Figure 5–17 The regulated sliding clamp that holds DNA polymerase on the DNA. (A) The structure of the clamp protein from E. coli, as determined by x-ray crystallography, with a DNA helix added to indicate how the protein fits around DNA (Movie 5.3). (B) Schematic illustration showing how the clamp (with red and yellow subunits) is loaded onto DNA to serve as a tether for a moving DNA polymerase molecule. The structure of the clamp loader (dark green) resembles a screw nut, (A) (B) sliding clamp clamp loader 5˜ATPADPPi ATPATP+ DNA + DNA polymerase 3˜5˜5˜3˜3˜RECYCLING OF RELEASED CLAMP LOADER ATP BINDING TO CLAMP LOADER OPENS SLIDING CLAMP DNA ENGAGED IN CLAMP ATP HYDROLYSIS LOCKS SLIDING CLAMP AROUND DNA AND RELEASES CLAMP LOADER DNA POLYMERASE BINDS TO SLIDING CLAMP + with its threads matching the grooves of double-stranded DNA. The loader binds to a free clamp molecule, forcing a gap in its ring of subunits so that this ring is able to slip around DNA. The clamp loader, thanks to its screw-nut structure, recognises the region of DNA that is double-stranded and latches onto it, tightening around the complex of a template strand with a freshly synthesized elongating (primer) strand. It carries the clamp along this double-stranded region until it encounters the 3ʹ end of the primer, at which point the loader hydrolyzes ATP and releases the clamp, allowing it to close around the DNA and bind to DNA polymerase. In the simplified reaction shown here, the clamp loader dissociates into solution once the clamp has been assembled. At a true replication fork, the clamp loader remains close to the polymerase so that, on the lagging strand, it is ready to assemble a new clamp at the start of each new Okazaki fragment (see Figure 5–18). (A, from  X.P. Kong et al., Cell 69:425–437, 1992. With permission from Elsevier; B, adapted from B.A. Kelch et al., Science 334:1675–1680, 2011. With permission from AAAS. PDB code: 3BEP.)
The Proteins at a Replication Fork Cooperate to Form a Replication Machine
Although we have discussed DNA replication as though it were performed by a mix of proteins all acting independently, in reality most of the proteins are held together in a large and orderly multienzyme complex that rapidly synthesizes DNA. This complex can be likened to a tiny sewing machine composed of protein parts and powered by nucleoside triphosphate hydrolysis. Like a sewing machine, the replication complex probably remains stationary with respect to its immediate surroundings; the DNA can be thought of as a long strip of cloth being rapidly threaded through it. Although the replication complex has been most intensively studied in E. coli and several of its viruses, a very similar complex also operates in eukaryotes, as we see below.