Figure 5–13 An assay for DNA helicase enzymes. A short DNA fragment is annealed to a long DNA single strand to form a region of DNA double helix. The double helix is melted as the helicase runs along the DNA single strand, releasing the short DNA fragment in a reaction that requires the presence of both the helicase protein and ATP. The rapid stepwise movement of the helicase is powered by its ATP hydrolysis (shown schematically in Figure 3–75A). As indicated, many DNA helicases are composed of six subunits.
Figure 5–14 The structure of a DNA helicase. (A) Diagram of the protein 5˜ as a hexameric ring drawn to scale with a replication fork. (B) Detailed structure of the bacteriophage T7 replicative helicase, as determined by x-ray diffraction. Six identical subunits bind and hydrolyze ATP in an ordered fashion to propel this molecule, like a rotary engine, along a DNA single strand that passes through the central hole. Red indicates bound ATP molecules in the structure (Movie 5.2). (PDB code: 1E0J.) finished synthesizing one Okazaki fragment on the lagging strand to be recycled quickly, so as to begin the synthesis of the next Okazaki fragment on the same strand. This rapid dissociation, however, would make it difficult for the polymerase to synthesize the long DNA strands produced at a replication fork were it not for an accessory protein (called PCNA in eukaryotes) that functions as a regulated sliding clamp. This clamp keeps the polymerase firmly on the DNA when it is moving, but releases it as soon as the polymerase runs into a double-strand region of DNA.
How can a sliding clamp prevent the polymerase from dissociating without at the same time impeding the polymerase’s rapid movement along the DNA molecule? The three-dimensional structure of the clamp protein, determined by x-ray diffraction, revealed it to be a large ring around the DNA double helix. One face of the ring binds to the back of the DNA polymerase, and the whole ring slides freely along the DNA as the polymerase moves. The assembly of the clamp around the DNA requires ATP hydrolysis by a special protein complex, the clamp loader, which hydrolyzes ATP as it loads the clamp on to a primer–template junction (Figure 5–17).
On the leading-strand template, the moving DNA polymerase is tightly bound to the clamp, and the two remain associated for a very long time. The DNA polymerase on the lagging-strand template also makes use of the clamp, but each time the polymerase reaches the 5ʹ end of the preceding Okazaki fragment, the polymerase releases itself from the clamp and dissociates from the template. This polymerase molecule then associates with a new clamp that is assembled on the RNA primer of the next Okazaki fragment.
single-stranded region of DNA template with short regions of base-paired “hairpins”
Figure 5–15 The effect of single-strand DNA-binding proteins (SSb proteins) on the structure of single-strand DNA. Because each protein molecule prefers to bind next to a previously bound molecule, long rows of this protein form on a DNA single strand. This cooperative binding straightens out the DNA template and facilitates the DNA polymerization process. The “hairpin helices” shown in the bare, single-strand DNA result from a chance matching of short regions of complementary nucleotide sequence; they are similar to the short helices that typically form in RNA molecules (see Figure 1–6).
Figure 5–16 Human single-strand binding protein bound to DNA. (A) Front view of the two DNA-binding domains of the protein (called RPA) which cover a total of eight nucleotides. Note that the DNA bases remain exposed in this protein–DNA complex. (B) Diagram showing the three-dimensional structure, with the DNA strand (orange) viewed end-on. (PDB code: 1JMC.)