1. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings PowerPoint Lectures for Biology, Seventh Edition Neil Campbell and Jane Reece Lectures by Chris Romero Chapter 16 The Molecular Basis of Inheritance

2. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Overview: Life’s Operating Instructions In 1953, James Watson and Francis Crick introduced an elegant double-helical model for the structure of deoxyribonucleic acid, or DNA DNA, the substance of inheritance, is the most celebrated molecule of our time Hereditary information is encoded in DNA and reproduced in all cells of the body This DNA program directs the development of biochemical, anatomical, physiological, and (to some extent) behavioral traits

3. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings The Search for the Genetic Material: Scientific Inquiry When 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

6. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Evidence That DNA Can Transform Bacteria The discovery of the genetic role of DNA began with research by Frederick Griffith in 1928 Griffith worked with two strains of a bacterium, a pathogenic “S” strain and a harmless “R” strain When he mixed heat-killed remains of the pathogenic strain with living cells of the harmless strain, some living cells became pathogenic He called this phenomenon transformation, now defined as a change in genotype and phenotype due to assimilation of foreign DNA

7. LE 16-2 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 Living S cells are found in blood sample Mouse healthy Mouse dies RESULTS

8. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings In 1944, Oswald Avery, Maclyn McCarty, and Colin MacLeod announced that the transforming substance was DNA Their conclusion was based on experimental evidence that only DNA worked in transforming harmless bacteria into pathogenic bacteria Many biologists remained skeptical, mainly because little was known about DNA

9. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Evidence That Viral DNA Can Program Cells More evidence for DNA as the genetic material came from studies of a virus that infects bacteria Such viruses, called bacteriophages (or phages), are widely used in molecular genetics research

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

11. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings In 1952, Alfred Hershey and Martha Chase performed experiments showing that DNA is the genetic material of a phage known as T2 To determine the source of genetic material in the phage, they designed an experiment showing that only one of the two components of T2 (DNA or protein) enters an E. coli cell during infection They concluded that the injected DNA of the phage provides the genetic information

12. LE 16-4 Bacterial cell Phage DNA Radioactive protein Empty protein shell Phage DNA Radioactivity (phage protein) in liquid Batch 1: Sulfur ( 35 S) Radioactive DNA Centrifuge Pellet (bacterial cells and contents) Pellet Radioactivity (phage DNA) in pellet Centrifuge Batch 2: Phosphorus ( 32 P)

13. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Additional Evidence That DNA Is the Genetic Material In 1947, 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 By the 1950s, it was already known that DNA is a polymer of nucleotides, each consisting of a nitrogenous base, a sugar, and a phosphate group

14. LE 16-5 Sugar–phosphate backbone 5 end Nitrogenous bases Thymine (T) Adenine (A) Cytosine (C) DNA nucleotide Phosphate 3 end Guanine (G) Sugar (deoxyribose)

15. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Building a Structural Model of DNA: Scientific Inquiry 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

16. LE 16-6 Franklin’s X-ray diffraction photograph of DNA Rosalind Franklin

17. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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

18. LE 16-7 5 end 3 end 5 end 3 end Space-filling modelPartial chemical structure Hydrogen bond Key features of DNA structure 0.34 nm 3.4 nm 1 nm

19. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 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 Instead, pairing a purine with a pyrimidine resulted in a uniform width consistent with the X-ray

20. LE 16-UN298 Purine + purine: too wide Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width consistent with X-ray data

21. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Watson and Crick reasoned that the pairing was more specific, dictated by the base structures They determined that adenine paired only with thymine, and guanine paired only with cytosine

22. LE 16-8 Adenine (A) Thymine (T) Guanine (G) Cytosine (C) Sugar

23. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Concept 16.2: Many proteins work together in 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

24. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 In DNA replication, the parent molecule unwinds, and two new daughter strands are built based on base-pairing rules

25. LE 16-9_1 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.

26. LE 16-9_2 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. The first step in replication is separation of the two DNA strands.

27. LE 16-9_3 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. The first step in replication is separation of the two DNA strands. Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand.

28. LE 16-9_4 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. The first step in replication is separation of the two DNA strands. Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand. The nucleotides are connected to form the sugar-phosphate back- bones of the new strands. Each “daughter” DNA molecule consists of one parental strand and one new strand.

29. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 and the dispersive model

30. LE 16-10 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 molecules contains a mixture of old and newly synthesized DNA. Parent cell First replication Second replication

31. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Experiments by Meselson and Stahl supported the semiconservative model They labeled the nucleotides of the old strands with a heavy isotope of nitrogen, while any new nucleotides were labeled with a lighter isotope The first replication produced a band of hybrid DNA, eliminating the conservative model A second replication produced both light and hybrid DNA, eliminating the dispersive model and supporting the semiconservative model

32. LE 16-11 Bacteria cultured in medium containing 15 N DNA sample centrifuged after 20 min (after first replication) DNA sample centrifuged after 40 min (after second replication) Bacteria transferred to medium containing 14 N Less dense More dense Conservative model First replication Semiconservative model Second replication Dispersive model

33. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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

34. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Getting Started: Origins of Replication 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 At the end of each replication bubble is a replication fork, a Y-shaped region where new DNA strands are elongating

35. LE 16-12 In eukaryotes, DNA replication begins at may sites along the giant DNA molecule of each chromosome. Two daughter DNA molecules Parental (template) strand Daughter (new) strand 0.25 µm Replication fork Origin of replication Bubble In this micrograph, three replication bubbles are visible along the DNA of a cultured Chinese hamster cell (TEM).

36. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Elongating a New DNA Strand Enzymes called DNA polymerases catalyze the elongation of new DNA at a replication fork Each nucleotide that is added to a growing DNA strand is a nucleoside triphosphate The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells

37. LE 16-13 New strand 5 end Phosphate Base Sugar Template strand 3 end 5 end 3 end 5 end 3 end 5 end 3 end Nucleoside triphosphate DNA polymerase Pyrophosphate

38. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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

39. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Along one template strand of DNA, called the leading strand, DNA polymerase can synthesize a complementary strand continuously, moving toward the replication fork 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

40. LE 16-14 Parental DNA 5 3 Leading strand 3 5 3 5 Okazaki fragments Lagging strand DNA pol III Template strand Leading strand Lagging strand DNA ligase Template strand Overall direction of replication

41. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Priming DNA Synthesis DNA polymerases cannot initiate synthesis of a polynucleotide; they can only add nucleotides to the 3 end The initial nucleotide strand is a short one called an RNA or DNA primer An enzyme called primase can start an RNA chain from scratch Only one primer is needed to synthesize the leading strand, but for the lagging strand each Okazaki fragment must be primed separately

42. LE 16-15_1 5 3 Primase joins RNA nucleotides into a primer. Template strand 5 3  Overall direction of replication

43. LE 16-15_2 5 3 Primase joins RNA nucleotides into a primer. Template strand 5 3  Overall direction of replication RNA primer 3 5 3 5 DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment.

44. LE 16-15_3 5 3 Primase joins RNA nucleotides into a primer. Template strand 5 3  Overall direction of replication RNA primer 3 5 3 5 DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. Okazaki fragment 3 5 5 3 After reaching the next RNA primer (not shown), DNA pol III falls off.

45. LE 16-15_4 5 3 Primase joins RNA nucleotides into a primer. Template strand 5 3  Overall direction of replication RNA primer 3 5 3 5 DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. Okazaki fragment 3 5 5 3 After reaching the next RNA primer (not shown), DNA pol III falls off. 3 3 5 5 After the second fragment is primed, DNA pol III adds DNA nucleotides until it reaches the first primer and falls off.

46. LE 16-15_5 5 3 Primase joins RNA nucleotides into a primer. Template strand 5 3  Overall direction of replication RNA primer 3 5 3 5 DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. Okazaki fragment 3 5 5 3 After reaching the next RNA primer (not shown), DNA pol III falls off. 3 3 5 5 After the second fragment is primed, DNA pol III adds DNA nucleotides until it reaches the first primer and falls off. 3 3 5 5 DNA pol I replaces the RNA with DNA, adding to the 3 end of fragment 2.

47. LE 16-15_6 5 3 Primase joins RNA nucleotides into a primer. Template strand 5 3  Overall direction of replication RNA primer 3 5 3 5 DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. Okazaki fragment 3 5 5 3 After reaching the next RNA primer (not shown), DNA pol III falls off. 3 3 5 5 After the second fragment is primed, DNA pol III adds DNA nucleotides until it reaches the first primer and falls off. 3 3 5 5 DNA pol I replaces the RNA with DNA, adding to the 3 end of fragment 2. 3 3 5 5 DNA ligase forms a bond between the newest DNA and the adjacent DNA of fragment 1. The lagging strand in the region is now complete.

48. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Other Proteins That Assist DNA Replication Helicase untwists the double helix and separates the template DNA strands at the replication fork 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

49. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Primase synthesizes an RNA primer at the 5 ends of the leading strand and the Okazaki fragments DNA pol III continuously synthesizes the leading strand and elongates Okazaki fragments DNA pol I removes primer from the 5 ends of the leading strand and Okazaki fragments, replacing primer with DNA and adding to adjacent 3 ends DNA ligase joins the 3 end of the DNA that replaces the primer to the rest of the leading strand and also joins the lagging strand fragments

50. LE 16-16 5 3 Parental DNA 3 5 Overall direction of replication DNA pol III Replication fork Leading strand DNA ligase Primase OVERVIEW Primer DNA pol III DNA pol I Lagging strand Lagging strand Leading strand Leading strand Lagging strand Origin of replication

51. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings The DNA Replication Machine as a Stationary Complex The proteins that participate in DNA replication form a large complex, a DNA replication “machine” The DNA replication machine is probably stationary during the replication process Recent studies support a model in which DNA polymerase molecules “reel in” parental DNA and “extrude” newly made daughter DNA molecules

52. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 In nucleotide excision repair, enzymes cut out and replace damaged stretches of DNA

53. LE 16-17 DNA ligase DNA polymerase DNA ligase seals the free end of the new DNA to the old DNA, making the strand complete. Repair synthesis by a DNA polymerase fills in the missing nucleotides. A nuclease enzyme cuts the damaged DNA strand at two points and the damaged section is removed. Nuclease A thymine dimer distorts the DNA molecule.