Figure 5–18 summarizes the functions of the subunits of the replication machine. At the front of the replication fork, DNA helicase opens the DNA helix. Two DNA polymerase molecules work at the fork, one on the leading strand and one on the lagging strand. Whereas the DNA polymerase molecule on the leading strand can operate in a continuous fashion, the DNA polymerase molecule on the lagging strand must restart at short intervals, using a short RNA primer made by a DNA primase molecule. The close association of all these protein components increases the efficiency of replication and is made possible by a folding back of the lagging strand as shown in Figure 5–18A. This arrangement also facilitates the loading of the polymerase clamp each time that an Okazaki fragment is synthesized: the clamp loader and the lagging-strand DNA polymerase molecule are kept in place as a part of the protein machine even when they detach from their DNA template. The replication proteins are thus linked together into a single large
Figure 5–18 A bacterial replication fork. (A) This schematic diagram shows a current view of the arrangement of replication proteins at a replication fork when DNA is being synthesized. The lagging-strand DNA is folded to bring the lagging-strand DNA polymerase molecule into a complex with the leading-strand DNA polymerase molecule. This folding also brings the 3ʹ end of each completed Okazaki fragment close to the start site for the next Okazaki fragment. Because the lagging-strand DNA polymerase molecule remains bound to the rest of the replication proteins, it can be reused to synthesize successive Okazaki fragments. In this diagram, it is about to let go of its completed DNA fragment and move to the RNA primer that is just being synthesized. Additional proteins (not shown) help to hold the different protein components of the fork together, enabling them to function as a well-coordinated protein machine (Movie 5.4 and Movie 5.5). (B) An electron micrograph showing the replication machine from the bacteriophage  T4 as it moves along a template synthesizing DNA behind it. (C) An interpretation of the micrograph is given in the sketch: note especially the DNA loop on the lagging strand. Apparently, the replication proteins became partly detached from the very front of the replication fork during the preparation of this sample for electron microscopy. (B, courtesy of Jack Griffith; see P.D. Chastain et al., J. Biol. Chem. 278:21276–21285, 2003.) unit (total molecular mass >106 daltons), enabling DNA to be synthesized on both sides of the replication fork in a coordinated and efficient manner.
On the lagging strand, the DNA replication machine leaves behind a series of unsealed Okazaki fragments, which still contain the RNA that primed their synthesis at their 5ʹ ends. As discussed earlier, this RNA is removed and the resulting gap is filled in by DNA repair enzymes that operate behind the replication fork (see Figure 5–11).
A Strand-Directed Mismatch Repair System Removes Replication Errors That Escape from the Replication Machine
As stated previously, bacteria such as E. coli are capable of dividing once every 30 minutes, making it relatively easy to screen large populations to find a rare mutant cell that is altered in a specific process. One interesting class of mutants consists of those with alterations in so-called mutator genes, which greatly increase the rate of spontaneous mutation. Not surprisingly, one such mutant makes a defective form of the 3ʹ-to-5ʹ proofreading exonuclease that is a part of the DNA polymerase enzyme (see Figures 5–8 and 5–9). The mutant DNA polymerase no longer proofreads effectively, and many replication errors that would otherwise have been removed accumulate in the DNA.