© 2011 Pearson Education, Inc. Chapter 15: DNA and the gene, synthesis & repair Learning objectives: Students should be able to…. Describe and interpret data from experiments that provided evidence that DNA is the hereditary material and that DNA replicates in a semiconservative manner. Explain the steps involved in DNA replication. Explain how errors in DNA replication are corrected. Explain how mutations in the excision repair process can result in the xeroderma pigmentosum phenotype.

© 2011 Pearson Education, Inc. Key Concepts Genes are made of DNA. When DNA is copied, each strand of a DNA double helix serves as the template for the synthesis of a complementary strand. DNA synthesis occurs in the 5 ′ → 3 ′ direction only and requires a large suite of specialized enzymes. The leading strand is synthesized continuously, but the lagging strand is synthesized as a series of fragments that are then linked together.

© 2011 Pearson Education, Inc. Key Concepts Specialized enzymes repair damages to DNA and fix mistakes in DNA synthesis. If these repair enzymes are defective, the mutation rate increases.

© 2011 Pearson Education, Inc. What Are Genes Made Of? While it was known and understood that chromosomes were comprised of (Deoxyribonucleic acid) DNA and protein, early biologists did not know whether genes were comprised of DNA or protein. The general consensus at the time supported the hypothesis that genes were comprised of proteins. –This was because of the relative complexity and variability of proteins compared to DNA which is comprised of only four different nucleotides.

© 2011 Pearson Education, Inc. DNA’s Primary Structure The primary structure of DNA has two major components: 1.A backbone made up of the sugar and phosphate groups of deoxyribonucleotides. 2.A series of nitrogen-containing bases that project from the backbone. DNA has directionality—one end has an exposed hydroxyl group on the 3 ′ carbon of deoxyribose, and the other end has an exposed phosphate group on a 5 ′ carbon. –The molecule thus has a 3 ′ end and a 5 ′ end.

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DNA’s Secondary Structure Watson and Crick proposed that two DNA strands line up in the opposite direction to each other, in what is called antiparallel fashion. The antiparallel strands twist to form a double helix. The secondary structure is stabilized by complementary base pairing: –Adenine (A) hydrogen bonds with thymine (T). –Guanine (G) hydrogen bonds with cytosine (C).

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DNA Strands Are Templates for DNA Synthesis Watson and Crick suggested that the existing strands of DNA served as a template (pattern) for the production of new strands, with bases being added to the new strands according to complementary base pairing. Biologists then proposed three alternative hypotheses for how the old and new DNA strands interacted during replication: –Semiconservative replication. –Conservative replication. –Dispersive replication.

© 2011 Pearson Education, Inc. How Do the Old and New DNA Strands Interact? In semiconservative replication, the parental DNA strands separate and each is used as a template for the synthesis of a new strand. Daughter molecules each consist of one old and one new strand. In conservative replication, the parental molecule serves as a template for the synthesis of an entirely new molecule. In dispersive replication, the parent molecule is cut into sections such that the daughter molecules contain old DNA interspersed with newly synthesized DNA.

© 2011 Pearson Education, Inc. The Meselson-Stahl Experiment Meselson and Stahl designed an experiment to provide more information about whether one of these hypotheses was correct. They grew E. coli in the presence of “heavy” nitrogen ( 15 N) to label the bacteria's DNA. After many generations, they moved the bacteria to a normal 14 N-containing medium and separated the DNA by density. The densities of the resulting DNA samples supported semiconservative DNA replication as the mechanism by which the hereditary material is duplicated.

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A Comprehensive Model for DNA Synthesis Meselson and Stahl showed that each parental DNA strand is copied in its entirety, but did not illustrate a mechanism for this process. The discovery of DNA polymerase, the enzyme that catalyzes DNA synthesis, cleared the way for understanding DNA replication reactions.

© 2011 Pearson Education, Inc. Characteristics of DNA Polymerases A critical characteristic of DNA polymerases is that they can only work in one direction. DNA polymerases can add deoxyribonucleotides to only the 3 ′ end of a growing DNA chain. As a result, DNA synthesis always proceeds in the 5 ′ → 3 ′ direction. DNA polymerization is exergonic because the monomers that act as substrates in the reaction are deoxyribonucleoside triphosphates (dNTPs), which have high potential energy because of their three closely packed phosphate groups.

© 2011 Pearson Education, Inc. How Does Replication Get Started? A replication bubble forms in a chromosome that is actively being replicated. –Replication bubbles grow as DNA replication proceeds, because synthesis is bidirectional. In bacterial chromosomes, the replication process begins at a single location, the origin of replication. Eukaryotes also have bidirectional replication but they have multiple origins of replication and thus have multiple replication bubbles. A replication fork is the Y-shaped region where the DNA is split into two separate strands for copying.

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How Is the Helix Opened and Stabilized? Several proteins are responsible for opening and stabilizing the double helix. –The enzyme helicase catalyzes the breaking of hydrogen bonds between the two DNA strands to separate them. –Then single-strand DNA-binding proteins (SSBPs) attach to the separated strands to prevent them from closing. Unwinding the DNA helix creates tension farther down the helix. –The enzyme topoisomerase cuts and rejoins the DNA downstream of the replication fork, relieving this tension.

© 2011 Pearson Education, Inc. How Is the Leading Strand Synthesized? DNA polymerase requires a primer—which consists of a few nucleotides bonded to the template strand—because it provides a free 3 ′ hydroxyl (OH) group that can combine with an incoming dNTP to form a phosphodiester bond. Primase, a type of RNA polymerase, synthesizes a short RNA segment that serves as a primer for DNA synthesis. The enzyme’s product is called the leading strand, or continuous strand, because it leads into the replication fork and is synthesized continuously in the 5 ′  3 ′ direction.

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The Lagging Strand The other DNA strand is called the lagging strand because it is synthesized discontinuously in the direction away from the replication fork and so lags behind the fork. –This occurs because DNA synthesis must proceed in the 5'  3' direction.

© 2011 Pearson Education, Inc. How Is the Lagging Strand Synthesized? As with the leading strand, synthesis of the lagging strand starts when primase synthesizes a short stretch of RNA that acts as a primer. DNA polymerase III then adds bases to the 3' end of the primer. DNA polymerase moves away from the replication fork, even though helicase continues to open the replication fork and expose single-strand DNA on the lagging strand.

© 2011 Pearson Education, Inc. The Discontinuous Replication Hypothesis The discontinuous replication hypothesis stated that once primase synthesizes an RNA primer on the lagging strand, DNA polymerase might synthesize short fragments of DNA along the lagging strand, and that these fragments would later be linked together to form a continuous whole. This hypothesis was tested by Okazaki and his colleagues.

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The Discovery of Okazaki Fragments The lagging strand is synthesized as short discontinuous fragments called Okazaki fragments. DNA polymerase I removes the RNA primer at the beginning of each Okazaki fragment and fills in the gap. The enzyme DNA ligase then joins the Okazaki fragments to form a continuous DNA strand. Because Okazaki fragments are synthesized independently and joined together later, the lagging strand is also called the discontinuous strand.

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Replicating the Ends of Linear Chromosomes Telomeres are the regions at the ends of linear chromosomes. As the replication fork reaches the end of a linear chromosome, there is no way to replace the RNA primer from the lagging strand with DNA, because there is no available primer for DNA synthesis. The primer is removed, leaving a section of single-stranded DNA (lagging strand) at one end of each new chromosome. This remaining single-stranded DNA is eventually degraded, resulting in shortening of the chromosome.

© 2011 Pearson Education, Inc. Replicating the Ends of Linear Chromosomes The region at the end of a linear chromosome is called a telomere. Replication of telomeres can be problematic. –While leading-strand synthesis results in a normal copy of the DNA molecule, the telomere on the lagging strand shortens during DNA replication.

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Replicating the Ends of Linear Chromosomes Telomeres do not contain genes, but consist of short repeating stretches of bases. The enzyme telomerase adds more repeating bases to the end of the lagging strand, catalyzing the synthesis of DNA from an RNA template that it carries with it. Primase then makes an RNA primer, which DNA polymerase uses to synthesize the lagging strand. Finally, ligase connects the new sequence. This prevents the lagging strand from getting shorter with each replication.

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Replication in Somatic Cells Somatic cells normally lack telomerase. –The chromosomes of somatic cells progressively shorten as the individual ages. This has led biologists to hypothesize that telomere shortening has a role in limiting the amount of time cells remain in an actively growing state.

© 2011 Pearson Education, Inc. Repairing Mistakes and Damage DNA replication is very accurate, with an average error rate of less than one mistake per billion bases. DNA polymerase is highly selective in matching complementary bases correctly. –As a result, DNA polymerase inserts the incorrect base only about once every 100,000 bases added. If mistakes remain after synthesis is complete or if DNA is damaged, repair enzymes can remove the defective bases and repair them.

© 2011 Pearson Education, Inc. How Does DNA Polymerase Proofread? DNA polymerase can proofread its work—it checks the match between paired bases, and can correct mismatched bases when they do occur. If the enzyme finds a mismatch, it pauses and removes the mismatched base that was just added. DNA polymerase III can do this because its  (epsilon) subunit acts as an exonuclease that removes deoxyribonucleotides from DNA. –This proofreading process reduces the error rate to about 1  10 –7.

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How Does DNA Polymerase Proofread? If—in spite of its proofreading ability—DNA polymerase leaves a mismatched pair behind in the newly synthesized strand, a battery of enzymes springs into action to correct the problem. Mismatch repair occurs when mismatched bases are corrected after DNA synthesis is complete. Mismatch repair enzymes recognize the mismatched pair, remove a section of the newly synthesized strand that contains the incorrect base, and fill in the correct bases.

© 2011 Pearson Education, Inc. Repairing Damaged DNA DNA can be broken or altered by various chemicals and types of radiation. For example, UV light can cause thymine dimers to form, causing a kink in the DNA strand. The nucleotide excision repair system recognizes such types of damage. Its enzymes then remove the single-stranded DNA in the damaged section. The presence of a DNA strand complementary to the damaged strand provides a template for resynthesis of the defective sequences.

© 2011 Pearson Education, Inc. Damage from UV (will need repair)

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