Purification and properties of the uvrA protein from Escherichia coli.

Abstract

The uvrA+ gene product from E. coli was purified to apparent homoge neity; the assay measured its ability to restore repair endonuclease activity in extracts from uvrA mutated cells. The uvrA protein is a 115,000 MW DNA-binding pr otein 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 Pi. The ATPase activity is not DNA dependent and has a Km of 0.23 mM, which corresponds to the Km for the ATP requirement of the UV-endonuclease reaction catalyzed by the combined uvrA+, uvrB+ and uvrC+ gene products. ADP and adenosine 5''-[.gamma.-thio]triphosphate both inhibit the uvrA ATPase and the uvrABC endonuclease and also prevent specific binding of the uvrA protein to UV-irradiated DNA. Both the DNA-binding property and the ATPase activity of the uvrA protein are essential for uvrABC endonuclease activity. The ATP requirement of the endonuclease reaction is determined by the uvrA ATPase.