1. Replication

2. Outline IV. Repair A. Mismatch (dam) B. Base Excision (single base)
I. Dogma
II. DNA structure (genes, chromosomes)-length of human genome
III. Replication
A. Initiation
1. Proteins involved
2. Enzymes involved
3. Sequence of events
B. Elongation
C. Termination
D. Proofreading
IV. Repair
A. Mismatch (dam)
B. Base Excision (single base)
C. Nucleotide excision (DNA segment)
V. Advanced Topics
A. Recombination
B. Transposons
0.34 nm/bp
[salt] for histone release
Prokaryote simpler
Compare and contrast eukaryotes to prokaryotes

3. Central Dogma of Information Flow
Historical overview of DNA as an informational molecule (?where did this dogma come from?)

4. Historical Basis I
Friedrich Miescher (1868) isolated “acidic component” from puss, now known to be DNA
“basic component” protein
Griffin (1928) DNA inserted into bacteria changed the behavior of bacteria

5. Historical Basis II: Avery, McLeod and McCarthy (1949) Fig
Historical Basis II: Avery, McLeod and McCarthy (1949) Fig (a-e) Lehninger POB 3rd Ed.

6. Historical Basis III Fig. 10-13 Lehninger POB 3rd Ed.
Hershey-Chase experiment
Shows that the protein of a bacteriophage does not enter bacteria.

7. Wagging the Dogma 1976 David Baltimore et al. RNA viruses such as HIV
Reverse transcriptase has a high error rate, makes (AIDS) hard to treat
1976 David Baltimore et al.
RNA viruses such as HIV
Pruisner prion hypothesis

8. DNA Structure
Last semester: ds, base-paired, anti-parallel -helix which could be melted
Now: levels of structure
1o structure: the sequence of nt
2o structure: the -helix
3o structure:chromosome
supersecondary structures
Human cell ~50 µm D, DNA strand 1-2 m long

9. Prokaryotic DNA Packing and Wiese’s Stupid Hose Trick

10. Eukaryotic DNA Packing - Histones
Lys/Arg rich protein
H1, H2a, H2b, H3, H4
Octamer forms: two each not H1

11. DNA Packing - The Nucleosome Fig. 24-24(c) Lehninger POB 3rd Ed.

12. DNA Packing - “Beads on a String” Fig. 24-23(b) Lehninger POB 3rd Ed.

13. DNA Packing – Solenoid Left: Fig. 24-27(a) Lehninger POB 3rd Ed
DNA Packing – Solenoid Left: Fig (a) Lehninger POB 3rd Ed. Right: Fig Harpers ROB 24th Ed.

14. DNA Packing - Looping From ?

15. DNA Packing - Minibands From ?

16. DNA Packing - Chromosome Fig. 24-7 Lehninger POB 3rd Ed.

17. Not All DNA is Packed Equally
DNase insensitive, DNase sensitive, DNase hypersensitive
Euchromatin- and heterochromatin- (facultative and constitutive)
Barr bodies-

18. Replication
Replication- the synthesis of DNA using itself as a template.
Three stages: Initiation, Elongation, Termination

19. Replication is Semiconservative
Meselson-Stahl Experiment
Grow bacteria on 15N source- get bottom band
Grow bacteria on 14N source- get top band
Grow bacteria on 15N source, then switch to 14N - get band halfway in between

20. Replication is 5’  3’ (Pulse-chase experiment)
Pulse with radioactive tracer
Chase with large mass unlabeled
One end of molecule labeled

21. Replication Begins at a Specific Place Each Time The Origin of replication (ori)
If starts at random If always starts same place

22. The Origin
Analogous to Fig Lehninger POB 3rd Ed
Not to scale

23. Consensus Sequence

24. Replication is Bidirectional (Usually) Fig. 25-3 Lehninger POB 3rd Ed.
Theta structure

25. Initiation

26. Ori C (AKA Dna C) Binds the Origin
~250 bp protected from DNase

27. DNA is Melted
Replication bubble

28. ssb (single strand binding) Proteins Binds Around ori C
Prevents DNA from reannealing
Prevents nuclease activity

29. Helicase Unwinds DNA

30. Primase Synthesizes an RNA Primer 10-200 nt Long

31. Elongation

32. There Are Multiple DNA Polymerases, With Different Functions
Prokaryote
I- fill in RNA gap, repair
II- ?
III- elongation
Eukaryote (19 ID’d)
- elongation -
- mitochdondrial
- elongation
- repair
All of these are multisubunit proteins

33. Function is Deduced From Activity
Nucleosomes interfere in eukaryote

34. Termination Least Well Understood
Crowding would have to occur
TBP- ter (termination site) binding protein - ?directs traffic?
Yeast: 5’ (TxGy)n
3’ (AxCy)n ~100 bp
DNA ligase seals ‘nick’ between fragments by mechanism shown on next slides

35. DNA Ligase is the Only Enzyme of Replication that “Requires” Energy

36. DNA Ligase is the Only Enzyme of Replication that “Requires” Energy

37. DNA Ligase is the Only Enzyme of Replication that “Requires” Energy
Mechanism analogous to attack on alpha phosphorous by polymerase

38. Review of Replication - Initiation
Ori bound by helicase and SSB (other proteins at site)
Replication bubble forms
primase and DNA polymerase III join complex
synthesis is 5’ to 3’
Okazaki fragments used on lagging strand
Newer concept: loop lagging strand

39. Termination Least Well Understood
Crowding would have to occur
TBP- ter (termination site) binding protein - ?directs traffic?
Yeast: 5’ (TxGy)n
3’ (AxCy)n ~100 bp
DNA ligase seals ‘nick’ between fragments
Topoisomerase functions by similar mechanism, except E attaches to DNA instead of AMP (ATP not required)

40. Review of Replication - Elongation
PPi product
polymerase I removes RNA and fills gap (in eukaryotes, Rnase H cuts out the RNA)

41. Review of Replication - Termination
Ligase seals nick (ATP dependent)
Topoisomerase supercoils DNA (cut, wrap, ligate)

42. Okazaki Fragments are Used to Synthesize the Lagging Strand Fig
Okazaki Fragments are Used to Synthesize the Lagging Strand Fig and Lehninger POB 3rd Ed.
synthesis is 5’ to 3’

43. Okazaki Fragments are Used to Synthesize the Lagging Strand Fig
Okazaki Fragments are Used to Synthesize the Lagging Strand Fig and Lehninger POB 3rd Ed.
Newer model: loop lagging strand

44. Proofreading 1 mistake every 105 - 106 bases during replication
(3’  5’ exonuclease activity)
In DNA, 1 mistake every bases
other repair mechanisms must exist

45. Four Types of Repair Mechanisms
Mismatch repair
Base Excision repair
Nucleotide Excision
Direct Repair

46. Mismatch Repair and dam
?which one is incorrect?: crapshoot with 50:50 odds
Upstream, GATC sequence, A methylated
Takes a while for methylation to occur

47. Base Excision Operates Where a Single Damaged Base Occurs
Uracil deglycosylase most common
AP site
AP endonuclease (variable specificity)

48. Nucleotide Excision Operates in More Heavily Damaged Areas Fig
Nucleotide Excision Operates in More Heavily Damaged Areas Fig Lehninger POB 3rd Ed.
Removes more than just damaged ?because surroundings also likely to be damaged?

49. Direct Repair of Thymidine Dimers by Photolyase Fig
Direct Repair of Thymidine Dimers by Photolyase Fig Lehninger POB 3rd Ed.

50. In Eukaryotes, the Major Differences are the Numbers and Names of the Molecules
Prokaryotes Eukaryotes
1 ori and ter multiple ori and ter
Polymerases III and I Pol’s d and a
Okazaki fragments ~1000 nt ~ nt
supercoiling histones

51. Types of Mutations I. Transition (Pu to other Pu)
a. Silent (no change in amino acids)
b. Missense (one amino acid converted to another)
c. Nonsense (early termination of protein)
II. Transversion (Pu to Py or vice versa)
a. Silent, Missense or Nonsense
III. Frame shift
a. Insertion
b. Deletion

52. Genetic Rearrangement
“Random genetic drift”

53. Sister Chromatid Exchange... Fig. 38-11 Harpers ROB 24th Ed.

54. …Occurs Through a Holliday Intermediate Fig. Lehninger POB Ed.

55. Immunoglobulins Arise Through Recombination Fig
Immunoglobulins Arise Through Recombination Fig Harpers ROB 24th Ed.

56. VDJC Sequence Heavy Chain Fig
VDJC Sequence Heavy Chain Fig. 5-4 Stites, Stobo, and Wells Basic and Clinical Immunology 6th Ed.

57. VDJC Sequence Light Chain Fig
VDJC Sequence Light Chain Fig. 5-1 Stites, Stobo, and Wells Basic and Clinical Immunology 6th Ed.

58. Transposons are “Jumping Genes”
Transposon short for transposable element
Gene + flanking regions
Flanking region consists of inverted repeats:

59. Transposon Structure and Function
Target site is simple and is cut to generate “sticky ends”:

60. Transposon Structure and Function
Transponson is inserted, gaps filled in and ligated
The insertion can lead to a gene being turned on (regulatory elements which will be discussed in transcription) or turned off (gibberish produced).