The Escherichia coli chromosomal replicase, DNA polymerase III holoenzyme, is highly processive during DNA synthesis. Underlying high processivity is a ringshaped protein, the ␤ clamp, that encircles DNA and slides along it, thereby tethering the enzyme to the template. The ␤ clamp is assembled onto DNA by the multiprotein ␥ complex clamp loader that opens and closes the ␤ ring around DNA in an ATP-dependent manner. This study examines the DNA structure required for clamp loading action. We found that the ␥ complex assembles ␤ onto supercoiled DNA (replicative form I), but only at very low ionic strength, where regions of unwound DNA may exist in the duplex. Consistent with this, the ␥ complex does not assemble ␤ onto relaxed closed circular DNA even at low ionic strength. Hence, a 3-end is not required for clamp loading, but a singlestranded DNA (ssDNA)/double-stranded DNA (dsDNA) junction can be utilized as a substrate, a result confirmed using synthetic oligonucleotides that form forked ssDNA/dsDNA junctions on M13 ssDNA. On a flush primed template, the ␥ complex exhibits polarity; it acts specifically at the 3-ssDNA/dsDNA junction to assemble ␤ onto the DNA. The ␥ complex can assemble ␤ onto a primed site as short as 10 nucleotides, corresponding to the width of the ␤ ring. However, a protein block placed closer than 14 base pairs (bp) upstream from the primer 3 terminus prevents the clamp loading reaction, indicating that the ␥ complex and its associated ␤ clamp interact with ϳ14 -16 bp at a ssDNA/dsDNA junction during the clamp loading operation. A protein block positioned closer than 20 -22 bp from the 3 terminus prevents use of the clamp by the polymerase in chain elongation, indicating that the polymerase has an even greater spatial requirement than the ␥ complex on the duplex portion of the primed site for function with ␤. Interestingly, DNA secondary structure elements placed near the 3 terminus impose similar steric limits on the ␥ complex and polymerase action with ␤. The possible biological significance of these structural constraints is discussed.Escherichia coli DNA polymerase III holoenzyme (pol III 1 holoenzyme) is a highly processive multisubunit replicase (1). Processivity is conferred to the polymerase by the ␤ subunit (2, 3). The ␤ subunit is a ring-shaped protein that completely encircles DNA and slides along the duplex (4, 5). The ␤ ring endows the polymerase with high processivity by binding directly to it, continuously tethering it to the template during synthesis (4). The ␤ ring does not assemble onto DNA by itself; for this, it requires the clamp loading action of the ␥ complex. The ␥ complex is composed of five different proteins (␥, ␦, ␦Ј, , and ) (1). Upon binding ATP, the ␥ complex opens the ␤ ring and positions it around the primed template (7,8). Hydrolysis of two molecules of ATP results in closing the ring around DNA and dissociation of the ␥ complex from the clamp (7, 49).Following the release of the clamp loader from the clamp, the catalytic core polymerase subassembly (pol ...