Dna Replication Is Similar in Eucaryotes and Bacteria

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Much of what we know about DNA replication was first derived from studies of purified bacterial and bacteriophage multienzyme systems capable of DNA replication in vitro. The development of these systems in the 1970s was greatly facilitated by the prior isolation of mutants in a variety of replication genes; these mutants were exploited to identify and purify the corresponding replication proteins. The first mammalian replication system that accurately replicated DNA in vitro was described in the mid-1980s, and mutations in genes encoding nearly all of the replication components have now been isolated and analyzed in the yeast Saccharomyces cerevisiae. As a result, a great deal is known about the detailed enzymology of DNA replication in eucaryotes, and it is clear that the fundamental features of DNA replication—including replication fork geometry and the use of a multiprotein replication machine—have been conserved during the long evolutionary process that separates bacteria and eucaryotes.

There are more protein components in eucaryotic replication machines than there are in the bacterial analogs, even though the basic functions are the same. Thus, for example, the eucaryotic single-strand binding (SSB) protein is formed from three subunits, whereas only a single subunit is found in bacteria. Similarly, the DNA primase is incorporated into a multisubunit enzyme called DNA polymerase α. The polymerase α begins each Okazaki fragment on the lagging strand with RNA and then extends the RNA primer with a short length of DNA, before passing the 3′ end of this primer to a second enzyme, DNA polymerase δ. This second DNA polymerase then synthesizes the remainder of each Okazaki fragment with the help of a clamp protein (Figure 5-28).

Figure 5-28

A mammalian replication fork. The fork is drawn to emphasize its similarity to the bacterial replication fork depicted in Figure 5-21. Although both forks use the same basic components, the mammalian fork differs in at least two important respects. First, (more...)

As we see in the next section, the eucaryotic 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 may explain why new Okazaki fragments are synthesized on the lagging strand at intervals of 100–200 nucleotides in eucaryotes, 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 eucaryotic replication forks move only one-tenth as fast as bacterial replication forks.

Summary

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DNA replication takes place at a Y-shaped structure called a replication fork. A self-correcting DNA polymerase enzyme catalyzes nucleotide polymerization in a 5′-to-3′ direction, copying a DNA template strand with remarkable fidelity. Since the two strands of a DNA double helix are antiparallel, this 5′-to-3′ 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.

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Copyright © 2002, Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter; Copyright © 1983, 1989, 1994, Bruce Alberts, Dennis Bray, Julian Lewis, Martin Raff, Keith Roberts, and James D. Watson .

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• Base-Pairing Underlies DNA Replication and DNA Repair

• The DNA Replication Fork Is Asymmetrical

• The High Fidelity of DNA Replication Requires Several Proofreading Mechanisms

• Only DNA Replication in the 5′-to-3′ Direction Allows Efficient Error Correction

• A Special Nucleotide-Polymerizing Enzyme Synthesizes Short RNA Primer Molecules on the Lagging Strand

• Special Proteins Help to Open Up the DNA Double Helix in Front of the Replication Fork

• A Moving DNA Polymerase Molecule Stays Connected to the DNA by a Sliding Ring

• The Proteins at a Replication Fork Cooperate to Form a Replication Machine

• A Strand-directed Mismatch Repair System Removes Replication Errors That Escape from the Replication Machine

• DNA Topoisomerases Prevent DNA Tangling During Replication

• DNA Replication Is Similar in Eucaryotes and Bacteria

• Summary