Chapter 16

1. Chapter 16 The Molecular Basis of Inheritance

2. 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

3. DNA, the substance of inheritance • Is the most celebrated molecule of our time • 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

4. 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

5. 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

6. Evidence That DNA Can Transform Bacteria • Frederick Griffith was studying Streptococcus pneumoniae (A bacterium that causes pneumonia in mammals) • He worked with two strains of the bacterium • A pathogenic strain [Smooth strain (S); encapsulated with a polysaccharide coat] and a nonpathogenic strain [Rough strain ( R); not encapsulated].

7. 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 EXPERIMENT 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 Heat-killed (control) S cells Mixture of heat-killed S cells and living R cells Living R (control) cells RESULTS Mouse dies Mouse healthy Mouse healthy Mouse dies Living S cells are found in blood sample. Griffith concluded that the living R bacteria had been transformed into pathogenic S bacteria by anunknown, heritable substance from the dead S cells. Figure 16.2 CONCLUSION

8. Griffith called the phenomenon transformation • Now defined as a change in genotype and phenotype due to the assimilation of external DNA by a cell

9. Evidence That Viral DNA Can Program Cells • Additional evidence for DNA as the genetic material Came from studies of a virus (T2 bacteriophage) that infects bacteria (Escherichiacoli, enteric bacteria)

10. Phage head Tail Tail fiber DNA 100 nm Bacterial cell Figure 16.3 • Viruses that infect bacteria, bacteriophages • Are widely used as tools by researchers in molecular genetics

11. Alfred Hershey and Martha Chase • Performed experiments showing that DNA is the genetic material of a phage known as T2

12. EXPERIMENT In their famous 1952 experiment, Alfred Hershey and Martha Chase used radioactive sulfur and phosphorus to trace the fates of the protein and DNA, respectively, of T2 phages that infected bacterial cells. CONCLUSION 3 2 4 1 Agitated in a blender to separate phages outside the bacteria from the bacterial cells. Centrifuged the mixture so that bacteria formed a pellet at the bottom of the test tube. Measured the radioactivity in the pellet and the liquid Mixed radioactively labeled phages with bacteria. The phages infected the bacterial cells. Radioactive protein Empty protein shell Radioactivity (phage protein) in liquid Phage Bacterial cell RESULTS DNA Batch 1: Phages were grown with radioactive sulfur (35S), which was incorporated into phage protein (pink). Phage DNA Centrifuge Radioactive DNA Pellet (bacterial cells and contents) Batch 2: Phages were grown with radioactive phosphorus (32P), which was incorporated into phage DNA (blue). Centrifuge Radioactivity (phage DNA) in pellet Pellet Phage proteins remained outside the bacterial cells during infection, while phage DNA entered the cells. When cultured, bacterial cells with radioactive phage DNA released new phages with some radioactive phosphorus. Figure 16.4 Hershey and Chase concluded that DNA, not protein, functions as the T2 phage’s genetic material. The Hershey and Chase experiment

13. Additional Evidence That DNA Is the Genetic Materia • Prior to the 1950s, it was already known that DNA • Is a polymer of nucleotides, each consisting of three components: a nitrogenous base, a sugar, and a phosphate group 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 Sugar (deoxyribose) 3 end DNA nucleotide 2 H 3 N Figure 16.5 H OH N H H Guanine (G)

14. Erwin Chargaff analyzed the base composition of DNA • From a number of different organisms • In 1947, Chargaff reported • That DNA composition varies from one species to the next; i.e. species-specific, in every species he studied, there was a regularity in base ratios. • The number of adenine (A) equals the number of thymines (T), and the number of guanines (G) equal the number of cytosines (C). • the A=T and G=C equalities became known as Chargaff’s rules. • This evidence of molecular diversity among species • Made DNA a more credible candidate for the genetic material

15. 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

16. Franklin’s X-ray diffraction Photograph of DNA (b) (a) Rosalind Franklin Figure 16.6 a, b • 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

17. G C A T T A 1 nm C G 3.4 nm C G A T G C T A T A A T T A G C 0.34 nm A T Figure 16.7a, c (a) Key features of DNA structure (c) Space-filling model • Watson and Crick deduced that DNA was a double helix • Through observations of the X-ray crystallographic images of DNA

18. Franklin had concluded that DNA • Was composed of two antiparallel sugar-phosphate backbones, with the nitrogenous bases paired in the molecule’s interior • The nitrogenous bases • Are paired in specific combinations: adenine with thymine, and cytosine with guanine. They were pointed toward the interior because they are hydrophobic.

19. 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

20. Watson and Crick reasoned that there must be additional specificity of pairing • 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

21. From X-ray photo of DNA taken by Rosalind Franklin, Watson and Crick deduced that : • DNA is a helix with a uniform width of 2nm suggesting it has two strands. • Purine and pyrimidine bases are stacked 0.32 nm apart. • The helix makes one full turn every 3.4 nm along its length. • There are 10 layers of nitrogenous base pairs in each turn of the helix.

22. Based on that, they put the base-pairing rule: • A must pair with T; Likewise, C must pair with G; There amount in a given DNA molecule will be nearly the same. • Bases from specific pair on one strand complement that on the other. • The sequence of bases can be highly variable which makes it suitable for coding genetic material.

23. 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 Figure 16.8 Cytosine (C) Guanine (G)

24. Concept 16.2: Many proteins work together in DNA replication and repair • The relationship between structure and function • Is manifest in the double helix

25. The Basic Principle: Base Pairing to a Template Strand • Since the two strands of DNA are complementary • Each strand acts as a template for building a new strand in replication

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 • In DNA replication • The parent molecule unwinds, and two new daughter strands are built based on base-pairing rules

27. Models of DNA replication • 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 • Question… is that true for all DNA replication product?

28. 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 A B c

29. Experiment performed by Meselson and Stahl • Supported the semiconservative model of DNA replication • 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.

30. 3 2 4 1 RESULTS EXPERIMENT Bacteria transferred to medium containing 14N Bacteria cultured in medium containing 15N Less dense DNA sample centrifuged after 20 min (after first replication) DNA sample centrifuged after 40 min (after second replication) 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. Figure 16.11

31. 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.

32. First replication Second replication Conservative model Semiconservative model Dispersive model Same bands New band generated Stays as one band

33. DNA Replication: A Closer Look • The copying of DNA (DNA replication) • The process of DNA replication is; • A.Complex: the helical molecule must untwist while it copies its own strands. More than a dozen enzymes and other proteins participate in DNA replication • B.Very rapid: in bacteria 500 nucleotides are added/sec. In human cells, 50 nucleotides /sec are added. • C.Accurate: only about one in a billion nucleotides is incorrectly paired.

34. Getting Started: Origins of Replication • The replication of a DNA molecule • Begins at special sites called origins of replication, have a specific sequence of nucleotides and where the two strands are separated. • specific proteins required to initiate replication. • The DNA double helix opens at the origin of replication and replication fork (The Y-shaped region of replicating DNA molecules where new strands are growing) spread in both directions, initiating a replication bubble. • Bacterial DNA have oneorigin of replication. • Eukaryotic DNA have hundreds or thousands of origins of replication.

35. 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

36. Elongation of new DNA at a replication fork Synthesis of the new DNA strand iscatalyzed by enzymes called DNA polymerases, which add nucleotides to the 3 end of a growing strand, grows in 5’—3’ direction. Each nucleotide that is added is actually a nucleoside (a sugar and a base) with three phosphate groups.

37. 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 Elongating a New DNA Strand • Elongation of new DNA at a replication fork needs energy • Energy required for building new strands comes from the hydrolysis of nucleoside triphosphate. Figure 16.13 DNA polymerase catalysis the addition of a nucleoside triphosphate to the 3’ end of a growing DNA strand DNA Polymerase Nucleoside triphosphate

38. Antiparallel Elongation • How does the antiparallel structure of the double helix affect replication?

39. DNA polymerases add nucleotides • Only to the free 3end of a growing strand • Along one template strand of DNA, the leading strand (the DNA strand which is synthesized as a single polymer in the 5’  3’ direction towards the replication fork) is synthesized. • DNA polymerase III can synthesize a complementary strand (leading strand) continuously, moving toward the replication fork.

40. To elongate the other new strand of DNA, the lagging strand(the DNA strand that is discontinuously synthesized against the overall direction of replication) is synthesized. • 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

41. 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

42. Priming DNA Synthesis • DNA polymerases cannot initiate the synthesis of a polynucleotide, they can only add nucleotides to the 3 end of already existing chain. • Before new DNA strand can be form, there must be a small pre-existing primers to start the addition of new nucleotides. • Primer is a short RNA segments that are complementary to a DNA segments and that is necessary to begin DNA replication. • Primers are polymerized by by an enzyme called: primase. • Only one primer is necessary for replication of the leading strand, but many are required to replicate the lagging strand. • An RNA primer must initiate the synthesis of each Okazaki fragments. • DNA polymerase removes the RNA primer and replaces it with DNA. • DNA ligase joins these fragments forming a continuous DNA strand

43. In summary; there are three types of proteins that function in DNA synthesis; • DNA polymerase • DNA ligase • DNA primase

44. Other proteins assist DNA replication There are three types of proteins also involved in DNA replication: • Helicase:enzyme which unwinds parental double helix at the replication fork (Strand separation)causing a tight twist for the DNA strand. • Topoisomerase;helps relief this strain (the tight twist). • Single-strand binding proteins:proteins which keep the separated strands apart and stabilize the unwound DNA until new complementary strand is synthesized.

45. 7 2 3 6 5 1 4 3 5 3 5 DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. Primase joins RNA nucleotides into a primer. Templatestrand 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

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

47. 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. 2 The leading strand is synthesized continuously in the 5 3 direction by DNA pol III. 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 ligase bonds the 3 end of the second fragment to the 5 end of the first fragment. 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. 5 6 7 Figure 16.16 • A summary of DNA replication

48. 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.

49. Proofreading and Repairing DNA • DNA replication is very accurate. Paring errors occurs at a rate of one to 10 billions in a complete DNA molecule. • DNA polymerases proofread newly made DNA by replacing any incorrect nucleotides • In Mismatchrepair of DNA: Repair enzymes correct errors in base pairing (DNA synthesis) In bacteria:DNA polymerase proofread each newly added nucleotide against its template. If polymerase founds an incorrectly paired nucleotide, the enzyme removes it and replaces it before continuing with synthesis. In eukaryotes: additional proteins as well as polymerase act in the mismatch repair.

50. 1 2 4 A thymine dimer distorts the DNA molecule. • Nucleotide excision repair : Enzymes cut out and replace damaged stretches of DNA. • The damaged segment is excised (cut) by an enzyme (Nuclease), and the remaining gap is filled in by base-pairing nucleotides. • DNA polymerase and Ligase are enzymes that catalyze the filling-in process. 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 3 DNA ligase DNA ligase seals the Free end of the new DNA To the old DNA, making the strand complete. Figure 16.17