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.
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.
Skaliter
R
R
Department of Biochemistry, Weizmann Institute of Science, Rehovot, Israel.
Bergstein
M
M
Livneh
Z
Z
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002850:Chromatography, Gel; D004258:DNA Polymerase III / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017931:DNA Primers; D015180:Electrophoresis, Gel, Two-Dimensional; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology / Q000528:radiation effects*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D014466:Ultraviolet Rays
D017931:DNA Primers; D000255:Adenosine Triphosphate; D004258:DNA Polymerase III