Abstract
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 beta and human proliferating cell nuclear antigen clamps for their ability to slide over various DNA secondary structures. The results 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 stem-loop, 28-nucleotide 5' tail, and 20-nucleotide bubble) the sliding motion of the beta 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 beta 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 beta 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.
Highlights
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]
On the discontinuous lagging strand, the polymerase must rapidly recycle from the end of one Okazaki fragment to a new upstream primed site synthesized by primase
Pol ␦ is thought to act on the lagging strand in similar fashion to the E. coli system, hopping among proliferating cell nuclear antigen (PCNA) clamps and leaving the used clamps behind on the DNA where they may interact with other proteins [24]
Summary
Pol III, DNA polymerase III; bp, base pair(s); Pol ␦, DNA polymerase ␦; PCNA, proliferating cell nuclear antigen; RF-C, replication factor C; ssDNA, single-stranded DNA; BSA, bovine serum albumin; DTT, dithiothreitol; kb, kilobase(s). The human DNA polymerase ␦ replicase (Pol ␦) relies on a ring shaped clamp and a clamp loader for processive synthesis [16, 17]. Sliding Clamp Dynamics a primed template, Pol ␦ binds the clamp for processivity in DNA synthesis (19 –23). Pol ␦ is thought to act on the lagging strand in similar fashion to the E. coli system, hopping among PCNA clamps and leaving the used clamps behind on the DNA where they may interact with other proteins [24]. Earlier studies have shown that  and PCNA slide freely over duplex DNA, consistent with their large inner diameters;  measures approximately 35 Å (not including extended side chains) [7], and the inner diameter of the PCNA ring measures approximately 34 Å [36]. We find here that upon encounter with a DNA structure that  is incapable of traversing, Pol III holoenzyme does not stop but instead strand displaces the oligonucleotide containing the obstacle
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