DNA: The Molecular Basis of Inheritance

1. Chapter 16 The Molecular Basis of Inheritance Edited by L. Bridge 2011

2. DNA, the substance of inheritance • Is the most celebrated molecule of our time • Chromosomes are composed of atoms arranged into molecules = field of “Molecular Genetics" • Hereditary information is encoded in the chemical language of DNA and reproduced in all the cells of your body • It is the DNA “program” that directs the development of many different types of traits

3. Concept 16.1: DNA is the genetic material • Early in the 20th century • The identification of the molecules of inheritance loomed as a major challenge to biologists • What was "The Language of Life”? early studies asked  • Proteins? (made up of 20 different AA’s) or DNA (made of only 4 bases)?  No one was sure back then...

4. The Nature of DNA: history • 1869- DNA First isolated by Swiss biologist, Friedrich Miescher, called it “nuclein” • His procedure: • http://www.dnalc.org/view/16003-Miescher-and-the-isolation-of-DNA.html • 1914- Robert Feulgenfound DNA staining with fuchsin (red dye)

5. Sugar-phosphate backbone Nitrogenous bases 5 end CH3 O– 5 O H CH2 O P O O 1 4 N O– N H H H H H O 2 3 H Thymine (T) O H H CH2 O O P N O N H O– H N H H H N N H H Adenine (A) H H O H N CH2 O O P H O O– N H N H H H O H Cytosine (C) O 5 H CH2 O P N O O O 1 4 O– H N H Phosphate H H N 2 H 3 DNA nucleotide N H OH N H Sugar (deoxyribose) 3 end H Figure 16.5 Guanine (G) The Nature of DNA • 1920- P.A. Levene- biochemically broke down DNA into: • 5-carbon sugar (deoxyribose) • Phosphate • 4 Nitrogen bases adenine/guanine= purines; thymine/cytosine= pyrimidines • Each unit = "Nucleotide“ • At the time, Levene (mistakenly) assumed a 1:1:1:1 ratio between the bases

6. Erwin Chargaff(1905-2002) • Analyzed the base composition of DNA from a number of different organisms: • In 1947, Chargaff reported: • DNA “Base Pairing Rules” • DNA composition varies from one species to the next http://www.dnai.org/text/mediashowcase/index2.html?id=569

7. The Search for the Genetic Material: Scientific Inquiry • The role of DNA in heredity • Was first worked out by studying bacteria and the viruses that infect them

8. Evidence That DNA Can Transform Bacteria • 1928, Frederick Griffithwas studying Streptococcus pneumoniae • A bacterium that causes pneumonia in mammals • He worked with two strains of the bacterium • A pathogenic strain (S) and a nonpathogenic (R) strain Streptococcus pneumoniae (pneumococci) growing as colonies on the surface of a culture medium. Left: The presence of a capsule around the bacterial cells gives the colonies a glistening, smooth (S) appearance. Right: Pneumococci lacking capsules have produced these rough (R) colonies. (Research photographs of Dr. Harriet Ephrussi-Taylor, courtesy of The Rockefeller University.)

9. EXPERIMENT RESULTS CONCLUSION Bacteria of the “S” (smooth) strain of Streptococcus pneumoniae are pathogenic because they have a capsule that protects them from an animal’s defense system. Bacteria of the “R” (rough) strain lack a capsule and are nonpathogenic. Frederick Griffith injected mice with the two strains as shown below: Living S (control) cells Living R (control) cells Heat-killed (control) S cells Mixture of heat-killed S cells and living R cells Mouse dies Mouse healthy Mouse healthy Mouse dies Living S cells are found in blood sample. Figure 16.2 Griffith concluded that the living R bacteria had been transformed into pathogenic S bacteria by an unknown, heritable substance from the dead S cells. “Sugar-coated microbes” • Griffith found that when he mixed heat-killed remains of the pathogenic strain • With living cells of the nonpathogenic strain, some of these living cells became pathogenic

10. The “Transformation Factor” From Griffith’s work, O.T. Avery:  • 1943, Avery teamed up with MacCleod and McCarty (Rockefeller Institute), and showed that DNA was the genetic material found in extracts from killed virulent bacteria that could make the living , harmless bacteria into the virulent type, and called the phenomenon “transformation” • defined as a “change in genotype and phenotype due to the assimilation of external DNA by a cell”

11. Further Evidence for DNA Alfred Mirsky – • Isolated DNA in the late 1940’s • Found that all somatic cells (of a species) contain same amount of DNA (gametes contain 1/2) • This evidence of molecular diversity among species • Made DNA a more credible candidate for the genetic material

12. Phage head Tail Tail fiber DNA 100 nm Bacterial cell Evidence That Viral DNA Can Program Cells • Additional evidence for DNA as the genetic material came from studies of a virus that infects bacteria • Viruses that infect bacteria, bacteriophages are widely used as tools by researchers in molecular genetics

13. The Bacteriophage Experiments Max Delbrück and Salvador Luria • Developed a simple model system using phage for studying how genetic information is transferred to host bacterial cells. • Studied a type of bacterial virus called the T phage that consists of a protein coat containing DNA. • 7 different phages attack E.coli bacteria, named T1 - T7 • Life cycle of the T2 phage narrated animation

14. Alfred Hershey and Martha Chase • Performed the famous "blender experiment“ showing that DNA, rather than protein, is the genetic material of a phage known as T2 • Advantages: small, cheap, easy to maintain in lab, replicated in 25 minutes

15. Hershey and Chase • Viruses made of: (1) protein coat “capsid” ; and (2) DNA • The question was, which part carried the info? • Labeled phage DNA with 32P, and the capsid protein with 35S. • Next allowed the labeled phage to begin to infect a bacterial colony. • In the infection process, the phage land on the bacterial wall and then inject their genetic material into the host cell. • Hershey allowed this to occur, but then at the crucial moment he whirred them in a Waring Blender, which he had discovered produced just the right shearing force to tear the phage particles from the bacterial walls while not rupturing the bacteria. • Upon examining the bacteria, Hershey found that only phage DNA, no detectable protein, had been inserted into them. • By process of elimination, they found it was indeed DNA that was inserted into the host cell, and thus was the “information” molecule. http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html#

16. The Nobel Prize in Physiology or Medicine 1969 Max Delbrück, Alfred D. Hershey, and Salvador E. Luria "for their discoveries concerning the replication mechanism and the genetic structure of viruses"

17. Building a Structural Model of DNA: Scientific Inquiry • Once most biologists were convinced that DNA was the genetic material, the challenge was to determine how the structure of DNA could account for its role in inheritance http://www.pbs.org/wgbh/nova/genome/dna.html What must DNA do? • Replicate to be passed on to the next generation • Store information • Undergo mutations to provide genetic diversity

18. Linus Pauling • As a result of studying X-ray photographs and constructing molecular models, Linus Pauling (and Robert Cory, in 1951) proposed that the protein structures were either in the form of an alpha helix or the beta pleated sheet. • the only person who has won two undivided Nobel Prizes

19. Franklin’s X-ray diffraction Photograph of DNA (b) (a) Rosalind Franklin Maurice Wilkins and Rosalind Franklin Were using a technique called X-ray crystallography to study molecular structure • Rosalind Franklin • Produced a picture of the DNA molecule using this technique

20. Figure 16.1 Overview: Life’s Operating Instructions In 1953, James Watson and Francis Crick shook the world • With an elegant double-helical model for the structure of deoxyribonucleic acid, or DNA

21. Watson and Crick deduced that DNA was a double helix 5¢ end 3¢ end purine base pyrimidine base P phosphate T A P S S S T A P P T C G S S 5¢ end 3¢ end P P P T T A 5¢ 2¢ 3¢ A S S S 1¢ 1¢ 4¢ 4¢ S S S P 2¢ 3¢ 5¢ P C G P S S S 5¢ C P O P T A 1¢ C C S 4¢ S S S S C C 3¢ 2¢ P deoxyribose 3¢ end 5¢ end a. Double helix b. Ladder structure c. One pair of bases

22. Figure 16.8 H N O H CH3 N N N N H Sugar N N O Sugar Adenine (A) Thymine (T) H O N H N N N H N Sugar N N N O H Sugar H Cytosine (C) Guanine (G) DNA Structure • The nitrogenous bases • Are paired in specific combinations: adenine with thymine, and cytosine with guanine • Dictated by the structure of the bases • Each base pair forms a different number of hydrogen bonds • Adenine and thymine form two bonds, cytosine and guanine form three bonds

23. 5 end O OH Hydrogen bond P 3 end –O O OH O A T O CH2 O O P O –O O– O P O H2C O O G C O O CH2 O P O O O –O O– O– O– O P P P O O O H2C O O O O C G O O CH2 O P –O O H2C A T O O CH2 OH 3 end (b) Partial chemical structure 5 end Figure 16.7b DNA Structure • Was composed of two antiparallel sugar-phosphate backbones, with the nitrogenous bases paired in the molecule’s interior

24. Narrated animation of DNA structure Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). You may see blank slides in the “Normal” or “Slide Sorter” views. All animations will appear after viewing in Presentation Mode and playing each animation. Most animations will require the latest version of the Flash Player, which is available at http://get.adobe.com/flashplayer.

25. DNA Replication • Concept 16.2: Many proteins work together in DNA replication and repair • Since the two strands of DNA are complementary • Each strand acts as a template for building a new strand in replication • http://highered.mcgraw-hill.com/sites/dl/free/0072835125/126997/animation16.html

26. T A A A A T A T T T T A G C G C C C C G G G G C A A T T T T A A A A T T T A A A A T A T T T T A C G G G G C G C C C C G (a) The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. (c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand. (d) The nucleotides are connected to form the sugar-phosphate backbones of the new strands. Each “daughter” DNA molecule consists of one parental strand and one new strand. (b) The first step in replication is separation of the two DNA strands. Figure 16.9 a–d Base Pairing to a Template Strand • In DNA replication • The parent molecule unwinds, and two new daughter strands are built based on base-pairing rules

27. First replication Second replication Parent cell Conservative model. The two parental strands reassociate after acting as templates for new strands, thus restoring the parental double helix. Semiconservative model. The two strands of the parental molecule separate, and each functions as a template for synthesis of a new, comple- mentary strand. Dispersive model. Each strand of both daughter mol- ecules contains a mixture of old and newly synthesized DNA. Figure 16.10 a–c DNA replication is semiconservative • Each of the two new daughter molecules will have one old strand, derived from the parent molecule, and one newly made strand (a) Experiments performed by Meselson and Stahl Supported the semiconservative model of DNA replication (1957) http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html# (b) (c)

28. EXPERIMENT Matthew Meselson and Franklin Stahl cultured E. coli bacteria for several generations on a medium containing nucleotide precursors labeled with a heavy isotope of nitrogen, 15N. The bacteria incorporated the heavy nitrogen into their DNA. The scientists then transferred the bacteria to a medium with only 14N, the lighter, more common isotope of nitrogen. Any new DNA that the bacteria synthesized would be lighter than the parental DNA made in the 15N medium. Meselson and Stahl could distinguish DNA of different densities by centrifuging DNA extracted from the bacteria. Bacteria cultured in medium containing 15N Bacteria transferred to medium containing 14N 3 2 4 1 RESULTS Less dense DNA sample centrifuged after 40 min (after second replication) DNA sample centrifuged after 20 min (after first replication) Figure 16.11 More dense The bands in these two centrifuge tubes represent the results of centrifuging two DNA samples from the flask in step 2, one sample taken after 20 minutes and one after 40 minutes. Meselson and Stahl experiment (1957) CONCLUSION: Meselson and Stahl concluded that DNA replication follows the semiconservative model by comparing their result to the results predicted by each of the three models in Figure 16.10. The first replication in the 14N medium produced a band of hybrid (15N–14N) DNA. This result eliminated the conservative model. A second replication produced both light and hybrid DNA, a result that eliminated the dispersive model and supported the semiconservative model.

29. DNA Replication: A Closer Look • The copying of DNA • Is remarkable in its speed and accuracy • More than a dozen enzymes and other proteins • Participate in DNA replication http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120076/bio23.swf::How%20Nucleotides%20are%20Added%20in%20DNA%20Replication

30. Getting Started: Origins of Replication • The replication of a DNA molecule • Begins at special sites called origins of replication, where the two strands are separated

31. Origin of replication Parental (template) strand 0.25 µm Daughter (new) strand 1 Replication begins at specific sites where the two parental strands separate and form replication bubbles. Bubble Replication fork 2 The bubbles expand laterally, as DNA replication proceeds in both directions. 3 Eventually, the replication bubbles fuse, and synthesis of the daughter strands is complete. Two daughter DNA molecules (a) In eukaryotes, DNA replication begins at many sites along the giant DNA molecule of each chromosome. (b) In this micrograph, three replication bubbles are visible along the DNA of a cultured Chinese hamster cell (TEM). Figure 16.12 a, b • A eukaryotic chromosome • May have hundreds or even thousands of replication origins

32. New strand Template strand 3 end 5 end 3 end 5 end Sugar A T A T Base Phosphate C G C G G G C C A T A T OH P P P P 3 end Pyrophosphate P C C OH 2 P 5 end 5 end Figure 16.13 Elongating a New DNA Strand • Elongation of new DNA at a replication fork • Is catalyzed by enzymes called DNA polymerases, which add nucleotides to the 3 end of a growing strand • The Energetics of DNA ReplicationPowered by extra phosphates (triphosphates) ex: adenine is added to DNA strand as"deoxyadenosine triphosphate " (dATP) and guanine added as "deoxyguanosine triphosphate" (dGTP) • As the phosphate bonds are made, to attach the nucleotide to the DNA molecule, the extra phosphates are removed (thus releasing energy to power the process) Nucleoside triphosphate

33. Antiparallel Elongation • How does the antiparallel structure of the double helix affect replication? • DNA is “built” in a 5’ to 3’ direction • DNA polymerases add nucleotides • Only to the free 3end of a growing strand

34. Leading and Lagging strands • Along one template strand of DNA, the leading strand • DNA polymerase III can synthesize a complementary strand continuously (one nucleotide at a time), moving toward the replication fork • To elongate the other new strand of DNA, the lagging strand • DNA polymerase III must work in the direction away from the replication fork • The lagging strand • Is synthesized as a series of segments called Okazaki fragments, which are then joined together by DNA ligase (enzyme)

35. 4 2 3 1 DNA pol Ill elongates DNA strands only in the 5 3 direction. 3 One new strand, the leading strand, can elongate continuously 5 3 as the replication fork progresses. 5 Parental DNA 5 3 Okazaki fragments The other new strand, the lagging strand must grow in an overall 3 5 direction by addition of short segments, Okazaki fragments, that grow 5 3 (numbered here in the order they were made). 2 3 1 5 DNA pol III Template strand DNA ligase joins Okazaki fragments by forming a bond between their free ends. This results in a continuous strand. Leading strand Lagging strand 3 1 2 Template strand DNA ligase Figure 16.14 Overall direction of replication • Synthesis of leading and lagging strands during DNA replication The whole process, animated http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120076/micro04.swf::DNA%20Replication%20Fork

36. Priming DNA Synthesis • DNA polymerases cannot initiate the synthesis of a polynucleotide • They can only add nucleotides to the 3 end • The initial nucleotide strand • Is an RNA or DNA primer

37. Only one primer is needed for synthesis of the leading strand • But for synthesis of the lagging strand, each Okazaki fragment must be primed separately • View animations of the whole process here: http://www.mcb.harvard.edu/Losick/images/TromboneFINALd.swf http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html#

38. 7 2 3 6 5 1 4 3 5 3 5 Templatestrand DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. Primase joins RNA nucleotides into a primer. RNA primer 3 5 3 1 5 After reaching the next RNA primer (not shown), DNA pol III falls off. Okazakifragment 3 3 5 1 5 After the second fragment is primed. DNA pol III adds DNAnucleotides until it reaches the first primer and falls off. 5 3 3 2 5 1 DNA pol 1 replaces the RNA with DNA, adding to the 3 end of fragment 2. 5 3 3 5 2 1 DNA ligase forms a bond between the newest DNAand the adjacent DNA of fragment 1. The lagging strand in this region is nowcomplete. 5 3 3 2 5 1 Figure 16.15 Overall direction of replication

39. Table 16.1 Other Proteins That Assist DNA Replication • Helicase, topoisomerase, single-strand binding protein • Are all proteins that assist DNA replication

40. Overall direction of replication Lagging strand Leading strand Helicase unwinds the parental double helix. Origin of replication 1 Molecules of single- strand binding protein stabilize the unwound template strands. The leading strand is synthesized continuously in the 5 3 direction by DNA pol III. 2 3 Leading strand Lagging strand OVERVIEW DNA pol III Leading strand 5 Replication fork DNA ligase DNA pol I 3 Primase 2 Parental DNA Lagging strand DNA pol III 1 Primer 3 Primase begins synthesis of RNA primer for fifth Okazaki fragment. 4 3 5 4 DNA pol I removes the primer from the 5 end of the second fragment, replacing it with DNA nucleotides that it adds one by one to the 3 end of the third fragment. The replacement of the last RNA nucleotide with DNA leaves the sugar- phosphate backbone with a free 3 end. DNA pol III is completing synthesis of the fourth fragment, when it reaches the RNA primer on the third fragment, it will dissociate, move to the replication fork, and add DNA nucleotides to the 3 endof the fifth fragment primer. DNA ligase bonds the 3 end of the second fragment to the 5 end of the first fragment. 5 6 7 Figure 16.16 Link to animations of processes discussed in this chapter • A summary of DNA replication

41. The DNA Replication Machine as a Stationary Complex • The various proteins that participate in DNA replication • Form a single large complex, a DNA replication “machine” • The DNA replication machine • Is probably stationary during the replication process

42. Proofreading and Repairing DNA • DNA polymerases proofread newly made DNA • Replacing any incorrect nucleotides • http://highered.mcgraw-hill.com/sites/dl/free/0072835125/126997/animation18.html • In mismatch repair of DNA • Repair enzymes correct errors in base pairing

43. 1 2 4 A thymine dimer distorts the DNA molecule. • In nucleotide excision repair • Enzymes cut out and replace damaged stretches of DNA A nuclease enzyme cuts the damaged DNA strand at two points and the damaged section is removed. Nuclease DNA polymerase Repair synthesis by a DNA polymerase fills in the missing nucleotides. 3 DNA ligase DNA ligase seals the Free end of the new DNA To the old DNA, making the strand complete. Figure 16.17

44. 1 µm Figure 16.19 Telomeres • Eukaryotic chromosomal DNA molecules • Have at their ends nucleotide sequences, called telomeres, that postpone the erosion of genes near the ends of DNA molecules

45. Telomeres: the ends of the chromosomes • If the chromosomes of germ cells became shorter in every cell cycle • Essential genes would eventually be missing from the gametes they produce • An enzyme called telomerase • Catalyzes the lengthening of telomeres in germ cells • In somatic cells, that MITOTICALLY divide, telomeres are thought to be involved in cell aging, as the ends wear down (like the wick on a candle) • http://highered.mcgraw-hill.com/sites/dl/free/0072835125/126997/animation19.html