1. The Molecular Basis of Inheritance
Campbell and Reece
Chapter 16

2. The Search for Genetic Material
once Morgan proved genes are in chromosomes big debate started:
Is the genetic material in chromosomes the DNA or the proteins?
@ first case for proteins seemed stronger
very heterogenous
great specificty

3. Evidence that DNA can Transform Bacteria
1928 Griffith studied Streptococcus pneumoniae

4. Transformation term coined by Griffith
change in genotype & phenotype due to the assimilation of external DNA by a cell
Avery spent the next 14 years identifying the “transforming agent”

5. Avery’s Experiment

6. Avery & his colleagues announced DNA was the transforming agent
many were skeptical
Was bacterial DNA anything like eukaryotic DNA?
Nothing much known about DNA
Most scientists held to belief that proteins had to be transforming agent

7. Bacteriophages are viruses that infect bacteria “phages” for short
Virus made of a protein coat covering genetic material
to produce more viruses it must invade a cell
& take over the cell’s metabolic machinery

9. Hershey & Chase Experiment

10. Additional Evidence that DNA is Genetic Material
Chargraff
already knew DNA made up of:
Deoxyribose
Phosphate group
Nitrogenous Base (A, G, C, T)
analyzed DNA from # of species
1950: base composition of DNA varies between species
made DNA more credible

11. Chargraff’s Rule
no matter what the source of DNA tested:

12. What is the structure of DNA?
early 1950’s: scientists convinced DNA carried genetic info
focus now on DNA’s structure
knew arrangement of DNA’s covalent bonds

13. Watson & Crick Cambridge, England 2 young, unknown scientists
same lab: Franklin & Wilkins doing x-ray crystallography on protein structure

15. X-Ray Crystallography of DNA
Rosalind Franklin had purified some DNA and showed results to Watkins who was familiar with pattern made by a helical structure

16. Watson & Crick began building models that satisfied:
known chemical properties of DNA
nitrogenous bases relatively hydrophobic
phosphate groups carry (-) charge
Chagraff’s rules
helical structure
how could this structure pass on genetic information?

18. DNA Structure
antiparallel: arrangement of sugar-phosphate backbones in a DNA double helix
means 1 strand runs 5’  3’ going “up” * the other runs 5’  3’ going “down”

19. DNA Structure
because of size differences in dbl ringed purines vs. single ringed pyrimidines Watson & Crick knew could not have a purine linked with itself or the other purine
also knew that adenine & thymine could form H bonds (2) with each other & cytosine & guanine could for 3 H bonds

21. Watson & Crick their 1 page paper published in Nature in April 1953
Watson, Crick, and Wilkins received Nobel Prize in 1962 (Franklin died in 1958)

23. DNA Replication
Watson and Crick’s 2nd paper stated their hypothesis on how DNA replicates:
DNA model is pair of complimentary templates
prior to replication H bonds broken & chains separate & unwind
each chain then acts as template for formation onto itself of a new complimentary chain
allows for exact duplication

24. DNA Replication

25. Watson & Crick’s Semiconservative Model of DNA Replication
predicts when a dbl helix replicates, each of the 2 daughter molecules will have 1 old strand and 1 new strand
Conservative Model: 1 new daughter molecule with 2 new strands & the original molecule
Dispersive Model: all 4 strands of DNA after replication have mixture of old & new parts

26. 3 Models of DNA Replication

27. Origins of Replication
DNA Replication
particular sites called:
Origins of Replication
short stretches of a specific sequence of nucleotides
many bacterial loops of DNA have single origin
proteins that initiate DNA replication recognize the sequence / attach to the DNA / separate the 2 strands by breaking H bonds creating “bubbles”

28. Prokaryotic Replication of DNA

29. Eukaryotic DNA Replication

30. Replication Bubble

31. Replication Forks @ each end of the replication bubble
Y-shaped region where DNA is unwinding
proteins that participate in the unwinding:
helicases
unwind double helix
single-strand binding proteins
bind to single strands prevents them from rewinding
topoisomerases
untwisting dbl helix puts strain on ahead of replication fork, these proteins relieve strain by breaking, swiveling, & rejoining DNA strands

32. Replication Forks

33. Replication of DNA
initial nucleotide chain made during DNA synthesis is actually a strand of RNA
this RNA chain called a primer which is made by an enzyme called primase (last slide)
primase starts a complementary RNA chain from a single RNA nucleotide then adds time

34. Primers
when primer 5 – 10 nucleotides long...new DNA strand will start from the 3’ end of the RNA primer

35. DNA Polymerase
enzyme that catalyzes the synthesis of new DNA by adding nucleotides to a pre-existing chain
2 major one in prokaryotes
11 different ones in eukaryotes
most require a primer & DNA template strand
rate: ~500 nucleotides/s in bacteria
~ 50 nucleotides/s in human cells

36. Source of Nucleotides
are in form of nucleoside triphosphates

37. dATP

38. Nucleoside Triphosphates
are chemically reactive (like ATP, except sugar is deoxyribose, not ribose)
as each nucleotide joins the growing end of a DNA strand 2 of the phosphate groups are lost as a molecule of inorganic phosphate in a couple exergonic reaction that drives the polymerization reaction

39. Polymerization Reaction

40. Antiparallel Elongation
each strand of DNA has directionality (1-way street)
& each strand oriented in opposite directions to each other
DNA polymerase III can add nucleotides only to the free 3’ end of a primer or growing DNA strand
along 1 template DNA polymerase synthesizes complementary strand continuously (5’  3’ direction)
called the Leading Strand

41. Antiparallel Elongation
along opposite strand because of orientation, DNA polymerase III must work in direction away from the replication fork
called Lagging Strand
synthesized in short segments called:
Okazaki Fragments
~ 1,000 – 2,000 nucleotides long in E. coli
~ 100 – 200 nucleotides long in eukaryotes

45. DNA Replication Complex
easy to think of DNA polymerase as a locomotive moving down template track but not really how it works:
various proteins that participate in DNA replication form a large complex
DNA replication complex doesn’t move, the DNA template moves thru the complex

46. DNA Replication Complex

47. Proofreading & Repairing DNA
~ 1/10 billion base pairs in completed DNA will be incorrect
but right after strands replicated errors ~ 100,000 times more common
DNA polymerases “proofread” each nucleotide against its template as soon as it is added
when error found, incorrect nucleotide removed, correct 1 inserted

49. Proofreading & Repairing
some errors evade DNA polymerase…other enzymes remove & replace incorrectly paired nucleotides
some errors arise after replication: damage to DNA relatively common: usually corrected by b/4 becoming permanent mutations
cells continuously monitor & repair damaged DNA

50. Repair Enzymes ~100 in E. coli ~ 130 in humans
most organisms use same mechanism to repair errors or damage
involves cutting out damaged area using DNA-cutting enzyme called nuclease
gap then filled with correct nucleotides done by a DNA polymerase & DNA ligase
1 of these systems called nucleotide excision repair

51. Nucleotide Excision Repair

52. Telomeres repetitive sequences @ ends of eukaryotic chromosomes
shorter as we age (with each round of DNA replication)
so preserves the ends of linear DNA & postpones erosion of genes
telomerase catalyzes the lengthening of telomeres in germ cells

53. Telomeres

55. Prokaryotic DNA usually loop of DNA some associated proteins
loop of DNA + proteins = nucleoid

56. Eukaryotic Chromatin
includes:
DNA
histones
other proteins

57. Nucleosomes
histones bind to each other & to the DNA to form nucleosomes: the most basic unit of DNA packing
histone tails extend outward from each bead-like nucleosome cone

58. additional coiling & folding  highly condensed chromosome as seen in mitosis

59. Euchromatin: term for the less coiled chromatin seen in interphase cells
easily accessible for transcription
Heterochromatin: portions of chromatin that remains highly condensed even in interphase
mostly inaccessible to transcription