Assembly of a Chromosomal Replication Machine: Two DNA Polymerases, a Clamp Loader, and Sliding Clamps in One Holoenzyme Particle.

Abstract

The nine-subunit DNA polymerase (Pol) III* coupled to its beta sliding clamp is a rapid and highly processive replicating machine. The multiple subunits are needed for the complicated task of duplicating the Escherichia coli chromosome. In this report, Pol III* was constituted from individual pure proteins, and its structure was studied. Constitution of the Pol III* particle requires an ordered addition of the subunits, and the final structure contains 14 polypeptides in the ratio alpha 2 epsilon 2 theta 2 tau 2 gamma 2 delta 1 delta' 1 chi 1 psi 1. The structure can be summarized as being composed of two core polymerases (alpha epsilon theta) held together by a dimer of tau and one gamma complex clamp loader (gamma 2 delta 1 delta' 1 chi 1 psi 1) for loading beta onto DNA. At the center of the structure, the related tau and gamma subunits form a heterotetramer upon which the two core polymerases and clamp loader proteins assemble. The single copy nature of the delta, delta', chi, and psi subunits confers a structural asymmetry with respect to the two polymerases, presumably for the different functions of replicating the leading and lagging strands.

Replication Factor C Clamp Loader Subunit Arrangement within the Circular Pentamer and Its Attachment Points to Proliferating Cell Nuclear Antigen

“…However, unlike RFC, ␥ complex has only one strong subunit contact to the ␤ clamp, mediated by the ␦ subunit (22). Although the ␥ subunits contact ␤, they do so only with very weak affinity, and there is no detectable interaction thus far between ␦Ј and ␤ (15).…”

“…The ␥ trimer can also bind ␤ and unload it, but it is feeble in these actions compared with ␦ (15). Although ␦ binds ␤ 2 tightly, the ␥ complex does not bind ␤ 2 in the absence of ATP, indicating that one or more subunits of ␥ complex block the ␦-to-␤ 2 interaction (22). ATP binding to the ␥ subunits promotes tight interaction between ␥ complex and ␤, implying that ATP binding induces a conformation change in ␥ complex that exposes ␦, and presumably ␥ subunits as well, for interaction with ␤ and opening of the ␤ 2 ring.…”

Structural analysis of the inactive state of the Escherichia coli DNA polymerase clamp-loader complex

“…7, which is published as supporting information on the PNAS web site). The rms deviation in C ␣ positions for domain I of the ␥-subunits [excluding the zinc-binding loop (residues 64-79) and an N-terminal region (residues [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] that is structurally a part of domain II] is Ϸ1.5 Å at 2.0 ns, and the deviation in the corresponding segments of ␦ A and ␦Ј E is similar (Fig. 7).…”

“…We reasoned that the binding energy of ATP might be used to pry apart the ␦ A -and ␦Ј E -subunits, allowing ␦ A to interact with and open the ␤ clamp (6). This model emphasized the cyclical release and sequestration of the ␦ A -subunit, because biochemical data demonstrate that although the isolated ␦-subunit binds to the ␤ clamp, the interaction of the assembled clamp-loader complex with the clamp is ATP-dependent (12,15). Recently, fluorescence resonance energy transfer experiments revealed little change in the distance between the N-terminal domains of ␦ A and ␦Ј E after nucleotide or ␤-clamp binding (16).…”

“…ATP binding to the clamp loader triggers a conformational change that allows the complex to bind to and open the clamp and load it onto DNA (11,12). In an intriguing control mechanism, the interaction with DNA stimulates the otherwise suppressed ATPase activity of the clamp loader, leading to ATP hydrolysis (11,13) and separation of the clamp-loader complex from the DNA-loaded clamp.…”