Chromosomal replicases of cellular organisms utilize a ring shaped protein that encircles DNA as a mobile tether for high processivity in DNA synthesis. These "sliding clamps" have sufficiently large linear diameters to encircle duplex DNA and are perhaps even large enough to slide over certain DNA secondary structural elements. This report examines the Escherichia coli ␤ and human proliferating cell nuclear antigen clamps for their ability to slide over various DNA secondary structures. The

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... ults show that these clamps are capable of traversing a 13-nucleotide ssDNA loop, a 4-base pair stem-loop, a 4-nucleotide 5 tail, and a 15-mer bubble within the duplex. However, upon increasing the size of these structures (20-nucleotide loop, 12-base pair stemloop, 28-nucleotide 5 tail, and 20-nucleotide bubble) the sliding motion of the ␤ and proliferating cell nuclear antigen over these elements is halted. Studies of the E. coli replicase, DNA polymerase III holoenzyme, in chain elongation with the ␤ clamp demonstrate that upon encounter with an oligonucleotide annealed in its path, it traverses the duplex and resumes synthesis on the 3 terminus of the oligonucleotide. This sliding and resumption of synthesis occurs even when the oligonucleotide contains a secondary structure element, provided the ␤ clamp can traverse the structure. However, upon encounter with a downstream oligonucleotide containing a large internal secondary structure, the holoenzyme clears the obstacle by strand displacing the oligonucleotide from the template. Implications of these protein dynamics to DNA transactions are discussed. The Escherichia coli chromosomal replicase, DNA polymerase III (Pol III) 1 holoenzyme, is rapid and processive, in keeping with the need to replicate a 4 million-bp chromosome within 30 min (1). This enzyme is a multisubunit complex made up of three subcomplexes: the catalytic Pol III core, the ␤ sliding clamp, and the ␥ complex clamp loader (2). The catalytic core, comprised of ␣ (polymerase), ⑀ (3Ј-5Ј exonuclease), and subunits (3-5), incorporates nucleotides into a growing nascent chain and proofreads the replicated sequence. Pol III core by itself is not processive; it extends a primed site