SUMMARY

• 34.1 DNA Is Replicated by Polymerases

  DNA polymerases are template-directed enzymes that catalyze the formation of phosphodiester linkages by the 3′-hydroxyl group’s nucleophilic attack on the innermost phosphorus atom of a deoxyribonucleoside 5′-triphosphate. They cannot start strands de novo; a primer with a free 3′-hydroxyl group is required. Many DNA polymerases proofread the nascent product: their 3′ → 5′ exonuclease activity potentially edits the outcome of each polymerization step. A mispaired nucleotide is excised before the next step proceeds. In E. coli, DNA polymerase I repairs DNA and participates in replication. Fidelity is further enhanced by an induced fit that results in a catalytically active conformation only when the complex of enzyme, DNA, and correct dNTP is formed.

  Helicases prepare the way for DNA replication by using ATP hydrolysis to separate the strands of the double helix. Single-strand-binding proteins bind to the separated strands of DNA to prevent them from reannealing. Separation of the DNA into single strands introduces torsional stress in the DNA ahead of the separation. Topoisomerase relieves the stress by introducing supercoils.

• 34.2 DNA Replication Is Highly Coordinated

  DNA replication in E. coli starts at a unique origin (oriC) and proceeds sequentially in opposite directions. More than 20 proteins are required for replication. An ATP-driven helicase unwinds the oriC region to create a replication fork. At this fork, both strands of parental DNA serve as templates for the synthesis of new DNA. A short stretch of RNA formed by primase, an RNA polymerase, primes DNA synthesis. One strand of DNA (the leading strand) is synthesized continuously, whereas the other strand (the lagging strand) is synthesized discontinuously, in the form of 1-kb Okazaki fragments. Both new strands are formed simultaneously by the concerted actions of the highly processive DNA polymerase III holoenzyme, an asymmetric dimer. The discontinuous assembly of the lagging strand enables 5′ → 3′ polymerization at the molecular level to give rise to overall growth of this strand in the 3′ → 5′ direction. The RNA primer stretch is hydrolyzed by the 5′ → 3′ nuclease activity of DNA polymerase I, which also fills gaps. Finally, nascent DNA fragments are joined by DNA ligase in a reaction driven by ATP.

  DNA synthesis in eukaryotes is more complex than in bacteria. Eukaryotes require thousands of origins of replication to complete replication in a timely fashion. A special RNA-dependent DNA polymerase called telomerase is responsible for the replication of the ends of linear chromosomes.