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DNA: The Genetic Material Chapter 14

Frederick Griffith – 1928 Studied Streptococcus pneumoniae, a pathogenic bacterium causing pneumonia 2 strains of Streptococcus S strain is virulent R strain is nonvirulent Griffith infected mice with these strains hoping to understand the difference between the strains

Griffith’s results Live S strain cells killed the mice Live R strain cells did not kill the mice Heat-killed S strain cells did not kill the mice Heat-killed S strain + live R strain cells killed the mice

Transformation Information specifying virulence passed from the dead S strain cells into the live R strain cells Our modern interpretation is that genetic material was actually transferred between the cells

Avery, MacLeod, & McCarty – 1944 Repeated Griffith’s experiment using purified cell extracts 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

Hershey & Chase –1952 Investigated bacteriophages Viruses that infect bacteria Bacteriophage was composed of only DNA and protein Wanted to determine which of these molecules is the genetic material that is injected into the bacteria

Bacteriophage DNA was labeled with radioactive phosphorus (32P) Bacteriophage protein was labeled with radioactive sulfur (35S) Radioactive molecules were tracked Only the bacteriophage DNA (as indicated by the 32P) entered the bacteria and was used to produce more bacteriophage Conclusion: DNA is the genetic material

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

NH2 N N N CH2 O Nitrogenous Base Nitrogenous base NH2 O N C N C C N C Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Nitrogenous Base Nitrogenous base NH2 NH2 O 7 N 6 5 N C N C C N C N H 1 H C H C Phosphate group 8 Purines C C 2 N C H N C NH2 N N O N 4 N H H 9 3 Adenine Guanine –O P O CH2 5´ O– O NH2 O O 1´ C C C 4´ H C N H3C C N H H C N H H C C O H C C O H C C O 3´ 2´ OH in RNA Pyrimidines N N N OH H H H H in DNA Sugar Cytosine (both DNA and RNA) Thymine (DNA only) Uracil (RNA only)

The chain of nucleotides has a 5′-to-3′ orientation 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 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5´ PO4 Base CH2 O C O Phosphodiester bond –O P O O Base CH2 O OH 3´

Chargaff’s Rules Erwin Chargaff determined that Amount of adenine = amount of thymine Amount of cytosine = amount of guanine Always an equal proportion of purines (A and G) and pyrimidines (C and T)

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

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 DNA Proposed a double helix structure

Double helix 2 strands are polymers of nucleotides Phosphodiester backbone – repeating sugar and phosphate units joined by phosphodiester bonds Wrap around 1 axis Antiparallel Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5´ Phosphate group P 5´ O 4´ 1´ Phosphodiester bond 3´ 2´ P 5´ O 4´ 1´ 3´ 2´ P 5´ O 4´ 1´ 5-carbon sugar 3´ 2´ Nitrogenous base P 5´ O 4´ 1´ 3´ 2´ OH 3´

Complementarity of bases A forms 2 hydrogen bonds with T Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Hydrogen bond H Complementarity of bases A forms 2 hydrogen bonds with T G forms 3 hydrogen bonds with C Gives consistent diameter H N O H N H N G N H N C H Sugar N N N H Sugar H Hydrogen bond H H N N H O CH3 N A N H N T H Sugar N N H Sugar

DNA Replication 3 possible models Conservative model Semiconservative model Dispersive model

Copyright © The McGraw-Hill Companies, Inc Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Conservative

Conservative Semiconservative 23 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 23 Conservative Semiconservative

Conservative Semiconservative Dispersive 24 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 24 Conservative Semiconservative Dispersive

Meselson and Stahl – 1958 Bacterial cells were grown in a heavy isotope of nitrogen, 15N All the DNA incorporated 15N Cells were switched to media containing lighter 14N DNA was extracted from the cells at various time intervals

Conservative model = rejected 2 densities were not observed after round 1 Semiconservative model = supported Consistent with all observations 1 band after round 1 2 bands after round 2 Dispersive model = rejected 1st round results consistent 2nd round – did not observe 1 band

DNA Replication Requires 3 things Something to copy Parental DNA molecule Something to do the copying Enzymes Building blocks to make copy Nucleotide triphosphates

DNA replication includes Initiation – replication begins Elongation – new strands of DNA are synthesized by DNA polymerase Termination – replication is terminated

All have several common features Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5´ 5´ 3´ 3´ 5´ 3´ 5´ RNA polymerase makes primer DNA polymerase extends primer DNA polymerase Matches existing DNA bases with complementary nucleotides and links them 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

Prokaryotic Replication E. coli model Single circular molecule of DNA Replication begins at one origin of replication Proceeds in both directions around the chromosome Replicon – DNA controlled by an origin

E. coli has 3 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′ exonuclase activity

Unwinding DNA causes torsional strain Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Supercoiling Replisomes No Supercoiling Replisomes DNA gyrase Unwinding DNA causes torsional strain Helicases – use energy from ATP to unwind DNA Single-strand-binding proteins (SSBs) coat strands to keep them apart Topoisomerase prevent supercoiling DNA gyrase is used in replication

Semidiscontinous DNA polymerase can synthesize only in 1 direction Leading strand synthesized continuously from an initial primer Lagging strand synthesized discontinuously with multiple priming events Okazaki fragments

Partial opening of helix forms replication fork DNA primase – RNA polymerase that makes RNA primer RNA will be removed and replaced with DNA

Leading-strand synthesis Single priming event Strand extended by DNA pol III Processivity –  subunit forms “sliding clamp” to keep it attached

Lagging-strand synthesis Discontinuous synthesis DNA pol III RNA primer made by primase for each Okazaki fragment All RNA primers removed and replaced by DNA DNA pol I Backbone sealed DNA ligase Termination occurs at specific site DNA gyrase unlinks 2 copies

Leading strand (continuous) Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5´ DNA ligase Lagging strand (discontinuous) RNA primer DNA polymerase I Okazaki fragment made by DNA polymerase III Primase Leading strand (continuous) 3´

Replisome Enzymes involved in DNA replication form a macromolecular assembly 2 main components Primosome Primase, helicase, accessory proteins Complex of 2 DNA pol III One for each strand

Copyright © The McGraw-Hill Companies, Inc Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. DNA polymerase III Leading strand 5´ 3´ Helicase 5´ 3´ Clamp loader DNA gyrase 5´ 3´ 5´ 3´ RNA primer First Okazaki fragment RNA primer β clamp 5´ Primase 3´ 5´ Second Okazaki fragment nears completion Single-strand binding proteins (SSB) Lagging strand 3´ Loop grows RNA primer 1. A DNA polymerase III enzyme is active on each strand. Primase synthesizes new primers for the lagging strand. 2. The “loop” in the lagging-strand template allows replication to occur 5´-to- 3´ on both strands, with the complex moving to the left. 5´ 3´ 5´ DNA polymerase III 3´ DNA polymerase I Lagging strand releases 5´ 3´ β clamp releases 3. When the polymerase III on the lagging strand hits the previously synthesized fragment, it releases the β clamp and the template strand. DNA polymerase I attaches to remove the primer.

4. The clamp loader attaches the β clamp and transfers this Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5´ 3´ Clamp loader 5´ 5´ 3´ 3´ DNA ligase patches “nick” Leading strand replicates continuously 5´ 3´ DNA polymerase I detaches after removing RNA primer 5´ 3´ Loop grows New bases 5´ 3´ 4. The clamp loader attaches the β clamp and transfers this to polymerase III, creating a new loop in the lagging-strand template. DNA ligase joins the fragments after DNA polymerase I removes the primers. 5. After the β clamp is loaded, the DNA polymerase III on the lagging strand adds bases to the next Okazaki fragment.

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Eukaryotic Replication Complicated by Larger amount of DNA in multiple chromosomes Linear structure Basic enzymology is similar Requires new enzymatic activity for dealing with ends only

Not sequence specific; can be adjusted Multiple replicons – multiple origins of replications for each chromosome Not sequence specific; can be adjusted Initiation phase of replication requires more factors to assemble both helicase and primase complexes onto the template, then load the polymerase with its sliding clamp unit Primase includes both DNA and RNA polymerase Main replication polymerase is a complex of DNA polymerase epsilon (pol ε) and DNA polymerase delta (pol δ)

Telomeres Specialized structures found on the ends of eukaryotic chromosomes Protect ends of chromosomes from nucleases and maintain the integrity of linear chromosomes Gradual shortening of chromosomes with each round of cell division Unable to replicate last section of lagging strand

Telomeres composed of short repeated sequences of DNA Telomerase – enzyme makes telomere of lagging strand using and internal RNA template (not the DNA itself) Leading strand can be replicated to the end Telomerase developmentally regulated Relationship between senescence and telomere length Cancer cells generally show activation of telomerase

DNA Repair Errors due to replication DNA polymerases have proofreading ability Mutagens – any agent that increases the number of mutations above background level Radiation and chemicals Importance of DNA repair is indicated by the multiplicity of repair systems that have been discovered

DNA Repair Falls into 2 general categories Specific repair Nonspecific Targets a single kind of lesion in DNA and repairs only that damage Nonspecific Use a single mechanism to repair multiple kinds of lesions in DNA

Photorepair Specific repair mechanism For one particular form of damage caused by UV light Thymine dimers Covalent link of adjacent thymine bases in DNA Photolyase Absorbs light in visible range Uses this energy to cleave thymine dimer

Excision repair Nonspecific repair Damaged region is removed and replaced by DNA synthesis 3 steps Recognition of damage Removal of the damaged region Resynthesis using the information on the undamaged strand as a template