DNA structure requirements for the Escherichia coli gamma complex clamp loader and DNA polymerase III holoenzyme

The Escherichia coli chromosomal replicase, DNA polymerase III holoenzyme, is highly processive during DNA synthesis. Underlying high processivity is a ring-shaped protein, the beta clamp, that encircles DNA and slides along it, thereby tethering the enzyme to the template. The beta clamp is assembl ed onto DNA by the multiprotein gamma complex clamp loader that opens and c loses the beta ring around DNA in an ATP-dependent manner. This study exami nes the DNA structure required for clamp loading action. We found that the gamma complex assembles beta 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 gamma complex does not assemble beta onto relaxed closed circular DNA even at low ionic strength. Hence, a 3'-e nd is not required for clamp loading, but a single-stranded DNA (ssDNA)/dou ble-stranded DNA (dsDNA) junction can be utilized as a substrate, a result confirmed using synthetic oligonucleotides that form forked ssDNA/dsDNA jun ctions on M13 ssDNA On a flush primed template, the gamma complex exhibits polarity; it acts specifically at the 3'-ssDNA/dsDNA junction to assemble b eta onto the DNA, The gamma complex can assemble beta onto a primed site as short as 10 nucleotides, corresponding to the width of the beta ring. Howe ver, a protein block placed closer than 14 base pairs (bp) upstream from th e primer 3' terminus prevents the clamp loading reaction, indicating that t he gamma complex and its associated beta clamp interact with similar to 14- 16 bp at a ssDNA/dsDNA junction during the clamp loading operation. A prote in 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 pol ymerase has an even greater spatial requirement than the gamma complex on t he duplex portion of the primed site for function with beta. Interestingly, DNA secondary structure elements placed near the 3' terminus impose simila r steric limits on the gamma complex and polymerase action with beta. The p ossible biological significance of these structural constraints is discusse d.