There are more protein components in eukaryotic replication machines than there are in the bacterial analogs, even though the basic functions are the same. Thus, for example, the eukaryotic single-strand binding (SSB) protein is formed from three subunits, whereas only a single subunit is found in bacteria. Similarly, the eukaryotic DNA primase is incorporated into a multisubunit enzyme that also contains a polymerase called DNA polymerase α-primase. This protein complex begins each Okazaki fragment on the lagging strand with RNA and then extends the RNA primer with a short length of DNA. At this point, the two main eukaryotic replicative DNA polymerases, Polδand Polε, come into play: Polδcompletes each Okazaki fragment on the lagging strand and Polε extends the leading strand. The increased complexity of eukaryotic replication machinery probably reflects reversal of the covalent attachment of the topoisomerase restores an intact orange double helix more elaborate controls. For example, the orderly maintenance of different cell types and tissues in animals and plants requires that DNA replication be tightly regulated. Moreover, eukaryotic DNA replication must be coordinated with the elaborate process of mitosis, as we discuss in Chapter 17.
As we see in the next section, the eukaryotic replication machinery has the added complication of having to replicate through nucleosomes, the repeating structural unit of chromosomes discussed in Chapter 4. Nucleosomes are spaced at intervals of about 200 nucleotide pairs along the DNA, which, as we will see, explains why new Okazaki fragments are synthesized on the lagging strand at intervals of 100–200 nucleotides in eukaryotes, instead of 1000–2000 nucleotides as in bacteria. Nucleosomes may also act as barriers that slow down the movement of DNA polymerase molecules, which may be why eukaryotic replication forks move only about one-tenth as fast as bacterial replication forks.
DNA replication takes place at a Y-shaped structure called a replication fork. A self-correcting DNA polymerase enzyme catalyzes nucleotide polymerization in a 5ʹ-to-3ʹ direction, copying a DNA template strand with remarkable fidelity. Since the two strands of a DNA double helix are antiparallel, this 5ʹ-to-3ʹ DNA synthesis can take place continuously on only one of the strands at a replication fork (the leading strand). On the lagging strand, short DNA fragments must be made by a “backstitching” process. Because the self-correcting DNA polymerase cannot start a new chain, these lagging-strand DNA fragments are primed by short RNA primer molecules that are subsequently erased and replaced with DNA.
DNA replication requires the cooperation of many proteins. These include (1) DNA polymerase and DNA primase to catalyze nucleoside triphosphate polymerization; (2) DNA helicases and single-strand DNA-binding (SSB) proteins to help in opening up the DNA helix so that it can be copied; (3) DNA ligase and an enzyme that degrades RNA primers to seal together the discontinuously synthesized lagging-strand DNA fragments; and (4) DNA topoisomerases to help to relieve helical winding and DNA tangling problems. Many of these proteins associate with each other at a replication fork to form a highly efficient “replication machine,” through which the activities and spatial movements of the individual components are coordinated.