Abstract
In Escherichia coli, the circular beta sliding clamp facilitates processive DNA replication by tethering the polymerase to primer-template DNA. When synthesis is complete, polymerase dissociates from beta and DNA and cycles to a new start site, a primed template loaded with beta. DNA polymerase cycles frequently during lagging strand replication while synthesizing 1-2-kilobase Okazaki fragments. The clamps left behind remain stable on DNA (t(12) approximately 115 min) and must be removed rapidly for reuse at numerous primed sites on the lagging strand. Here we show that delta, a single subunit of DNA polymerase III holoenzyme, opens beta and slips it off DNA (k(unloading) = 0.011 s(-)(1)) at a rate similar to that of the multisubunit gamma complex clamp loader by itself (0.015 s(-)(1)) or within polymerase (pol) III* (0.0065 s(-)(1)). Moreover, unlike gamma complex and pol III*, delta does not require ATP to catalyze clamp unloading. Quantitation of gamma complex subunits (gamma, delta, delta', chi, psi) in E. coli cells reveals an excess of delta, free from gamma complex and pol III*. Since pol III* and gamma complex occur in much lower quantities and perform several DNA metabolic functions in replication and repair, the delta subunit probably aids beta clamp recycling during DNA replication.
Highlights
Sliding clamps are ring-shaped proteins that completely encircle DNA and slide freely along the double helix
In vitro rolling circle assays performed with pol III* detect increasing amounts of  on the lagging strand DNA as replication proceeds, confirming that clamps left behind by the polymerase remain on the DNA [14]
We examined the clamp unloading activities of both ␥ complex and the ␦ protein, in order to determine whether ␦ can serve as the physiological clamp unloader in E. coli
Summary
Loading and unloading activities must be regulated such that ␥ complex performs the right function at the right time. A minimal subcomplex of ␥2–4␦1␦Ј1 without and appears sufficient for clamp loading [27], their presence facilitates holoenzyme activity under high ionic conditions [28] This effect may be due to a stabilizing interaction between the subunit of ␥ complex and single-stranded DNA-binding protein (SSB) on DNA [29]. The mechanism of ␥ complex-catalyzed clamp unloading has not been defined in detail, it is an ATP-dependent reaction; the initial steps are probably similar to those of clamp loading In this case, ATP binding to ␥ subunits and consequent conformational changes expose the ␦ subunit, which binds  on DNA and opens the ring, but instead of closing the ring back around DNA, ␥ complex allows it to slip free from DNA.
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