the ATP requirement of the UV-endonuclease reaction catalyzed by the combined uvrA+, uvrB+, and uvrC+ gene products. ADP and adenosine 5'-[γ-thio]triphosphate both inhibit the uvrA ATPase as wel as the uvrABC endonuclease and also prevent specific binding of the uvrA protein to UV-irradiated DNA. These results indicate that both the DNA-binding property and the ATPase activity of the uvrA protein are essential for uvrABC endonuclease activity and that the ATP requirement of the endonuclease reaction is determined by the uvrA ATPase.