DNA and the Gene: Synthesis and Repair DNA and the Gene: Synthesis and Repair

Chapter 15 Opening Roadmap. © 2017 Pearson Education, Inc.

What Are Genes Made Of? Researchers knew that chromosomes were comprised of DNA and protein Are genes made of DNA or protein? Most biologists thought genes were made of proteins They are relatively more complex They are more variable DNA is comprised of only four different nucleotides

The Hershey–Chase Experiment Hershey and Chase studied how the T2 virus infects the bacterium Escherichia coli T2 infection of E. coli begins when The virus injects its genes into the cell These genes direct production of new virus particles The virus’s protein coat, or capsid, is left behind on the outside of the cell Does protein or DNA enter the cell?

(b) The virus’s capsid stays outside the cell. Figure 15.1 (a) Virus capsid Virus genes Host cell 1. Start of infection. 2. Production of new virus particles. 3. End of infection. (b) The virus’s capsid stays outside the cell. Figure 15.1 Viruses Inject Genes into Bacterial Cells and Leave a Capsid Behind. Virus capsid Virus genes 100 nm © 2017 Pearson Education, Inc.

The Hershey–Chase Experiment Hershey and Chase grew the virus in the presence of one of two radioactive isotopes: 32P, which is incorporated into DNA, or 35S, which is incorporated into proteins Labeled viruses were used to infect E. coli cells Only the radioactive DNA was found inside the cells Therefore, genes must be composed of DNA

Figure 15.2 Do viral genes consist of DNA or protein? Viral genes consist of DNA. Viral genes consist of protein. 1. Label viruses. Viral DNA is radioactive. Viral protein is radioactive. 2. Infect bacteria. E. coli E. coli Viral capsids outside 3. Agitate cultures. Genes inside 4. Centrifuge solutions. Viral capsids in solution Figure 15.2 Experimental Evidence Showed that DNA Is the Hereditary Material. Viral genes in cells in pellet Radioactive DNA will be located within pellet. Radioactive protein will be located within pellet. Radioactive protein is in solution Radioactive DNA is in pellet DNA Viral genes consist of DNA. Protein © 2017 Pearson Education, Inc.

The Secondary Structure of DNA The primary structure of each strand of DNA has A backbone made up of the sugar and phosphate groups of deoxyribonucleotides A series of nitrogen-containing bases that project from the backbone DNA has directionality The 3′ end has an exposed hydroxyl group attached to the 3′ carbon of deoxyribose The 5′ end has an exposed phosphate group attached to a 5′ carbon

(a) Structure of a deoxyribonucleotide (b) Primary structure of DNA Figure 15.3 (a) Structure of a deoxyribonucleotide (b) Primary structure of DNA 5′ end of strand Phosphate group attached to 5′ carbon of the sugar Bases project from the backbone Could be adenine (A), thymine (T), guanine (G), cytosine (C) Sugar–phosphate backbone of DNA strand Hydroxyl (OH) group on 3′ carbon of the sugar Phosphodiester bond links deoxyribonucleotides Figure 15.3 DNA’s Primary Structure Has a Sugar-Phosphate Backbone. 3′ end of strand © 2017 Pearson Education, Inc.

The Secondary Structure of DNA Watson and Crick proposed that Two DNA strands line up in the opposite direction to each other in 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)

(a) Complementary base pairing (b) The double helix Figure 15.4 (a) Complementary base pairing (b) The double helix 3′ 5′ 5′ 3′ Sugar– phosphate backbone of DNA Complementary base pairs held together by hydrogen bonding Figure 15.4 DNA’s Secondary Structure Is the Double Helix. Antiparallel strands (their 5′ → 3′ polarities run in opposite directions) 5′ 5′ 3′ 3′ © 2017 Pearson Education, Inc.

The Secondary Structure of DNA Because of complementary base pairing Existing strands of DNA can serve as a template (pattern) for the production of new strands Three hypotheses for how parental strands and daughter strands act during replication: Semiconservative replication Conservative replication Dispersive replication

Testing Early Hypotheses about DNA Synthesis Semiconservative replication—Parental strands separate, and each is a template for a new strand Each daughter has one old and one new strand Conservative replication—The parental molecule serves as a template for an entirely new molecule One daughter has both old strands; the other has both new strands Dispersive replication—The parent molecule is cut into sections Each daughter has old and new DNA interspersed

The Meselson–Stahl Experiment Meselson and Stahl grew E. coli in the presence of “heavy” nitrogen (15N) to label the bacteria’s DNA Bacteria copy their entire complement of DNA, or genome, before every cell division Then they moved the bacteria to a normal 14N-containing medium Allowed the cells to divide once Separated the DNA by density The results supported semiconservative replication

Figure 15.5 Is replication semiconservative, conservative, or dispersive? Replication is semiconservative. Replication is conservative. Replication is dispersive. Generation 0 DNA sample Generation 1 DNA sample Generation 2 DNA sample 15N 14N 14N Cell transfer 1. Grow E. coli cells in medium with 15N as sole source of nitrogen for many generations. Collect sample and purify DNA. 2. Transfer cells to medium containing 14N. After cells divide once, collect sample and purify DNA. 3. After cells have divided a second time in 14N medium, collect sample and purify DNA. 4. Centrifuge the three samples separately. Compare the locations of the DNA bands in each sample to determine density. Semiconservative replication Conservative replication Dispersive replication Generation 0 15N 15N 15N Generation 1 Hybrid Hybrid 15N 14N Hybrid Hybrid Generation 2 Figure 15.5 The Meselson–Stahl Experiment Settled a Key Question about Replication. Hybrid 14N Hybrid 14N 15N 14N Hybrid After 2 generations: 1/2 low-density DNA (14N) 1/2 intermediate-density DNA (hybrid) After 2 generations: 1/4 high-density DNA (15N) 3/4 low-density DNA (14N) After 2 generations: All intermediate-density DNA (hybrid) Top of centrifuge tube (lower density) After 2 generations: 1/2 low-density DNA 1/2 intermediate-density DNA 14N Hybrid Bottom of centrifuge tube (higher density) 15N 1 2 Generation Data from generation 1 conflict with conservative replication hypothesis. Data from generation 2 conflict with dispersive replication hypothesis. Replication is semiconservative. © 2017 Pearson Education, Inc.

A Model for DNA Synthesis The enzyme that catalyzes DNA synthesis is called DNA polymerase There are several types of DNA polymerases They can work only in one direction Can add deoxyribonucleotides only to the 3′ end of a growing DNA chain Therefore, DNA synthesis always proceeds in the 5′ → 3′ direction

A Model for DNA Synthesis DNA polymerization should be endergonic The monomers are deoxyribonucleoside triphosphates (dNTPs) Have high potential energy because of their three closely packed phosphate groups They have enough potential energy to make the formation of phosphodiester bonds exergonic

Figure 15.6 DNA Synthesis Proceeds in Only One Direction. Parental strand Parental strand 3′ end 3′ end Daughter strand Daughter strand 5′ end 5′ end Phosphodiester bond 3′ end Figure 15.6 DNA Synthesis Proceeds in Only One Direction. 5′ end 5′ end Synthesis reaction 3′ end dNTP © 2017 Pearson Education, Inc.

Where Does Replication Start? A replication bubble forms when DNA is being synthesized Forms at a specific sequence called the origin of replication Bacteria have one and form one replication bubble Eukaryotic cells have many on each chromosome Each replication bubble has two replication forks Because synthesis is bidirectional Replication bubbles grow in two directions

(a) DNA being replicated Figure 15.7 (a) DNA being replicated (b) Bacterial chromosomes have a single origin of replication. Old DNA New DNA Replication proceeds 5′ → 3′ in both directions Origin of replication 0.25 μm (c) Eukaryotic chromosomes have multiple origins of replication. Replication fork Figure 15.7 DNA Replication Forks Move in Two Directions from an Origin of Replication. Replication bubble Old DNA New DNA Replication proceeds 5′ → 3′ in both directions from each starting point © 2017 Pearson Education, Inc.

How Is the Helix Opened and Stabilized? Several proteins are responsible for opening and stabilizing the double helix DNA helicase breaks hydrogen bonds between the two DNA strands to separate them 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 Topoisomerase cuts and rejoins the DNA to relieve this tension

How Is the Leading Strand Synthesized? DNA polymerase has two parts: A sliding clamp that forms a ring around the DNA A part that grips the DNA strand DNA polymerase Works only in the 5′ → 3′ direction on a single-stranded template Requires a 3′ end to extend from

How Is the Leading Strand Synthesized? The 3′ end for DNA polymerase to add to is supplied by a primer An RNA strand about a dozen nucleotides long Base-pairs with the DNA template strand Provides a 3′ hydroxyl (OH) group that can combine with a dNTP to form a phosphodiester bond

How Is the Leading Strand Synthesized? Primers are made by an enzyme called primase A type of RNA polymerase Does not require a free 3′ end to begin synthesis DNA polymerase adds dNTPs to the primer’s 3′ end Since DNA strands are antiparallel, the synthesis process differs for each strand The strand that is synthesized toward the replication fork is the leading strand, or continuous strand It is synthesized continuously in the 5′ → 3′ direction

Primase synthesizes RNA primer Figure 15.8 Primase synthesizes RNA primer Topoisomerase relieves twisting forces 1. DNA is opened, unwound, and primed. Helicase opens double helix Single-strand DNA–binding proteins (SSBPs) stabilize single strands Sliding clamp holds DNA polymerase in place DNA polymerase synthesizes leading strand in 5′ → 3′ direction RNA primer 2. Synthesis of leading strand begins. Figure 15.8 Leading-Strand Synthesis. Leading strand © 2017 Pearson Education, Inc.

How Is the Lagging Strand Synthesized? The strand synthesized away from the replication fork is the lagging strand or discontinuous strand It occurs because DNA synthesis must proceed in the 5′ → 3′ direction The discontinuous replication hypothesis Primase synthesizes new RNA primers on the lagging strand as the replication fork opens DNA polymerase synthesizes short fragments of DNA along the lagging strand Fragments are then linked into a continuous strand

Leading strand synthesized 5′ → 3′ DNA unwinds Time 1 Lagging strand Figure 15.9 Leading strand synthesized 5′ → 3′ DNA unwinds Time 1 Lagging strand synthesized 5′→ 3′ Figure 15.9 The Lagging Strand Is Synthesized Away from the Replication Fork. Time 2 Region of single- stranded DNA © 2017 Pearson Education, Inc.

The Discovery of Okazaki Fragments This hypothesis was confirmed by Okazaki The lagging strand is synthesized as short discontinuous fragments called Okazaki fragments DNA polymerase I removes the RNA primers and replaces them with DNA The enzyme DNA ligase joins the Okazaki fragments

Figure 15.10 Lagging-Strand Synthesis. The leading strands are faded out to help you focus on synthesis of the lagging strand RNA primer 1. Primer added. Topoisomerase SSBPs Helicase Primase Okazaki fragment 2. First fragment synthesized. Sliding clamp DNA polymerase III 2nd Okazaki fragment 1st Okazaki fragment 3. Second fragment synthesized. Figure 15.10 Lagging-Strand Synthesis. DNA polymerase I 4. Primer replaced. DNA ligase 5. Gap closed. © 2017 Pearson Education, Inc.

Table 15.1 Proteins Required for DNA Synthesis in Bacteria. © 2017 Pearson Education, Inc.

The Discovery of Okazaki Fragments The replisome Contains the enzymes responsible for DNA synthesis around the replication fork Joined into one large, multi-enzyme machine

Sliding clamp DNA polymerase III Topoisomerase Leading strand Helicase Figure 15.11 Sliding clamp DNA polymerase III Topoisomerase Leading strand Helicase Primase Figure 15.11 The Replisome Is a DNA-Synthesizing Machine. Lagging strand SSBPs DNA ligase © 2017 Pearson Education, Inc.

Web Activity: DNA Synthesis

Replicating the Ends of Linear Chromosomes Replication of telomeres (ends of linear chromosomes) can be problematic The leading strand is synthesized all the way to the end On the lagging strand, primase adds an RNA primer close to the end of the chromosome The final Okazaki fragment is made and the primer is removed DNA polymerase cannot add to the end with no primer

The End Replication Problem A single-stranded DNA is left at the end of the lagging strand The single-stranded DNA is eventually degraded This would shorten the chromosome by 50 to 100 nucleotides each time replication occurs Over time, linear chromosomes would vanish Telomeres do not contain genes Consist of short, repeating stretches of bases

Figure 15.12 Chromosomes Shorten during Normal DNA Replication. DNA polymerase End of chromosome Leading strand Sliding clamp 1. DNA unwinding completed. Lagging strand Helicase 2. Leading strand completed. RNA primer Primase 3. Lagging strand nears completion. Figure 15.12 Chromosomes Shorten during Normal DNA Replication. DNA polymerase Last Okazaki fragment 4. Lagging strand too short. No primer for DNA polymerase; unreplicated end is eventually lost, shortening chromosome Unreplicated end © 2017 Pearson Education, Inc.

Telomerase Solves the End Replication Problem The enzyme telomerase replicates telomeres, using an RNA template that it carries The 3′ end of the lagging strand forms a single-stranded “overhang” Telomerase binds to the overhang and uses the RNA that it carries as a template for DNA synthesis Telomerase continues to move down the new strand, adding more short DNA sequences to the end Once the overhang is long enough, normal DNA synthesis can occur

Missing DNA on lagging strand Figure 15.13 Missing DNA on lagging strand 1. End is unreplicated. Telomerase with its own RNA template 2. Telomerase extends unreplicated end. 3. Telomerase repeats activity. Figure 15.13 Telomerase Prevents Shortening of Telomeres during Replication. RNA primer 4. Extended single-strand DNA acts as template. DNA polymerase Sliding clamp © 2017 Pearson Education, Inc.

Effect of Telomere Length on Cell Division Somatic cells normally lack telomerase Chromosomes of somatic cells progressively shorten This occurs as the individual ages Greider proposed that the number of cell divisions is limited by the initial length of a cell’s telomeres Once chromosome are shortened to a threshold length, further divisions are shut down In culture, telomere length correlates to number of cell divisions

Number of cell divisions 26–50 0–25 Figure 15.14 Donor age >75 51–75 Number of cell divisions 26–50 0–25 Cells with initially longer telomeres divided more times than cells with initially shorter telomeres, regardless of donor age Figure 15.14 Telomere Length Predicts the Number of Divisions before Cells Stop Dividing. Initial telomere length (base pairs) © 2017 Pearson Education, Inc.

Effect of Telomere Length on Cell Division Adding telomerase to cells in culture allows them to continue dividing longer Most cancer cells have active telomerase May allow unlimited division of cancer cells Could inhibiting telomerase slow or stop cancer?

Correcting Mistakes in DNA Synthesis DNA replication is very accurate Error rate is about one mistake per billion bases DNA polymerase matches bases with high accuracy Correct bases are the most energetically favorable Correct base pairs have a distinct shape Inserts an incorrect base about once every 100,000 bases Repair enzymes remove defective bases and replace them with the correct one

DNA Polymerase Proofreads DNA polymerase can proofread its work Mismatched bases have a distinct shape DNA polymerase will add a nucleotide only if the previous base pair is correct If the enzyme finds a mismatch Its ε (epsilon) subunit removes the mismatched base This proofreading process reduces the error rate to about one mistake in 10 million base pairs

(a) DNA polymerase adds a mismatched deoxyribonucleotide ... Figure 15.15 (a) DNA polymerase adds a mismatched deoxyribonucleotide ... (b) ... but detects the mistake and corrects it. Figure 15.15 DNA Polymerase Can Proofread. © 2017 Pearson Education, Inc.

Mismatch Repair DNA polymerase sometimes leaves a mismatched pair behind 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 Fill in the correct bases

Repairing Damaged DNA DNA can be damaged by sunlight, X-rays, and many chemicals Organisms have DNA damage-repair systems For example, UV light and some chemicals can cause thymine dimers to form These dimers produce a kink in the DNA strand This blocks DNA replication

adjacent thymine bases Figure 15.16 UV light Kink Figure 15.16 UV Light Damages DNA. Thymine dimer DNA strand with adjacent thymine bases Damaged DNA strand © 2017 Pearson Education, Inc.

Repairing Damaged DNA The nucleotide excision repair system recognizes such types of damage A protein complex recognizes the kink Removes the damaged single-stranded DNA Uses the intact strand as a template for new DNA DNA ligase links the repaired strand to the original undamaged DNA

1. Error detection. 2. Nucleotide excision. 3. Nucleotide replacement. Figure 15.17 Damaged bases Cut Cut 1. Error detection. 2. Nucleotide excision. 3. Nucleotide replacement. Figure 15.17 Nucleotide Excision Repair Removes and Replaces Defective Bases. Repaired damage 4. Nucleotide linkage. © 2017 Pearson Education, Inc.

Xeroderma Pigmentosum: A Case Study Xeroderma pigmentosum (XP) A rare autosomal recessive disease in humans Causes extreme sensitivity to UV light Increases chance of skin cancer by 1000−2000 times XP is caused by mutations in nucleotide excision repair systems The cells of people with XP cannot repair DNA damaged by ultraviolet radiation Can result from mutations in any of eight genes

Unaffected cells XP cells Amount of radioactive nucleotide Figure 15.18 Amount of radioactive nucleotide incorporated (counts per minute) Unaffected cells Figure 15.18 DNA Damage from UV Light IS Not Repaired Properly in Individuals with XP. XP cells Dose of UV light (ergs/mm2) © 2017 Pearson Education, Inc.

Xeroderma Pigmentosum: A Case Study Defects in the genes required for DNA repair are frequently associated with cancer If mutations in the genes involved in the cell cycle go unrepaired The cell may begin to grow in an uncontrolled manner This growth can result in the formation of a tumor If the overall mutation rate in a cell is elevated Because of defects in DNA repair genes Then the mutations that trigger cancer become more likely