DnaX Complex of Escherichia coli DNA Polymerase III Holoenzyme THE χ·ψ

Abstract

An artificial operon that contains tandem holC-holD genes was used to overproduce a complex of the [IMG]_11043_tex2html_wrap78.xbm"> and [IMG]_11043_tex2html_wrap118.xbm"> subunits of the DNA polymerase III holoenzyme. Normally insoluble by itself, [IMG]_11043_tex2html_wrap118.xbm"> forms a tight soluble complex with [IMG]_11043_tex2html_wrap78.xbm">. A purification procedure that yields pure, active [IMG]_11043_tex2html_wrap78.xbm">[bullet][IMG]_11043_tex2html_wrap118.xbm"> complex in 100-mg quantities suitable for biophysical studies is reported. Sedimentation equilibrium studies demonstrate that [IMG]_11043_tex2html_wrap78.xbm">[bullet][IMG]_11043_tex2html_wrap118.xbm"> is a 1:1 heterodimer. The presence of [IMG]_11043_tex2html_wrap78.xbm">[bullet][IMG]_11043_tex2html_wrap118.xbm"> dramatically lowers the level of [IMG]_11043_tex2html_wrap104.xbm">[bullet][IMG]_11043_tex2html_wrap104.xbm">` required to reconstitute holoenzyme to levels expected in vivo. That [IMG]_11043_tex2html_wrap78.xbm">[bullet][IMG]_11043_tex2html_wrap118.xbm"> accomplishes this by binding to [IMG]_11043_tex2html_wrap96.xbm"> or [IMG]_11043_tex2html_wrap98.xbm"> and increasing their affinity for [IMG]_11043_tex2html_wrap104.xbm">[bullet][IMG]_11043_tex2html_wrap104.xbm">` was demonstrated by surface plasmon resonance using a Pharmacia BIAcore[IMG]_11043_tex2html_wrap106.xbm"> instrument. In the absence of [IMG]_11043_tex2html_wrap104.xbm">[bullet][IMG]_11043_tex2html_wrap104.xbm">`, [IMG]_11043_tex2html_wrap78.xbm">[bullet][IMG]_11043_tex2html_wrap118.xbm"> binds to either the [IMG]_11043_tex2html_wrap96.xbm"> or [IMG]_11043_tex2html_wrap98.xbm"> DnaX protein with K[IMG]_11043_tex2html_wrap124.xbm"> = 2 nM.