Beta*, a UV-inducible shorter form of the beta subunit of DNA polymerase III of Escherichia coli. II. Overproduction, purification, and activity as a polymerase processivity clamp.

Abstract:

Control elements located inside the coding sequence of dnaN, the gene encoding the beta subunit of DNA polymerase III holoenzyme, direct the synthesis of a shorter and UV-inducible form of the beta subunit (Skaliter, R., Paz-Elizur, T., and Livneh, Z. (1996) J. Biol. Chem. 271, 2278-2281, and Paz-Elizur, T., Skaliter, R., Blumenstein, S., and Livneh, Z. (1996) J. Biol. Chem. 271, 2282-2290). The protein, termed beta*, was overproduced using the phage T7 expression system, leading to its accumulation as inclusion bodies at 5-10% of the total cellular proteins. beta* was purified in denatured form, followed by refolding to yield a preparation > 95% pure. Denatured beta* had a molecular mass of 26 kDa and contained two isoforms when analyzed by two-dimensional gel electrophoresis. The major isoform had a pI of 5.45, and comigrated with cellular beta*. Size exclusion high performance liquid chromatography under nondenaturing conditions and chemical cross-linking experiments indicate that beta* is a homotrimer. DNA synthesis by DNA polymerase III* was stimulated up to 10-fold by beta*, primarily due to an increase in the processivity of polymerization. It is suggested that beta* functions as an alternative sliding DNA clamp in a process associated with DNA synthesis in UV-irradiated cells.

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