1. Chapter 15: Molecular Basis of Inheritance

3. DNA is the genetic material Early in the 20th century, the identification of the molecules of inheritance loomed as a major challenge to biologists

4. The Search for the Genetic Material When T. H. Morgan’s group showed that genes are located on chromosomes, the two components of chromosomes—DNA and protein—became candidates for the genetic material The key factor in determining the genetic material was choosing appropriate experimental organisms The role of DNA in heredity was first discovered by studying bacteria and the viruses that infect them

5. DNA is Genetic Material The discovery of the genetic role of DNA began with research by Frederick Griffith in 1928 Griffith worked with two strains of a bacterium, one pathogenic and one harmless

6. Living S cells (control) Living R cells (control) Heat-killed S cells (control) Mixture of heat-killed S cells and living R cells Mouse dies Mouse healthy Living S cells RESULTS EXPERIMENT

7. Frederick Griffith (1928) -Gave mice the bacterium that causes pneumonia (2 strains) -Smooth strain killed the mice (DEADLY) -Rough strain did not kill the mice (HARMLESS) -Killed smooth strain with heat, did not kill the mice -Mixed the harmless strain and the heat-treated deadly strain -He found that the harmless rough strain, TRANSFORMED into a deadly form. -He discovered the TRANSFORMING principle which is now DNA

8. Oswald Avery(1944) -Wanted to test if protein was the transforming factor -Treated Griffith’s mixture of heat-treated deadly strain and live harmless strain with protein-destroying enzymes. -The bacterial colonies grown from the mixture were still transformed -Then treated the mixture with DNA destroying enzymes. -The bacterial colonies failed to transform. -CONCLUSION: DNA IS THE GENETIC MATERIAL OF THE CELL

9. Additional Evidence Viruses: small infectious agent that can only reproduce in living cells More evidence for DNA as the genetic material came from studies of bacteriophages Bacteriophages- viruses that infect bacteria

10. Fig. 16-3 Bacterial cell Phage head Tail sheath Tail fiber DNA 100 nm

11. Alfred Hershey and Martha Chase(1952) -Conducted experiments using viruses to be sure DNA was the transforming factor, not protein. -A virus is DNA wrapped in a protein. They reproduce by infecting a living cell with genetic materal. A virus that infects bacteria is a bacteriophage. -Concluded that the phage’s DNA entered the bacterial cell during infection but the proteins did not.

12. Fig. 16-4-3 EXPERIMENT Phage DNA Bacterial cell Radioactive protein Radioactive DNA Batch 1: radioactive sulfur ( 35 S) Batch 2: radioactive phosphorus ( 32 P) Empty protein shell Phage DNA Centrifuge Pellet Pellet (bacterial cells and contents) Radioactivity (phage protein) in liquid Radioactivity (phage DNA) in pellet

13. Additional Evidence That DNA Is the Genetic Material It was known that DNA is a polymer of nucleotides, each consisting of a nitrogenous base, a sugar, and a phosphate group In 1950, Erwin Chargaff reported that DNA composition varies from one species to the next This evidence of diversity made DNA a more credible candidate for the genetic material

14. Chargaff’s rules state that in any species there is an equal number of A and T bases, and an equal number of G and C bases

15. Sugar–phosphate backbone 5’ end Nitrogenous bases Thymine (T) Adenine (A) Cytosine (C) Guanine (G) DNA nucleotide Sugar (deoxyribose) 3’ end Phosphate

16. Building a Structural Model of DNA\ After most biologists became convinced that DNA was the genetic material, the challenge was to determine how its structure accounts for its role Maurice Wilkins and Rosalind Franklin were using a technique called X-ray crystallography to study molecular structure Franklin produced a picture of the DNA molecule using this technique

17. Franklin’s X-ray crystallographic images of DNA enabled Watson to deduce that DNA was helical The X-ray images also enabled Watson to deduce the width of the helix and the spacing of the nitrogenous bases The width suggested that the DNA molecule was made up of two strands, forming a double helix –2 strands, that are twisted

18. Nucleotides Monomers of DNA- nucleotides Made up of: 1.Pentose Sugar 2.Phosphate group 3.Nitrogenous Base

19. Nucleotides join together Joined together by covalent bonds that connect sugar of one nucleotide to the phosphate group of the next The repeating pattern of sugar-phosphate is called a sugar-phosphate “backbone”

20. Fig. 16-7a Hydrogen bond 3’ end 5’ end 3.4 nm 0.34 nm 3’ end 5’ end (b) Partial chemical structure(a) Key features of DNA structure 1 nm

21. Fig. 16-7b (c) Space-filling model

22. Watson and Crick built models of a double helix to conform to the X-rays and chemistry of DNA Franklin had concluded that there were two antiparallel sugar-phosphate backbones, with the nitrogenous bases paired in the molecule’s interior Antiparallel- run side by side in opposite directions

23. At first, Watson and Crick thought the bases paired like with like (A with A, and so on), but such pairings did not result in a uniform width

24. Watson and Crick reasoned that the pairing was more specific, dictated by the base structures They determined that adenine (A) paired only with thymine (T), and guanine (G) paired only with cytosine (C) The Watson-Crick model explains Chargaff’s rules: in any organism the amount of A = T, and the amount of G = C

25. Purine: Adenine and Guanine 2 fused rings Pyrimidine: Thymine and Cytosine 1 ring

26. Fig. 16-UN1 Purine + purine: too wide Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width consistent with X-ray data

27. Fig. 16-8 Cytosine (C) Adenine (A)Thymine (T) Guanine (G)

28. DNA replication and repair The relationship between structure and function is manifest in the double helix Watson and Crick noted that the specific base pairing suggested a possible copying mechanism for genetic material

29. 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 In DNA replication, the parent molecule unwinds, and two new daughter strands are built based on base-pairing rules How does DNA act as a template?

30. Fig. 16-9-1 A T G C TA TA G C (a) Parent molecule

31. Fig. 16-9-2 A T G C TA TA G C A T G C T A T A G C (a) Parent molecule (b) Separation of strands

32. Fig. 16-9-3 A T G C TA TA G C (a) Parent molecule AT GC T A T A GC (c) “Daughter” DNA molecules, each consisting of one parental strand and one new strand (b) Separation of strands A T G C TA TA G C A T G C T A T A G C

33. Watson and Crick’s semiconservative model of replication predicts that when a double helix replicates, each daughter molecule will have one old strand (derived or “conserved” from the parent molecule) and one newly made strand Competing models were the conservative model (the two parent strands rejoin) and the dispersive model (each strand is a mix of old and new)

34. Fig. 16-10 Parent cell First replication Second replication (a) Conservative model (b) Semiconserva- tive model (c) Dispersive model

35. DNA Replication The copying of DNA is remarkable in its speed and accuracy More than a dozen enzymes and other proteins participate in DNA replication

36. Getting Started 1. Replication begins at special sites called origins of replication, where the two DNA strands are separated, opening up a replication “bubble” A eukaryotic chromosome may have hundreds or even thousands of origins of replication Replication proceeds in both directions from each origin, until the entire molecule is copied

37. Fig. 16-12 Origin of replication Parental (template) strand Daughter (new) strand Replication fork Replication bubble Two daughter DNA molecules (a) Origins of replication in E. coli Origin of replicationDouble-stranded DNA molecule Parental (template) strand Daughter (new) strand Bubble Replication fork Two daughter DNA molecules (b) Origins of replication in eukaryotes 0.5 µm 0.25 µm Double- stranded DNA molecule

38. Fig. 16-12b 0.25 µm Origin of replicationDouble-stranded DNA molecule Parental (template) strand Daughter (new) strand Bubble Replication fork Two daughter DNA molecules (b) Origins of replication in eukaryotes

39. Fig. 16-13 Topoisomerase Helicase Primase Single-strand binding proteins RNA primer 5’5’ 5’5’ 5’5’3’3’ 3’3’ 3’3’

40. 2. At the end of each replication bubble is a replication fork, a Y-shaped region where new DNA strands are elongating

41. Helicases are enzymes that untwist the double helix at the replication forks Single-strand binding protein binds to and stabilizes single-stranded DNA until it can be used as a template Topoisomerase corrects “overwinding” ahead of replication forks by breaking, swiveling, and rejoining DNA strands Enzymes/Proteins Involved in the beginning…. http://www.youtube.com/watch? v=EYGrElVyHnU

42. Antiparallel Elongation The antiparallel structure of the double helix (two strands oriented in opposite directions) affects replication DNA polymerases add nucleotides only to the free 3’  end of a growing strand; therefore, a new DNA strand can elongate only in the 5’  to  3’  direction

43. Synthesizing a New DNA Strand Enzymes called DNA polymerases catalyze the elongation of new DNA at a replication fork (adding new nucleotides) The rate of elongation is about per second in bacteria and 50 per second in human cells500 nucleotides

44. Along one template strand of DNA, the DNA polymerase synthesizes a leading strand continuously, moving toward the replication fork

45. DNA polymerases can only add nucleotides to a FREE 3’ end THERE IS NO FREE 3’ end on lagging strand Uses a short “primer” to initiate synthesis The initial nucleotide strand is a short RNA primer- used on lagging strand

46. An enzyme called primase can start an RNA chain from scratch and adds RNA nucleotides one at a time using the parental DNA as a template The primer is short (5–10 nucleotides long), and the 3’ end serves as the starting point for the new DNA strand Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

47. Fig. 16-13 Topoisomerase Helicase Primase Single-strand binding proteins RNA primer 5’5’ 5’5’ 5’5’3’3’ 3’3’ 3’3’

48. To elongate the other new strand, called the lagging strand, DNA polymerase 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 joined together by DNA ligase

49. Table 16-1

50. Fig. 16-17 Overview Origin of replication Leading strand Lagging strand Overall directions of replication Leading strand Lagging strand Helicase Parental DNA DNA pol III PrimerPrimase DNA ligase DNA pol III DNA pol I Single-strand binding protein 5 3 5 5 5 5 3 3 3 3 1 3 2 4

51. Proofreading and Repairing DNA DNA polymerases proofread newly made DNA, replacing any incorrect nucleotides In mismatch repair of DNA, repair enzymes correct errors in base pairing DNA can be damaged by chemicals, radioactive emissions, X-rays, UV light, and certain molecules (in cigarette smoke for example) In nucleotide excision repair, a nuclease cuts out and replaces damaged stretches of DNA Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

52. Fig. 16-18 Nuclease DNA polymerase DNA ligase

53. Replicating the Ends of DNA Molecules Limitations of DNA polymerase create problems for the linear DNA of eukaryotic chromosomes The usual replication machinery provides no way to complete the 5’  ends, so repeated rounds of replication produce shorter DNA molecules Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

54. Fig. 16-19 Ends of parental DNA strands Leading strand Lagging strand Last fragment Previous fragment Parental strand RNA primer Removal of primers and replacement with DNA where a 3 end is available Second round of replication New leading strand New lagging strand Further rounds of replication Shorter and shorter daughter molecules 5 3 3 3 3 3 5 5 5 5 http://www.youtube.com/watch? v=AJNoTmWsE0s http://www.youtube.com/watch? v=x1zw6uRxKYU

55. Eukaryotic chromosomal DNA molecules have at their ends nucleotide sequences called telomeres Telomeres do not prevent the shortening of DNA molecules, but they do postpone the erosion of genes near the ends of DNA molecules It has been proposed that the shortening of telomeres is connected to aging Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

56. If 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 Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

57. The shortening of telomeres might protect cells from cancerous growth by limiting the number of cell divisions There is evidence of telomerase activity in cancer cells, which may allow cancer cells to persist Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

58. The bacterial chromosome is a double- stranded, circular DNA molecule associated with a small amount of protein Eukaryotic chromosomes have linear DNA molecules associated with a large amount of protein In a bacterium, the DNA is “supercoiled” and found in a region of the cell called the nucleoid Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

59. Chromatin is a complex of DNA and protein, and is found in the nucleus of eukaryotic cells Histones are proteins that are responsible for the first level of DNA packing in chromatin Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

60. Fig. 16-21a DNA double helix (2 nm in diameter) Nucleosome (10 nm in diameter) Histones Histone tail H1 DNA, the double helixHistones Nucleosomes, or “beads on a string” (10-nm fiber)

61. Fig. 16-21b 30-nm fiber Chromatid (700 nm) LoopsScaffold 300-nm fiber Replicated chromosome (1,400 nm) 30-nm fiber Looped domains (300-nm fiber) Metaphase chromosome