SUMMARY

28.1 DNA Replication Proceeds by the Polymerization of Deoxyribonucleoside Triphosphates Along a Template

DNA polymerases are template-directed enzymes that catalyze the formation of phosphodiester bonds by nucleophilic attack by the 3′-hydroxyl group on the innermost phosphorus atom of a deoxyribonucleoside 5′-triphosphate. The complementarity of shape between correctly matched nucleotide bases is crucial to ensuring the fidelity of base incorporation. DNA polymerases cannot start chains de novo; a primer with a free 3′-hydroxyl group is required. Thus, DNA synthesis is initiated by the synthesis of an RNA primer, the task of a specialized primase enzyme. After serving as a primer, the RNA is degraded and replaced by DNA. DNA polymerases always synthesize a DNA strand in the 5′-to-3′ direction. So that both strands of the double helix can be synthesized in the same direction simultaneously, one strand is synthesized continuously while the other is synthesized in fragments called Okazaki fragments. Gaps between the fragments are sealed by DNA ligases. ATP-driven helicases prepare the way for DNA replication by separating the strands of the double helix.

28.2 DNA Unwinding and Supercoiling Are Controlled by Topoisomerases

A key topological property of DNA is its linking number (Lk), which is defined as the number of times one strand of DNA winds around the other in the right-hand direction when the DNA axis is constrained to lie in a plane. Molecules differing in linking number are topoisomers of one another and can be interconverted only by cutting one or both DNA strands; these reactions are catalyzed by topoisomerases. Changes in linking number generally lead to changes in both the number of turns of double helix and the number of turns of superhelix. Topoisomerase II catalyzes the ATP-driven introduction of negative supercoils, which leads to the compaction of DNA and renders it more susceptible to unwinding. Supercoiled DNA can be relaxed by topoisomerase I or topoisomerase II. Topoisomerase I acts by transiently cleaving one strand of DNA in a double helix, whereas topoisomerase II transiently cleaves both strands simultaneously.

28.3 DNA Replication Is Highly Coordinated

Replicative DNA polymerases are processive; that is, they catalyze the addition of many nucleotides without dissociating from the template. A major contributor to processivity is the DNA sliding clamp, such as the dimeric β subunit of the E. coli replicative polymerase. The sliding clamp has a ring structure that encircles the DNA double helix and keeps the enzyme and DNA associated. The DNA polymerase holoenzyme is a large DNA-copying machine formed by two DNA polymerase enzymes, one to act on each template strand, associated with other subunits including a sliding clamp and a clamp loader.

The synthesis of the leading and lagging strands of a double-stranded DNA template is coordinated. As a replicative polymerase moves along a DNA template, the leading strand is copied smoothly while the lagging strand forms loops that change length in the course of the synthesis of each Okazaki fragment. The mode of action is referred to as the trombone model.

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DNA replication is initiated at a single site within the E. coli genome. A set of specific proteins recognizes this origin of replication and assembles the enzymes needed for DNA synthesis, including a helicase that promotes strand separation. The initiation of replication in eukaryotes is more complex. DNA synthesis is initiated at thousands of sites throughout the genome. Complexes homologous to those in E. coli, but more complicated, are assembled at each eukaryotic origin of replication. A special polymerase called telomerase that relies on an RNA template synthesizes specialized structures called telomeres at the ends of linear chromosomes.

28.4 Many Types of DNA Damage Can Be Repaired

A wide variety of DNA damage can occur. For example, mismatched bases may be incorporated in the course of DNA replication or individual bases may be damaged by oxidation or alkylation after DNA replication. Other forms of damage are the formation of cross-links and the introduction of single- or double-stranded breaks in the DNA backbone. Several different repair systems detect and repair DNA damage. Repair begins with the process of proofreading in DNA replication: mismatched bases that were incorporated in the course of synthesis are excised by exonuclease activity present in replicative polymerases. Some DNA lesions such as thymine dimers can be directly reversed through the action of specific enzymes. Other DNA-repair pathways act through the excision of single damaged bases (base-excision repair) or short segments of nucleotides (nucleotide-excision repair). Double-stranded breaks in DNA can be repaired by homologous or nonhomologous end-joining processes. Defects in DNA-repair components are associated with susceptibility to many different sorts of cancer. Such defects are a common target of cancer treatments. Many potential carcinogens can be detected by their mutagenic action on bacteria (the Ames test).

28.5 DNA Recombination Plays Important Roles in Replication, Repair, and Other Processes

Recombination is the exchange of segments between two DNA molecules. Recombination is important in some types of DNA repair as well as other processes such as meiosis, the generation of antibody diversity, and the life cycles of some viruses. Some recombination pathways are initiated by strand invasion, in which a single strand at the end of a DNA double helix forms base pairs with one strand of DNA in another double helix and displaces the other strand. A common intermediate formed in other recombination pathways is the Holliday junction, which consists of four strands of DNA that come together to form a crosslike structure. Recombinases promote recombination reactions through the introduction of specific DNA breaks and the formation and resolution of Holliday-junction intermediates.