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

Abstract

The Escherichia coli chromosomal replicase, DNA polymerase III holoenzyme, is highly processive during DNA synthesis. Underlying high processivity is a ring-shaped protein, the β clamp, that encircles DNA and slides along it, thereby tethering the enzyme to the template. The β clamp is assembled onto DNA by the multiprotein γ complex clamp loader that opens and closes the β ring around DNA in an ATP-dependent manner. This study examines the DNA structure required for clamp loading action. We found that the γ complex assembles β onto supercoiled DNA (replicative form I), but only at very low ionic strength, where regions of un-wound DNA may exist in the duplex. Consistent with this, the γ complex does not assemble β onto relaxed closed circular DNA even at low ionic strength. Hence, a 3'-end is not required for clamp loading, but a single-stranded 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 γ complex exhibits polarity; it acts specifically at the 3'-ssDNA/dsDNA junction to assemble β onto the DNA. The γ complex can assemble onto a primed site as short as 10 nucleotides, corresponding to the width of the β 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 γ complex and its associated β clamp interact with ~14-16 hp at a ssDNA/dsDNA junction during the clamp loading operation. A protein block positioned closer than 20-22 hp 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 γ complex on the duplex portion of the primed site for function with β. Interestingly, DNA secondary structure elements placed near the 3' terminus impose similar steric limits on the γ complex and polymerase action with β. The possible biological significance of these structural constraints is discussed.