DNA Replication and Recombination

DNA Replication and Recombination

Chapter 11 Contents 11.1 DNA Is Reproduced by Semiconservative Replication 11.2 DNA Synthesis in Bacteria Involves Five Polymerases, as well as Other Enzymes 11.3 Many Complex Issues Must Be Resolved during DNA Replication 11.4 A Coherent Model Summarizes DNA Replication 11.5 Replication Is Controlled by a Variety of Genes Continued © 2012 Pearson Education, Inc.

Chapter 11 Contents 11.6 Eukaryotic DNA Replication Is Similar to Synthesis in Prokaryotes, but Is More Complex 11.7 The Ends of Linear Chromosome Are Problematic during Replicate 11.8 DNA Recombination, Like DNA Replication, Is Directed by Specific Enzymes 11.9 Gene Conversion Is a Consequence of DNA Recombination © 2012 Pearson Education, Inc.

11.1 DNA Is Reproduced by Semiconservative Replication
© 2012 Pearson Education, Inc.

Figure 11-1 Generalized model of semiconservative replication of DNA
Figure 11-1 Generalized model of semiconservative replication of DNA. New synthesis is shown in teal. Figure 11.1 © 2012 Pearson Education, Inc.

Three modes of DNA replication are possible:
Section 11.1 Three modes of DNA replication are possible: Conservative Original helix is conserved and two newly synthesized strands come together Semiconservative Each replicated DNA molecule consists of one "old" strand and one new strand Dispersive Parental strands are dispersed into two new double helices (Figure 11.2) © 2012 Pearson Education, Inc.

Section 11.1 Meselson and Stahl (1958), using 15N-labeled E. coli grown in medium containing 14N, demonstrated that DNA replication is semiconservative in prokaryotes each new DNA molecule consists of one old strand and one newly synthesized strand (Figure 11.3 and Figure 11.4) © 2012 Pearson Education, Inc.

Figure 11-3 The Meselson–Stahl experiment.

Figure 11-5 The Taylor–Woods–Hughes experiment, demonstrating the semiconservative mode of replication of DNA in root tips of Vicia faba. A portion of the plant is shown in the top photograph. (a) An unlabeled chromosome proceeds through the cell cycle in the presence of 3H-thymidine. As it enters mitosis, both sister chromatids of the chromosome are labeled, as shown, by autoradiography. After a second round of replication (b), this time in the absence of 3H-thymidine, only one chromatid of each chromosome is expected to be surrounded by grains. Except where a reciprocal exchange has occurred between sister chromatids (c), the expectation was upheld. The micrographs are of the actual autoradiograms obtained in the experiment. Figure 11.5 © 2012 Pearson Education, Inc.

Figure 11-5a The Taylor–Woods–Hughes experiment, demonstrating the semiconservative mode of replication of DNA in root tips of Vicia faba. A portion of the plant is shown in the top photograph. (a) An unlabeled chromosome proceeds through the cell cycle in the presence of 3H-thymidine. As it enters mitosis, both sister chromatids of the chromosome are labeled, as shown, by autoradiography. After a second round of replication (b), this time in the absence of 3H-thymidine, only one chromatid of each chromosome is expected to be surrounded by grains. Except where a reciprocal exchange has occurred between sister chromatids (c), the expectation was upheld. The micrographs are of the actual autoradiograms obtained in the experiment. Figure 11.5a © 2012 Pearson Education, Inc.

Figure 11-5b The Taylor–Woods–Hughes experiment, demonstrating the semiconservative mode of replication of DNA in root tips of Vicia faba. A portion of the plant is shown in the top photograph. (a) An unlabeled chromosome proceeds through the cell cycle in the presence of 3H-thymidine. As it enters mitosis, both sister chromatids of the chromosome are labeled, as shown, by autoradiography. After a second round of replication (b), this time in the absence of 3H-thymidine, only one chromatid of each chromosome is expected to be surrounded by grains. Except where a reciprocal exchange has occurred between sister chromatids (c), the expectation was upheld. The micrographs are of the actual autoradiograms obtained in the experiment. Figure 11.5b © 2012 Pearson Education, Inc.

Figure 11-5c The Taylor–Woods–Hughes experiment, demonstrating the semiconservative mode of replication of DNA in root tips of Vicia faba. A portion of the plant is shown in the top photograph. (a) An unlabeled chromosome proceeds through the cell cycle in the presence of 3H-thymidine. As it enters mitosis, both sister chromatids of the chromosome are labeled, as shown, by autoradiography. After a second round of replication (b), this time in the absence of 3H-thymidine, only one chromatid of each chromosome is expected to be surrounded by grains. Except where a reciprocal exchange has occurred between sister chromatids (c), the expectation was upheld. The micrographs are of the actual autoradiograms obtained in the experiment. Figure 11.5c © 2012 Pearson Education, Inc.

DNA replication begins at the origin of replication
Section 11.1 DNA replication begins at the origin of replication Where replication is occurring, the strands of the helix are unwound, creating a replication fork Replication is bidirectional; therefore, there are two replication forks (Figure 11.6) © 2012 Pearson Education, Inc.

Figure 11-6 Bidirectional replication of the E. coli chromosome
Figure 11-6 Bidirectional replication of the E. coli chromosome. The thin black arrows identify the advancing replication forks. The micrograph is of a bacterial chromosome in the process of replication, comparable to the figure next to it. Figure 11.6 © 2012 Pearson Education, Inc.

Section 11.1 The length of DNA that is replicated following one initiation event at a single origin is called a replicon Bacteria have a single circular DNA, and DNA synthesis originates at a single point, the origin of replication, called OriC The entire bacterial chromosome constitutes one replicon (6.6 million base pairs) © 2012 Pearson Education, Inc.

11.2 DNA Synthesis in Bacteria Involves Five Polymerases, as well as Other Enzymes

Figure 11-7 The chemical reaction catalyzed by DNA polymerase I
Figure 11-7 The chemical reaction catalyzed by DNA polymerase I. During each step, a single nucleotide is added to the growing complement of the DNA template, using a nucleoside triphosphate as the substrate. The release of inorganic pyrophosphate drives the reaction energetically. Figure 11.7 © 2012 Pearson Education, Inc.

Section 11.2 Chain elongation occurs in the 5' to 3' direction by addition of one nucleotide at a time to the 3' end (Figure 11.8) As the nucleotide is added, the two terminal phosphates are cleaved off, providing a newly exposed 3'-OH group that can participate in the addition of another nucleotide as DNA synthesis proceeds © 2012 Pearson Education, Inc.

Figure 11-8 Demonstration of to synthesis of DNA.

Section 11.2 DNA polymerases I, II, and III can elongate an existing DNA strand (called a primer) but cannot initiate DNA synthesis (Table 11.2) All three possess 3' to 5' exonuclease activity, allowing them to proofread newly synthesized DNA and remove and replace incorrect nucleotides Only DNA polymerase I demonstrates 5' to 3' exonuclease activity, excising primers and filling in the gaps left behind © 2012 Pearson Education, Inc.

Table 11-2 Properties of Bacterial DNA Polymerases I, II, and III

Its 3' to 5' exonuclease activity allows proofreading
Section 11.2 DNA polymerase III is the enzyme responsible for the 5' to 3' polymerization essential in vivo Its 3' to 5' exonuclease activity allows proofreading © 2012 Pearson Education, Inc.

Section 11.2 DNA polymerase III is a complex enzyme (holoenzyme) made up of 10 subunits whose functions are shown in Table 11.3 The holoenzyme and some other proteins at the replication fork form a complex called the replisome © 2012 Pearson Education, Inc.

Table 11-3 Subunits of the DNA Polymerase III Holoenzyme

11.3 Many Complex Issues Must Be Resolved during DNA Replication

Section 11.3 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 continued © 2012 Pearson Education, Inc.

Section 11.3 continued Removal of the RNA primers
Joining of the gap-filling DNA to the adjacent strand Proofreading © 2012 Pearson Education, Inc.

Figure 11-9 Helical unwinding of DNA during replication as accomplished by DnaA, DnaB, and DnaC proteins. Initial binding of many monomers of DnaA occurs at DNA sites containing repeating sequences of 9 nucleotides, called 9mers. Not illustrated are 13mers, which are also involved. Figure 11.9 © 2012 Pearson Education, Inc.

Section 11.3 Subsequent binding of DnaB and DnaC further opens and destabilizes the helix Proteins such as these, which require the energy normally supplied by the hydrolysis of ATP to break hydrogen bonds and denature the double helix, are called helicases Single-stranded binding proteins (SSBPs) stabilize the open conformation © 2012 Pearson Education, Inc.

Section 11.3 Unwinding produces supercoiling that is relieved by DNA gyrase, a member of a larger group of enzymes referred to as DNA topoisomerases Gyrase makes single- or double-stranded cuts to undo the twists and knots created during supercoiling, which are then resealed © 2012 Pearson Education, Inc.

DNA polymerase I removes the primer and replaces it with DNA
Section 11.3 To elongate a polynucleotide chain, DNA polymerase III requires a primer with a free 3'-hydroxyl group Primase synthesizes an RNA primer that provides the free 3'-hydroxyl required by DNA polymerase III (Figure 11.10) DNA polymerase I removes the primer and replaces it with DNA Priming is a universal phenomenon during initiation of DNA synthesis © 2012 Pearson Education, Inc.

Figure 11-10 The initiation of DNA synthesis
Figure The initiation of DNA synthesis. A complementary RNA primer is first synthesized, to which DNA is added. All synthesis is in the to direction. Eventually, the RNA primer is replaced with DNA under the direction of DNA polymerase I. Figure 11.10 © 2012 Pearson Education, Inc.

Section 11.3 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 (Figure 11.11) © 2012 Pearson Education, Inc.

Figure Opposite polarity of DNA synthesis along the two strands, necessary because the two strands of DNA run antiparallel to one another and DNA polymerase III synthesizes only in one direction ( to ). On the lagging strand, synthesis must be discontinuous, resulting in the production of Okazaki fragments. On the leading strand, synthesis is continuous. RNA primers are used to initiate synthesis on both strands. Figure 11.11 © 2012 Pearson Education, Inc.

Section 11.3 The lagging strand is synthesized as Okazaki fragments, each with an RNA primer DNA polymerase I removes the primers on the lagging strand, and the fragments are joined by DNA ligase © 2012 Pearson Education, Inc.

Figure Illustration of how concurrent DNA synthesis may be achieved on both the leading and lagging strands at a single replication fork. The lagging template strand is "looped" in order to invert the physical direction of synthesis, but not the biochemical direction. The enzyme functions as a dimer, with each core enzyme achieving synthesis on one or the other strand. Figure 11.12 © 2012 Pearson Education, Inc.

Section 11.3 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 © 2012 Pearson Education, Inc.

11.4 A Coherent Model Summarizes DNA Replication

DNA synthesis at a single replication fork involves
Section 11.4 DNA synthesis at a single replication fork involves DNA polymerase III single-stranded binding proteins DNA gyrase DNA helicase RNA primers (Figure 11.13) © 2012 Pearson Education, Inc.

Figure 11-13 Summary of DNA synthesis at a single replication fork
Figure Summary of DNA synthesis at a single replication fork. Various enzymes and proteins essential to the process are shown. Figure 11.13 © 2012 Pearson Education, Inc.

11.5 Replication Is Controlled by a Variety of Genes

Table 11-4 Some of the Various

Section 11.5 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 © 2012 Pearson Education, Inc.

11. 6 Eukaryotic DNA Replication Is
11.6 Eukaryotic DNA Replication Is Similar to Replication in Prokaryotes, but Is More Complex © 2012 Pearson Education, Inc.

Section 11.6 Eukaryotic DNA replication shares many features with replication in bacteria Double-stranded DNA unwound at replication origins Replication forks are formed Bidirectional snythesis creates leading and lagging strands Eukaryotic polymerases require four deoxyribonucleoside triphosphates, a template, and a primer © 2012 Pearson Education, Inc.

This makes DNA replication more complex in eukaryotes than in bacteria
Section 11.6 However, eukaryotic DNA replication is more complex due to several features of eukaryotic DBA: 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 © 2012 Pearson Education, Inc.

Figure A demonstration of the multiple origins of replication along a eukaryotic chromosome. Each origin is apparent as a replication bubble along the axis of the chromosome. Arrows identify some of these replication bubbles. Figure 11.14 © 2012 Pearson Education, Inc.

Section 11.6 Yeast genomes contain 250–400 origins and are called autonomously replicating sequences (ARSs) These sequences contain an 11-bp consensus sequence flanked by other short sequences involved in efficient initiation © 2012 Pearson Education, Inc.

Eukaryotic origins also control timing of DNA replication
Section 11.6 Eukaryotic origins also control timing of DNA replication The prereplication complex (pre-Rc) assembles at replication origins In early G1 phase of the cell cycle, replication origins are recognized by a six-protein complex, the origin recognition complex (ORC), which tags the origin as the site of initiation © 2012 Pearson Education, Inc.

Three DNA polymerases are involved in replication of nuclear DNA
Section 11.6 Three DNA polymerases are involved in replication of nuclear DNA One involves mitochondrial DNA replication Others are involved in repair processes (Table 11.5) © 2012 Pearson Education, Inc.

Table 11-5 Properties of Eukaryotic DNA Polymerases

Section 11.6 Pol , , and ɛ are the major forms of the enzyme involved in initiation and elongation Pol  possesses low processivity, a term that reflects the length of DNA that is synthesized by an enzyme before it dissociates from the template © 2012 Pearson Education, Inc.

Section 11.6 Pol  functions in synthesis of the RNA primers during initiation on the leading and lagging strands Once the primer is in place, polymerase switching occurs: Pol  and ɛ are replaced by Pol  for elongation Pol  synthesizes the lagging strand and Pol ɛ synthesizes the leading strand © 2012 Pearson Education, Inc.

11.7 The Ends of Linear Chromosome Are Problematic during Replication

Section 11.7 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 (Figure 11.16) © 2012 Pearson Education, Inc.

Figure Diagram illustrating the difficulty encountered during the replication of the ends of linear chromosomes. A gap (- -b- -) is left following synthesis on the lagging strand. Figure 11.16 © 2012 Pearson Education, Inc.

Section 11.7 Telomerase directs synthesis of the telomere repeat sequence to fill the gap (Figure 11.17) This enzyme is a ribonucleoprotein with an RNA that serves as the template for the synthesis of its DNA complement Reverse transcription © 2012 Pearson Education, Inc.

Figure The predicted solution to the problem posed in Figure 11–16. The enzyme telomerase directs synthesis of the TTGGGG sequences, resulting in the formation of a hairpin structure. The gap can now be filled, and, following cleavage of the hairpin structure, the process averts the creation of a gap during replication of the ends of linear chromosomes. Figure 11.17 © 2012 Pearson Education, Inc.

In most eukaryotic somatic cells, telomerase is not active
Section 11.7 In most eukaryotic somatic cells, telomerase is not active With each successive cell division, telomeres shorten and erode, causing further cell division to stop Malignant cells maintain telomerase activity and are immortalized © 2012 Pearson Education, Inc.

11.8 DNA Recombination, Like DNA Replication, Is Directed by Specific Enzymes

Genetic recombination involves
Section 11.8 Genetic recombination involves endonuclease nicking strand displacement and pairing with complement ligation branch migration duplex separation to generate the characteristic Holliday structure (Figure 11.18) © 2012 Pearson Education, Inc.

Figure Model depicting how genetic recombination can occur as a result of the breakage and rejoining of heterologous DNA strands. Each stage is described in the text. The electron micrograph shows DNA in a -form structure similar to the diagram in (g); the DNA is an extended Holliday structure, derived from the ColE1 plasmid of E. coli. David Dressler, Oxford University, England Figure 11.18 © 2012 Pearson Education, Inc.

Figure 11-18a Model depicting how genetic recombination can occur as a result of the breakage and rejoining of heterologous DNA strands. Each stage is described in the text. The electron micrograph shows DNA in a -form structure similar to the diagram in (g); the DNA is an extended Holliday structure, derived from the ColE1 plasmid of E. coli. David Dressler, Oxford University, England Figure 11.18a © 2012 Pearson Education, Inc.

Figure 11-18b Model depicting how genetic recombination can occur as a result of the breakage and rejoining of heterologous DNA strands. Each stage is described in the text. The electron micrograph shows DNA in a -form structure similar to the diagram in (g); the DNA is an extended Holliday structure, derived from the ColE1 plasmid of E. coli. David Dressler, Oxford University, England Figure 11.18b © 2012 Pearson Education, Inc.

Figure 11-18c Model depicting how genetic recombination can occur as a result of the breakage and rejoining of heterologous DNA strands. Each stage is described in the text. The electron micrograph shows DNA in a -form structure similar to the diagram in (g); the DNA is an extended Holliday structure, derived from the ColE1 plasmid of E. coli. David Dressler, Oxford University, England Figure 11.18c © 2012 Pearson Education, Inc.

Figure 11-18d Model depicting how genetic recombination can occur as a result of the breakage and rejoining of heterologous DNA strands. Each stage is described in the text. The electron micrograph shows DNA in a -form structure similar to the diagram in (g); the DNA is an extended Holliday structure, derived from the ColE1 plasmid of E. coli. David Dressler, Oxford University, England Figure 11.18d © 2012 Pearson Education, Inc.

Figure 11-18f Model depicting how genetic recombination can occur as a result of the breakage and rejoining of heterologous DNA strands. Each stage is described in the text. The electron micrograph shows DNA in a -form structure similar to the diagram in (g); the DNA is an extended Holliday structure, derived from the ColE1 plasmid of E. coli. David Dressler, Oxford University, England Figure 11.18f © 2012 Pearson Education, Inc.

Figure 11-18g Model depicting how genetic recombination can occur as a result of the breakage and rejoining of heterologous DNA strands. Each stage is described in the text. The electron micrograph shows DNA in a -form structure similar to the diagram in (g); the DNA is an extended Holliday structure, derived from the ColE1 plasmid of E. coli. David Dressler, Oxford University, England Figure 11.18g © 2012 Pearson Education, Inc.

Figure 11-18h Model depicting how genetic recombination can occur as a result of the breakage and rejoining of heterologous DNA strands. Each stage is described in the text. The electron micrograph shows DNA in a -form structure similar to the diagram in (g); the DNA is an extended Holliday structure, derived from the ColE1 plasmid of E. coli. David Dressler, Oxford University, England Figure 11.18h © 2012 Pearson Education, Inc.

Figure 11-18i Model depicting how genetic recombination can occur as a result of the breakage and rejoining of heterologous DNA strands. Each stage is described in the text. The electron micrograph shows DNA in a -form structure similar to the diagram in (g); the DNA is an extended Holliday structure, derived from the ColE1 plasmid of E. coli. David Dressler, Oxford University, England Figure 11.18i © 2012 Pearson Education, Inc.

Section 11.8 Genetic exchange at equivalent positions along two chromosomes with substantial DNA sequence homology is referred to as general, or homologous, recombination © 2012 Pearson Education, Inc.

Section 11.8 The RecA protein in E. coli promotes the exchange of reciprocal single-stranded DNA molecules and enhances hydrogen bond formation during strand displacement © 2012 Pearson Education, Inc.

Figure A proposed mechanism that accounts for the phenomenon of gene conversion. A base-pair mismatch occurs in one of the two homologs (bearing the mutant allele) during heteroduplex formation, which accompanies recombination in meiosis. During excision repair, one of the two mismatches is removed and the complement is synthesized. In one case, the mutant base pair is preserved. When it is subsequently included in a recombinant spore, the mutant genotype will be maintained. In the other case, the mutant base pair is converted to the wild-type sequence. When included in a recombinant spore, the wild-type genotype will be expressed, leading to a nonreciprocal exchange ratio. Figure 11.19 © 2012 Pearson Education, Inc.

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