1. Chapter 16 The Molecular Basis of Inheritance

2. But first… Bessbugs! (Bess beetles)

3. Overview: Life’s Operating Instructions In 1953, Watson and Crick introduced an elegant double-helical model for the structure of deoxyribonucleic acid, or DNA 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

4. Each nucleotide consists of a: 1. pentose sugar 2. nitrogenous base 3. phosphate group Nucleic acid 1869: Friedrich Miescher isolated DNA from pus! DNA and RNA are nucleic acids.

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

6. What is the genetic material? When genes were shown to be located on chromosomes, the 2 components of chromosomes- DNA and protein- became candidates for the genetic material –Protein seemed more complex than DNA 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

7. Evidence That DNA Can Transform Bacteria Discovery of genetic role of DNA began w/ research by Frederick Griffith in 1928, who worked with two strains of a bacterium, Streptococcus pneumoniae 1.pathogenic “S” (smooth) strain 2.harmless “R” (rough) 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

8. Evidence That Viral DNA Can Program Cells In 1944, Oswald Avery, Maclyn McCarty, and Colin MacLeod announced that the transforming substance was DNA –Avery-MacLeod-McCarty experiment 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

9. Phage DNA Bacterial cell Radioactive protein Radioactive DNA Batch 1: radioactive sulfur ( 35 S) Batch 2: radioactive phosphorus ( 32 P) radioactive sulfur labels protein radioactive phosphorus labels DNA In 1952, Hershey and Chase performed experiments showing that only one of the two components (DNA/ protein) of a phage T2 enters an E. coli cell during infection 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 radioactive sulfur labels protein radioactive phosphorus labels DNA In 1952, Hershey and Chase performed experiments showing that only one of the two components (DNA/ protein) of a phage T2 enters an E. coli cell during infection shake loose phages that remained outside the bacteria 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 radioactive sulfur labels protein radioactive phosphorus labels DNA In 1952, Hershey and Chase performed experiments showing that only one of the two components (DNA/ protein) of a phage T2 enters an E. coli cell during infection They concluded that the injected DNA of the phage provides the genetic information shake loose phages that remained outside the bacteria

10. Building a Structural Model of DNA Clue #1 Erwin Chargaff In 1950, 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 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 AT GC

11. Building a Structural Model of DNA Clue #2 Wilkins and Franklin were using a technique called X-ray crystallography to study molecular structure Franklin produced a picture of the DNA molecule using this technique

12. Rosalind Franklin (1920-1958)

13. The X-ray crystallographic images of DNA enabled Watson to deduce that: 1. DNA was helical 2. the width of the helix and the spacing of the nitrogenous bases –suggested that the DNA molecule was made up of two strands, forming a double helix

14. 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

15. Adenine (A) Thymine (T) Guanine (G) Cytosine (C) Sugar They determined: adenine (A) paired only with thymine (T) guanine (G) paired only with cytosine (C) 2 hydrogen bonds 3 hydrogen bonds

17. DNA Replication Watson and Crick noted that the specific base pairing suggested a possible copying mechanism for genetic material 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 2 new daughter strands are built based on base-pairing rules 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. AT CG TA A T GC 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. AT CG TA A T GCG AT CG TA A T C The first step in replication is separation of the two DNA strands. 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. AT CG TA A T GCG AT CG TA A T G A C T A C T G A T C A C T A G T G A T C 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 of one new strand. AT CG TA A T GCG AT CG TA A T G A C T A C T G A T C A C T A G T G A T CG A C T A T G A T CG A C T A T G A T C

18. April 23, 2012

19. Updated syllabus Homework

20. Ch 16 so far The history –Griffith (1928) –Avery-MacLeod-McCarty (1944) –Hershey and Chase (1952) –Francis and Crick (and Franklin) (1953) – Meselson and Stahl (1958) DNA Replication Chromosome molecular structure

21. 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 dispersive model 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

22. 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 Meselson & Stahl- To test the 3 models 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 hybridhybrid light ( 14 N) hybridhybrid hybridhybrid heavy ( 15 N) light ( 14 N) heavy ( 15 N) light ( 14 N) hybridhybrid hybridhybrid mostly light ( 14 N)

24. DNA replication overview

25. 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 replication Double-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 DNA Replication begins at special sites called origins of replication,  A eukaryotic chromosome may have 100-1,000’s The two DNA strands are separated, opening up a replication “bubble” consisting of 2 replication forks, Y-shaped regions where new DNA strands are elongating at each end replication fork

26. Topoisomerase Helicase Single-strand binding proteins RNA primer (5-10 nucleotides) 5 5 5 3 3 3 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, & rejoining DNA strands Primase- synthesizes the initial nucleotide strand, a short RNA primer

27. Elongating a New DNA Strand The rate of elongation is ~ 500 nucleotides/ sec in bacteria & 50 nucleotides/ sec in human cells 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 enzymes that catalyze the elongation of new DNA at a replication fork Each nucleotide that is added to a growing DNA strand is a nucleoside triphosphate

28. 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

29. Leading strand Overview Origin of replication Lagging strand Leading strandLagging strand Primer Overall directions of replication Origin of replication RNA primer “Sliding clamp” DNA poll III Parental DNA 5 3 3 3 3 5 5 5 5 5 Along one template strand of DNA, called the leading strand, DNA polymerase can synthesize a complementary strand continuously, moving toward the replication fork 5 5 3 3

30. 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

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

32. REVIEWREVIEW

33. April 25, 2012 Self Description

34. Announcements Homework due tomorrow (Apr 26) Old exams Exam III to hand back at the end of class

35. 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

36. The DNA Replication Machine The proteins that participate in DNA replication form a large complex, a 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

37. Proofreading & Repairing DNA DNA polymerases proofread newly made DNA, replacing any incorrect nucleotides DNA can be damaged by:  chemicals  X-rays  UV light  toxic chemicals- cigarettes mismatch repair of DNA- repair enzymes correct errors in base pairing nucleotide excision repair- enzymes cut out and replace damaged stretches of DNA 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. often caused by UV nucleotide excision repair

38. 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 with uneven ends End of parental DNA strands 5 3 Lagging strand 5 3 Last fragment RNA primer Leading strand Lagging strand Previous fragment Primer removed but cannot be replaced with DNA because no 3 end available for DNA polymerase 5 3 Removal of primers and replacement with DNA where a 3 end is available Second round of replication 5 3 5 3 Further rounds of replication New leading strand Shorter and shorter daughter molecules

39. 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 telomeres TTAGGG 100 - 1,000 x

40. If chromosomes of germ cells became shorter in every cell cycle, essential genes would eventually be missing from gametes they produce An enzyme called telomerase catalyzes the lengthening of telomeres in germ cells There is evidence of telomerase activity in cancer cells, which may allow cancer cells to persist It has been proposed that the shortening of telomeres is connected to aging –The shortening of telomeres might protect cells from cancerous growth by limiting the number of cell divisions

41. Chromosome structure 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 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

42. 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)  DNA winds around histones to form nucleosome “beads”  Nucleosomes are strung together like beads on a string by linker DNA

43. 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 Interactions between nucleosomes cause the thin fiber to coil or fold into this thicker fiber The 30-nm fiber forms looped domains that attach to proteins

44. Most chromatin is loosely packed in the nucleus during interphase and condenses prior to mitosis euchromatin- loosely packed chromatin heterochromatin- highly condensed chromatin during interphase a few regions of chromatin (centromeres and telomeres) Dense packing of the heterochromatin makes it difficult for the cell to express genetic information coded in these regions

45. Painted Chromosomes At interphase, some chromatin is organized into a 10-nm fiber, but much is compacted into a 30-nm fiber, through folding and looping Interphase chromosomes are not highly condensed, but still occupy specific restricted regions in nucleus