Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 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 2

3 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

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6 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

7 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

8 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

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

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11 DNA Structure DNA is a nucleic acid Composed of nucleotides –5-carbon sugar called deoxyribose –Phosphate group (PO 4 ) Attached to 5′ carbon of sugar –Nitrogenous base Adenine, thymine, cytosine, guanine –Free hydroxyl group (—OH) Attached at the 3′ carbon of sugar

12 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Purines Pyrimidines Adenine Guanine NH 2 C C N N N C H N C CH O H H OC NC H N C H C H O O C N C H N C H3CH3C C H H O O C N C H N C H C H C C N N N C H N C CH H Nitrogenous Base 4´4´ 5´5´ 1´1´ 3´3´2´2´ O P O–O– –O–O Phosphate group Sugar Nitrogenous base O CH 2 N N O N NH 2 OH in RNA Cytosine (both DNA and RNA) Thymine (DNA only) Uracil (RNA only) OH H in DNA

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 13 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Base CH 2 O 5´5´ 3´3´ O P O OH CH 2 –O–OO C Base O PO 4 Phosphodiester bond

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) 14

15 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

16 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 17 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5´5´ 3´3´ P P P P OH 5-carbon sugar Nitrogenous base Phosphate group Phosphodiester bond O O O O 4´4´ 5´5´ 1´1´ 3´3´ 2´2´ 4´4´ 5´5´ 1´1´ 3´3´ 2´2´ 4´4´ 5´5´ 1´1´ 3´3´ 2´2´ 4´4´ 5´5´ 1´1´ 3´3´ 2´2´

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Complementarity of bases A forms 2 hydrogen bonds with T G forms 3 hydrogen bonds with C Gives consistent diameter 19 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. A H Sugar T G C N H N O H CH 3 H H N N N H N N N H H H N O H H H N NH N N H N N Hydrogen bond Hydrogen bond

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

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

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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 23 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5´5´ 3´3´ 5´5´ 5´5´ 5´5´ 3´3´ 3´3´ RNA polymerase makes primerDNA polymerase extends primer

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 24

25 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

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 26 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Supercoiling Replisomes No Supercoiling Replisomes DNA gyrase

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 27

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29 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

30 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 31

32 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

33 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

34 DNA Repair Falls into 2 general categories 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

35 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

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Excision repair Nonspecific repair Damaged region is removed and replaced by DNA synthesis 3 steps 1.Recognition of damage 2.Removal of the damaged region 3.Resynthesis using the information on the undamaged strand as a template 37

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