Purification of the rep Protein of Escherichia COG AN ATPase WHICH SEPARATES DUPLEX DNA STRANDS IN ADVANCE

The product of the rep gene o f Escherichia coli catalytically separa tes 4x174 duplex DNA s t rands in advance of the i r replication, uti l izing ATP in the process (Scott, J. F., Eisenberg, S., Bertsch, L. L., and Kornberg, A. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 193-197). The enzyme has now been purified to near-homogeneity. Relatively large quant i ties were obtained f rom ColEl-plasmid-containing cells in which t h e enzyme level was 7 to 10 t imes above wild type. The assay for rep protein was based on i t s essential role, with phage-induced cistron A protein, in enzymat ic synthesis of phage 4 x 1 7 4 (+) strands, using duplex c i rcu lar DNA as template. The protein exhibits a molecular weight of 65,000 under dena tur ing a n d reducing conditions. T h e tu rn over number of t h e enzyme i s approximately 6800 ATP moleculeslmin in s t rand separa t ion as measured by extent of replication, or in an uncoupled reaction us ing singlestranded DNA effector.