Connecting Concepts Across Chapters H

This chapter has built on a central concept introduced in Chapter 2, that cell division is preceded by replication of the genetic material. In Chapter 2, we saw that DNA replication takes place in the S phase of the cell cycle and that several checkpoints ensure that division does not take place in the absence of DNA replication. The current chapter examined the process of DNA synthesis.

DNA is sometimes said to be a self-replicating molecule, but nothing could be farther from the truth. Replication requires much more than a DNA template; a large number of proteins and enzymes also are necessary. Despite this complexity, a few rules summarize the process: (1) all replication is semiconservative, (2) new DNA molecules always elongate at the 3' end (replication is 5':3'), (3) replication begins at sequences called origins and requires RNA primers for initiation, (4) DNA synthesis takes place continuously on one strand and dis-continuously on the other, and (5) newly synthesized nucleotide strands are antiparallel and complementary to their template strands.

As we have seen, replication takes place with a high degree of accuracy; this accuracy is essential to maintain the integrity of genetic information as DNA molecules are copied again and again. The accuracy of replication is maintained by several different mechanisms, including precision in nucleotide selection, the ability of DNA polymerases to proofread and correct mistakes, and the detection and repair of residual mismatches after replication (mismatch repair).

An understanding of DNA replication provides a foundation for several topics that will be introduced in later chapters of this book. Chapter 18 (on recombinant DNA technology) examines the polymerase chain reaction and other techniques (DNA sequence analysis and cloning) that require an understanding of DNA synthesis. In Chapter 17 (on gene mutation and DNA repair), we learn that, in spite of the accuracy of DNA synthesis, errors do arise and sometimes lead to mutations. These errors are addressed by mechanisms of DNA repair, many of which require DNA synthesis. The movement of transposable genetic elements (Chapter 11) also requires DNA synthesis.

CONCEPTS SUMMARY]_

• Replication is semiconservative: DNA's two nucleotide strands separate and each serves as a template on which a new strand is synthesized

• A replicon is a unit of replication that contains an origin of replication.

• In theta replication of DNA, the two nucleotide strands of a circular DNA molecule unwind, creating a replication bubble; within each replication bubble, DNA is normally synthesized on both strands and at both replication forks, producing two circular DNA molecules.

• Rolling-circle replication is initiated by a nick in one strand of circular DNA, which produces a 3'-OH group to which new nucleotides are added while the 5' end of the broken strand is displaced from the circle. Replication proceeds around the circle, producing a circular DNA molecule and a single-stranded linear molecule.

• Linear eukaryotic DNA contains many origins of replication. At each origin, the DNA unwinds, producing two nucleotide strands that serve as templates. Unwinding and replication take place on both templates at both ends of the replication bubble until adjacent replicons meet, resulting in two linear DNA molecules.

• DNA synthesis requires a single-stranded DNA template, deoxyribonucleoside triphosphates; and a group of enzymes and proteins that carry out replication.

• All DNA synthesis is in the 5 ': 3' direction. Because the two nucleotide strands of DNA are antiparallel, replication takes place continuously on one strand (the leading strand) and discontinuously on the other (the lagging strand).

• Replication begins when an initiator protein binds to a replication origin and unwinds a short stretch of DNA, to which DNA helicase attaches. DNA helicase unwinds the DNA at the replication fork, single-strand-binding proteins bind to single nucleotide strands to prevent them from reannealing, and DNA gyrase (a topoisomerase) removes the strain ahead of the replication fork that is generated by unwinding.

• During replication, primase synthesizes short primers of RNA nucleotides, providing a 3'-OH group to which DNA polymerase can add DNA nucleotides.

• DNA polymerase adds new nucleotides to the 3' end of a growing polynucleotide strand. Bacteria have two DNA polymerases that have primary roles in replication: DNA polymerase III, which synthesizes new DNA on the leading and lagging strands; and DNA polymerase I, which removes and replaces primers.

• DNA ligase seals nicks that remain in the sugar - phosphate backbones when the RNA primers are replaced by DNA nucleotides.

• Several mechanisms ensure the high rate of accuracy in replication, including precise nucleotide selection, proofreading, and mismatch repair.

Eukaryotic replication is similar to bacterial replication, although eukaryotes have multiple origins of replication and different DNA polymerases.

Precise replication at multiple origins is ensured by a licensing factor that must attach to an origin before replication can begin. The licensing factor is removed after replication is initiated and renewed after cell division.

Eukaryotic nucleosomes are quickly assembled on new molecules of DNA; newly assembled nucleosomes consist of a random mixture of old and new histone proteins.

The ends of linear eukaryotic DNA molecules are replicated by the enzyme telomerase.

Replication in archaeal bacteria has a number of features in common with eukaryotic replication.

Homologous recombination takes place through the exchange of genetic material between homologous DNA molecules. In the Holliday model, homologous recombination begins with single-strand breaks in both DNA molecules, followed by strand displacement, branch migration, and Holliday junction resolution. In the double-strand break model, it begins with a double-strand-break, followed by strand displacement, DNA synthesis, and resolution of two Holliday junctions.

Homologous recombination in E. coli requires a number of enzymes, including RecA, RecBCD, resolvase, single-strand-binding proteins, ligase, DNA polymerases, and gyrase.

(important terms_

semiconservative replication (p. 000) equilibrium density gradient centrifugation (p. 000) replicon (p. 000) replication origin (p. 000) theta replication (p. 000) replication bubble (p. 000) replication fork (p. 000) bidirectional replication (p. 000) rolling-circle replication (p. 000) DNA polymerase (p. 000)

continuous replication (p. 000) leading strand (p. 000) discontinuous replication (p. 000) lagging strand (p. 000) Okazaki fragments (p. 000) initiator protein (p. 000) DNA helicase (p. 000) single-strand-binding protein

(SSB) (p. 000) DNA gyrase (p. 000) primase (p. 000)

primer (p. 000) DNA polymerase III (p. 000) DNA polymerase I (p. 000) DNA ligase (p. 000) proofreading (p. 000) mismatch repair (p. 000) autonomously replicating sequence (p. 000) replication licensing factor (p. 000) DNA polymerase a (p. 000) DNA polymerase 8 (p. 000)

DNA polymerase ß (p. 000) DNA polymerase 7 (p. 000) DNA polymerase € (p. 000) telomerase (p. 000) homologous recombination (p. 000) heteroduplex DNA (p. 000) Holliday junction (p. 000) branch migration (p. 000) Holliday intermediate (p. 000) double-strand-break model (p. 000)

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