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Copyright © 2009 Pearson Education, Inc.
Chapter 11 DNA Replication and Recombination
Copyright © 2009 Pearson Education, Inc.
Copyright © 2009 Pearson Education, Inc.
11.1 DNA is reproduced by Semiconservative Replication
The complementarity of DNA strands allows
each strand to serve as a template for synthesis of the other
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Copyright © 2009 Pearson Education, Inc.
Three possible modes of DNA replication are possible
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• The Meselson-Stahl experiment demonstrated that: • DNA replication is semiconservative • each new DNA molecule consists of one old
strand and one newly synthesized strand
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Copyright © 2009 Pearson Education, Inc.
Copyright © 2009 Pearson Education, Inc.
The Taylor-Woods-Hughes experiment demonstrated that DNA replication is semiconservative in eukaryotes.
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DNA replication begins at the origin of replication and is bidirectional rather than unidirectional. A replicon is the length of DNA that is replicated following one initiation event at a single origin.
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11.2 DNA Synthesis in Bacteria Involves Five Polymerases, as well as Other Enzymes
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DNA polymerase catalyzes DNA synthesis and requires a DNA template and all four dNTPs.
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• Chain elongation occurs in the 5' to 3' direction by addition of one nucleotide at a time to the 3' end.
• As the nucleotide is added, the two terminal phosphates are cleaved off.
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• DNA polymerases I, II, and III can elongate an existing DNA strand but cannot initiate DNA synthesis.
• All three possess 3' to 5' exonuclease activity. • But only DNA polymerase I demonstrates 5' to 3'
exonuclease activity.
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• DNA polymerase III is the enzyme responsible for the 5' to 3' polymerization essential in vivo. Its 3' to 5' exonuclease activity allows proofreading.
• Polymerase I is believed to be responsible for removing the primer and the synthesis that fills gaps produced during synthesis
• DNA polymerases I, II, IV, and V are involved in various aspects of repair of damaged DNA.
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• DNA polymerase III has 10 subunits whose functions are shown.
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Copyright © 2009 Pearson Education, Inc.
11.3 Many Complex Tasks Must Be Performed during DNA Replication
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There are seven key issues that must be resolved during DNA replication
• unwinding of the helix • reducing increased coiling generated during unwinding • synthesis of a primer for initiation • discontinuous synthesis of the second strand • removal of the RNA primers • joining of the gap-filling DNA to the adjacent strand • proofreading
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• DnaA binds to the origin of replication and is responsible for the initial steps in unwinding the helix.
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• Subsequent binding of DnaB and DnaC further opens and destabilizes the helix.
• Single-stranded binding proteins (SSBPs) stabilize the open conformation.
• Helicases require the energy normally supplied by the hydrolysis of ATP to break hydrogen bonds and denature the double helix.
• Unwinding produces supercoiling that is relieved by DNA gyrase, a member of a larger group of enzymes referred to as DNA topoisomerases.
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• To elongate a polynucleotide chain, DNA polymerase III requires a primer with a free 3'-hydroxyl group.
• The enzyme primase synthesizes an RNA primer that provides the free 3'-hydroxyl required by DNA polymerase III.
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• As the replication fork moves, only one strand can serve as a template for continuous DNA synthesis—the leading strand.
• The opposite lagging strand undergoes discontinuous DNA synthesis.
• The lagging strand is synthesized as Okazaki fragments, each with an RNA primer.
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• DNA polymerase I removes the primers on the lagging strand and the fragments are joined by DNA ligase.
• Both DNA strands are synthesized concurrently by looping the lagging strand to invert the physical but not biological direction of synthesis
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• The β-subunit clamp prevents the core enzyme from falling off the template during DNA synthesis.
• Proofreading and error correction are an integral part of DNA replication.
• All of the DNA polymerases have 3' to 5' exonuclease activity that allows proofreading.
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11.4 A Summary of DNA Replication in Prokaryotes
DNA synthesis at a single replication fork involves DNA polymerase III, single-stranded binding proteins DNA gyrase, DNA helicase, RNA primers
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11.5 Replication in Prokaryotes is Controlled by a Variety of Genes
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• A number of genes involved in DNA replication have been identified by conditional mutations
• A temperature-sensitive mutation is an example of a conditional mutation.
• It may not be expressed at a particular permissive temperature, but when mutant cells are grown at a restrictive temperature, the mutant phenotype is expressed and can be studied.
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11.6 Eukaryotic DNA Synthesis Is Similar to Synthesis in Prokaryotes, but More Complex
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• In eukaryotic cells • there is more DNA than prokaryotic cells. • the chromosomes are linear. • the DNA is complexed with proteins.
• This makes DNA replication more complex in
eukaryotes than in bacteria.
• Eukaryotic chromosomes contain multiple origins of replication to allow the genome to be replicated in a few hours.
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Multiple origins of replication along a eukaryotic chromosome
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• Yeast autonomously replicating sequences (ARSs) contain an 11-bp consensus sequence flanked by other short sequences involved in efficient initiation.
• The ARSs are initially bound by a group of proteins to form the origin recognition complex (ORC).
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• Three DNA polymerases are involved in replication of nuclear DNA.
• One involves mitochondrial DNA replication. • Others are involved in repair processes.
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• Pol α and δ are the major forms of the enzyme involved in initiation and elongation.
• Pol α possesses low processivity, a term that reflects the length of DNA that is synthesized by an enzyme before it dissociates from the template.
• Pol α functions in synthesis of the RNA primers during initiation on the leading and lagging strands.
• Polymerase switching occurs, and Pol α is replaced by Pol δ for elongation.
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11.7 Telomeres Provide Structural Integrity at Chromosome Ends but Are Problematic to Replicate
• Telomeres at the ends of linear chromosomes consist of long stretches of short repeating sequences and preserve the integrity and stability of chromosomes.
• Lagging strand synthesis at the end of the chromosome is a problem because once the RNA primer is removed, there is no free 3'-hydroxyl group from which to elongate.
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Copyright © 2009 Pearson Education, Inc.
• Telomerase directs synthesis of the telomere repeat sequence to fill the gap.
• This enzyme is a ribonucleoprotein with an RNA that serves as the template for the synthesis of its DNA complement.
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Copyright © 2009 Pearson Education, Inc.
11.8 DNA Recombination, Like DNA Replication, Is Directed by Specific Enzymes
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• Genetic recombination involves • endonuclease nicking • strand displacement • ligation • branch migration • duplex separation to generate the
characteristic Holliday structure
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Copyright © 2009 Pearson Education, Inc.
• Genetic exchange at equivalent positions along two chromosomes with substantial DNA sequence homology is referred to as general, or homologous, recombination.
• The RecA protein in E. coli promotes the exchange of reciprocal single-stranded DNA molecules.
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• Gene Conversion Is a Consequence of DNA Recombination.
• Gene conversion is characterized by nonreciprocal genetic exchange between two closely linked genes.