Chapter 14 DNA - Tagged PDF

Because learning changes everything. ® Chapter 14 DNA: The Genetic Material BIOLOGY Thirteenth Edition Raven, Johnson, Mason, Losos, Duncan © 2023 McGraw Hill, LLC. All rights reserved. Authorized only for instructor use in the classroom. No reproduction...

Because learning changes everything. ® Chapter 14 DNA: The Genetic Material BIOLOGY Thirteenth Edition Raven, Johnson, Mason, Losos, Duncan © 2023 McGraw Hill, LLC. All rights reserved. Authorized only for instructor use in the classroom. No reproduction or further distribution permitted without the prior written consent of McGraw Hill, LLC.  Lecture Outline 14.1 The Nature of the Genetic Material 14.2 DNA Structure 14.3 Basic Characteristics of DNA Replication 14.4 Prokaryotic Replication 14.5 Eukaryotic Replication 14.6 DNA Repair Molekuul/SPL/age fotostock © McGraw Hill, LLC 2 © McGraw Hill, LLC 3  Learning Objectives 14.1 The Nature of the Genetic Material 1. Describe the experiments of Griffith and Avery. 2. Evaluate the evidence for DNA as genetic material. 14.2 DNA Structure 3. Explain how the Watson–Crick structure rationalized the data available to them. 4. Evaluate the significance of complementarity for DNA structure and function. 14.3 Basic Characteristics of DNA Replication 5. Illustrate the products of semiconservative replication. 6. Describe the requirements for DNA replication. © McGraw Hill, LLC 4  What are genes made of? Scientists knew chromosomes were primarily made of protein and DNA, but did not know which actually made up genes DNA composed of four nucleotides while proteins contained 20 distinct amino acids, suggesting proteins had greater capacity for storing information A series of experiments in 1920’s to 1950’s determined DNA is the genetic material © McGraw Hill, LLC 5  Frederick Griffith – 1928 Studied Streptococcus pneumoniae, a pathogenic bacterium causing pneumonia Two strains of Streptococcus S strain is virulent R strain is nonvirulent Griffith infected mice with both strains hoping to understand the difference between the strains © McGraw Hill, LLC 6  Griffith’s Results 1 Injection of: Live virulent (S) strain cells killed the mice Live nonvirulent (R) strain cells did not kill the mice Access the text alternative for slide images. © McGraw Hill, LLC 7  Griffith’s Results 2 Injection of: Heat-killed virulent (S) strain cells did not kill the mice Heat-killed virulent (S) strain + live nonvirulent (R) strain cells killed the mice Access the text alternative for slide images. © McGraw Hill, LLC 8  Injection Outcome 1. Live S cells Mice die, live S cells in Shows S cells are blood virulent 2. Live R cells Mice live, no cells in Shows R cells are not blood virulent 3. Heat killed S cells Mice live, no cells in Shows live S cells are blood required 4. Live R cells with heat Mice die, live S cells in Dead S cells transform killed S cells blood R cells to S!!!! © McGraw Hill, LLC  Transformation Griffith called the transfer of virulence from the dead S strain cells into the live R strain cells transformation Did not know the mechanism for movement of genetic information Modern interpretation is that genetic material was physically transferred between the cells © McGraw Hill, LLC 10  Avery, MacLeod, & McCarty – 1944 Repeated Griffith’s experiment using purified cell extracts Treat S cells, then mix with R cells and inject the mice Used the following treatments: Protein digesting enzymes DNA digesting enzymes © McGraw Hill, LLC 11  Avery, MacLeod, & McCarty – 1944 Removal of all protein from the transforming material did not destroy its ability to transform R strain cells DNA-digesting enzymes destroyed all transforming ability Supported DNA as the genetic material, at least in bacteria © McGraw Hill, LLC 12  Hershey & Chase –1952 Investigated genetic material using bacteriophages (also called phages), viruses that infect bacteria Bacteriophages are composed of only DNA and protein Wanted to determine which of these molecules is the genetic material that is injected into the bacteria © McGraw Hill, LLC 13  Hershey & Chase experiment Bacteriophage DNA was labeled with radioactive 32 phosphorus ( P) Bacteriophage protein was labeled with radioactive sulfur (35S) Radioactive molecules were tracked 32 Only the bacteriophage DNA (as indicated by the P) entered the bacteria and was used to produce more bacteriophage Conclusion: DNA is the genetic material © McGraw Hill, LLC 14  Hershey and Chase demonstrate that phage genetic material is DNA Access the text alternative for slide images. © McGraw Hill, LLC 15  DNA Structure DNA is a nucleic acid composed of nucleotides: 5-carbon sugar called deoxyribose Phosphate group (PO4) Attached to 5′ carbon of sugar Nitrogenous base Adenine, thymine, cytosine, guanine Free hydroxyl group (—OH) Attached at the 3′ carbon of sugar © McGraw Hill, LLC 16  Nucleotide subunits of DNA and RNA Access the text alternative for slide images. © McGraw Hill, LLC 17  Phosphodiester bond Bond between adjacent nucleotides Formed between the phosphate group of one nucleotide and the 3′ —OH of the next nucleotide The chain of nucleotides has a 5′-to-3′ orientation Access the text alternative for slide images. © McGraw Hill, LLC 18  Chargaff’s Rules Erwin Chargaff determined that: Always an equal proportion of two-ringed purines (A and G) and single-ringed pyrimidines (C and T) Amount of adenine = amount of thymine Amount of cytosine = amount of guanine The ratio of A-T and G-C varies by species © McGraw Hill, LLC 19  Working in the Amazon River, a biologist isolated DNA from two unknown organisms, P and Q. He discovered that the adenine content of P was 15% and the cytosine content of Q was 42%. This means that: A. The amount of guanine in P is 15% B. The amount of guanine and cytosine combined in P is 70% C. The amount of adenine in Q is 42% D. The amount of thymine in Q is 21% © McGraw Hill, LLC  Rosalind Franklin Performed X-ray diffraction studies to identify the 3-D structure Discovered that DNA is helical Using Maurice Wilkins’ DNA fibers, discovered that the molecule has a diameter of 2 nm and makes a complete turn of the helix every 3.4 nm (a) Jewish Chronicle Archive/Heritage Image Partnership Ltd/Alamy Stock Photo; (b) Omikron/Science Source Access the text alternative for slide images. © McGraw Hill, LLC 21  James Watson and Francis Crick – 1953 Deduced the structure of DNA using evidence from Chargaff, Franklin, and others Did not perform a single experiment themselves related to DN A Key insight of their model was each DNA molecule was made of two intertwined chains of nucleotides, that is a double helix structure © McGraw Hill, LLC 22  Structure of a single strand of DNA Phosphodiester backbone – repeating sugar and phosphate units joined by phosphodiester bonds A single strand extends in a 5′ to 3′ direction Access the text alternative for slide images. © McGraw Hill, LLC 23  The double helix Two strands arranged as a double helix Forms two grooves, the larger major groove and the smaller minor groove Strands connected via hydrogen bonds between bases on opposite strands Result is specific base-pairs: A-T and G-C Helix has a consistent diameter, is stable because of additive property of thousands of low-energy hydrogen bonds Access the text alternative for slide images. © McGraw Hill, LLC 24  Base-pairing Pattern of base-paring is complementary A forms two hydrogen bonds with T G forms three hydrogen bonds with C Two strands of single DNA molecule are not identical, each strand specifies the other by base-pair complementarity Access the text alternative for slide images. © McGraw Hill, LLC 25  Antiparallel configuration Each phosphodiester strand has inherent polarity based on orientation of sugar-phosphate backbone One end terminates in 3′ OH One end terminates in 5′ PO4 Strands are referred as having either 5′-to-3′ or 3′-to-5′ polarity The two strands of a single DNA molecule have opposite polarity to one another © McGraw Hill, LLC 26  DNA Double Helix © McGraw Hill, LLC  Watson and Crick Observed: “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” What does this imply regarding the sequence of bases on the two strands that make up a DNA molecule? © McGraw Hill, LLC  Learning Objectives 14.4 Prokaryotic Replication 1. Describe the functions of E. coli DNA polymerases. 2. Explain why replication is discontinuous on one strand. 3. Diagram the functions found at the replication fork. 14.5 Eukaryotic Replication 4. Compare eukaryotic replication with prokaryotic. 5. Explain the function of telomeres. 6. Evaluate the role of telomerase in cell division. 14.6 DNA Repair 7. Explain why DNA repair is critical for cells. © McGraw Hill, LLC 29  Three Possible Models of DNA Replication 1 1. Conservative model – both strands of parental DNA remain intact; new DNA copies consist of all new molecules 2. Semiconservative model – daughter strands each consist of one parental strand and one new strand 3. Dispersive model – new DNA is dispersed throughout each strand of both daughter molecules after replication © McGraw Hill, LLC 30  Three Possible Models of DNA Replication 2 Access the text alternative for slide images. © McGraw Hill, LLC 31  Meselson and Stahl – 1958 Bacterial cells were grown in a heavy isotope of nitrogen, 15 N After several generations, the DNA of these bacteria was denser than normal DNA Cells were switched to media containing lighter 14 N DNA was extracted from the cells at various time intervals and centrifuged to separate out by weight © McGraw Hill, LLC 32  Messleson Stahl Predictions Start with heavy DNA (N15) then allow one round of replication. If the mechanism is conservative, what are the densities of the two daughter double helices produced? If the mechanism is semiconservative, what are the densities of the two daughter double helices produced? If the mechanism is dispersive, what are the densities of the two daughter double helices produced? © McGraw Hill, LLC 33  The Meselson–Stahl experiment Meselson, M., Stahl, F., (1958) “The replication of DNA in Escherichia coli,” PNAS, 44(7):671-682, Fig. 4a Access the text alternative for slide images. © McGraw Hill, LLC 34  Meselson and Stahl’s Results Conservative model = rejected Two density bands were not observed after round 1 Semiconservative model = supported Consistent with all observations One band after round 1 Two bands after round 2 Dispersive model = rejected 1st round results consistent 2nd round – did not observe one band © McGraw Hill, LLC 35  DNA Replication Requires three things: 1. Something to copy Parental DNA molecule 2. Something to do the copying Enzymes 3. Building blocks to make copy Nucleotide triphosphates © McGraw Hill, LLC 36  Stages of DNA replication Initiation – replication begins Elongation – new strands of DNA are synthesized by DNA polymerase Termination – replication is terminated © McGraw Hill, LLC 37  Action of DNA polymerase Access the text alternative for slide images. © McGraw Hill, LLC 38  DNA polymerase DNA polymerases match existing DNA bases with complementary nucleotides and links them, that is build new DNA strands All have several common features Add new bases to 3′ end of existing strands Synthesize in 5′-to-3′ direction Require a primer of RNA © McGraw Hill, LLC 39  Prokaryotic Replication E. coli used as model system for understanding universal attributes of replication Single circular molecule of DNA Replication begins at the origin of replication Proceeds in both directions around the chromosome Replicon – DNA controlled by an origin © McGraw Hill, LLC 40  Replication is bidirectional from a unique origin Access the text alternative for slide images. © McGraw Hill, LLC 41  E. coli has three DNA polymerases DNA polymerase I (Pol I) Acts on lagging strand to remove primers and replace them with DNA DNA polymerase II (Pol II) Involved in DNA repair processes DNA polymerase III (Pol III) Main replication enzyme All 3 have 3′-to-5′ exonuclease activity – proofreading DNA Pol I has 5′-to-3′ exonuclease activity – removing RNA primers © McGraw Hill, LLC 42  DNA polymerase activity In addition to adding nucleotides to a growing DNA strand, some polymerase molecules can remove nucleotides, acting as nucleases Can be endonucleases (cut DNA internally) or exonucleases (remove nucleotides from end of DNA) All three E. coli DNA polymerases have 3′-to-5′ exonuclease activity – proofreading DNA Pol I has 5′-to-3′ exonuclease activity – removing RNA primers © McGraw Hill, LLC 43  Enzymes unwind DNA Helicases – use energy from ATP to unwind DNA Single-strand-binding proteins (SSBs) coat strands to keep them apart Unwinding of DNA introduces torsional strain in the molecule that can lead to additional twisting of the helix, called supercoiling Topoisomerases are enzymes that prevent supercoiling DNA gyrase is the topoisomerase involved in DNA replication that relieves the torsional strain © McGraw Hill, LLC 44  Unwinding the helix causes torsional strain Access the text alternative for slide images. © McGraw Hill, LLC 45  Replication is semi discontinuous DNA polymerase can only synthesize in the 5’-to-3’ direction Antiparallel nature of DNA means new DNA strands must be synthesized in opposite directions Leading strand synthesized continuously from an initial primer Lagging strand synthesized discontinuously with multiple priming events DNA fragments on the lagging strand are called Okazaki fragments, must be connected together © McGraw Hill, LLC 46  The lagging strand is synthesized in pieces Access the text alternative for slide images. © McGraw Hill, LLC 47  Synthesis occurs at the replication fork Replication fork is partial opening of helix formed where double stranded DNA is being unwound DNA primase – RNA polymerase that makes RNA primer RNA will be removed and replaced with DNA later © McGraw Hill, LLC 48  Leading-strand synthesis Single priming event Strand extended by DNA Pol III Processivity – the ability of a polymerase to stay attached β subunit forms “sliding clamp” to keep DNA Pol III attached to DNA (high processivity) 3Dciencia/SPL/Science Source © McGraw Hill, LLC 49  Lagging-strand synthesis requires additional enzymes Discontinuous synthesis, requires multiple enzymes: DNA Pol III (like leading strand) Primase - Makes RNA primer for each Okazaki fragment DNA Pol I - Removes all RNA primers and replaces with DNA DNA ligase – joins Okazaki fragments to form complete strands Termination occurs at specific site: DNA gyrase unlinks two copies © McGraw Hill, LLC 50  Lagging-strand synthesis Access the text alternative for slide images. © McGraw Hill, LLC 51  Replisome Replisome is a macromolecular assembly of enzymes involved in DNA replication Two main components Primosome Primase, helicase, accessory proteins Complex of two DNA Pol III One for each strand © McGraw Hill, LLC 52  A model for the structure of the replication fork Access the text alternative for slide images. © McGraw Hill, LLC 53  https://www.youtube.com/watch?v=3ltc21PfR9M © McGraw Hill, LLC 54  DNA synthesis by the replisome: Stage 1 Access the text alternative for slide images. © McGraw Hill, LLC 55  DNA synthesis by the replisome: Stage 2 Access the text alternative for slide images. © McGraw Hill, LLC 56  DNA synthesis by the replisome: Stage 3 Access the text alternative for slide images. © McGraw Hill, LLC 57  DNA synthesis by the replisome: Stage 4 Access the text alternative for slide images. © McGraw Hill, LLC 58  DNA synthesis by the replisome: Stage 5 Access the text alternative for slide images. © McGraw Hill, LLC 59  Eukaryotic Replication More complex than in prokaryotes due primarily to: Larger amount of DNA in multiple chromosomes Linear structure (versus circular chromosomes) Don W. Fawcett/Science Source © McGraw Hill, LLC 60  Eukaryotic replication uses multiple origins Basic enzymology is similar Requires new enzymatic activity for dealing with ends only Multiple replicons – multiple origins of replications for each chromosome Not sequence specific; can be adjusted Example: early in development when cells divide rapidly, more origins can be used © McGraw Hill, LLC 61  The eukaryotic replication fork is more complex Before S phase, helicases are loaded onto possible replication origins, but not activated During S phase, a subset of these are activated, and the rest of the replisome assembled Priming uses a complex of both DNA polymerase α and primase DNA polymerase epsilon (Pol ε) synthesizes leading strand DNA polymerase delta (Pol δ) synthesizes lagging strand © McGraw Hill, LLC 62  Archaeal and eukaryotic replication proteins are evolutionarily related Enzymes that are similar between eukaryotes and archaea, but different from those in prokaryotes: DNA polymerases Replicative helicases Primases © McGraw Hill, LLC 63  Linear chromosomes have specialized ends Telomeres Specialized structures found on the ends of eukaryotic chromosomes Composed of specific repeat sequences Protect ends of chromosomes from nucleases Maintain the integrity of linear chromosomes Not made by replication complex © McGraw Hill, LLC 64  Replication of the end of linear DNA presents a problem Access the text alternative for slide images. © McGraw Hill, LLC 65  Telomere maintenance The last primer removed from the 3′ end of the lagging strand cannot be replaced Result would be shortening of chromosomes with each round of cell division Telomerase is an enzyme that synthesizes the telomere repeat sequences at the ends of strand Uses an internal RNA template (not the DNA itself) © McGraw Hill, LLC 66  Action of telomerase Access the text alternative for slide images. © McGraw Hill, LLC 67  Regulation of telomerase Telomerase activity is developmentally regulated High in early development/childhood Low in most somatic adult cells Exceptions: cells that must continue dividing (for example lymphocytes) Telomerase plays a role in senescence/aging Mice that completely lack telomerase activity had nonviable offspring after six generations Telomerase contributes to cancer Cancer cells generally show activation of telomerase to maintain telomere length © McGraw Hill, LLC 68  DNA damage constantly occurs Random errors during to replication DNA polymerases have proofreading ability to counteract Mutagens – any agent that increases the number of mutations above background level Examples: radiation and chemicals Importance of DNA repair is indicated by the multiplicity of repair systems that have been discovered © McGraw Hill, LLC 69  Two categories of DNA repair 1. Specific repair Targets a single kind of lesion in DNA and repairs only that damage 2. Nonspecific Use a single mechanism to repair multiple kinds of lesions in DNA © McGraw Hill, LLC 70  Mismatch repair Mismatch repair (MMR) removes incorrect bases incorporated during DNA replication Replaces them with the correct base by copying the template strand Must distinguish between the template strand and the newly synthesized strand In E. coli, this involves methylation of the A in the sequence 5′ GATC 3′ The newly synthesized strand will be unmethylated for a brief window during which MMR can identify strands © McGraw Hill, LLC 71  Photorepair Specific repair mechanism Example: thymine dimers caused by UV light where adjacent thymines become covalently linked together Photolyase enzyme: Absorbs light in visible range Uses this energy to cleave thymine dimer Access the text alternative for slide images. © McGraw Hill, LLC 72  Excision repair Nonspecific repair Damaged region removed and replaced by DNA synthesis: 1. Recognition of damage 2. Removal of the damaged region 3. Resynthesis using the information on the undamaged strand as a template In E. coli, proteins encoded by uvr-A, -B, and -C genes form a complex to carry out excision repair © McGraw Hill, LLC 73  Repair of damaged DNA by excision repair Access the text alternative for slide images. © McGraw Hill, LLC 74  Additional repair pathways Most DNA repair mechanisms are error-free, some are actually error-prone ”SOS response” system in E. coli is error prone, thought to be a last-ditch effort should there be too much damage for normal pathways to handle Repair of damage with actual breaks in DNA uses systems with enzymes related to those used during meiosis recombination Thought that recombination enzymes originally evolved for DNA repair © McGraw Hill, LLC 75  End of Main Content Because learning changes everything. ® www.mheducation.com © 2023 McGraw Hill, LLC. All rights reserved. Authorized only for instructor use in the classroom. No reproduction or further distribution permitted without the prior written consent of McGraw Hill, LLC.