1. DNA Replication, Repair, and Recombination

2. DNA Maintenance Mutation rate are extremely low
1 mutation out of 109 nucleotides per generation

3. DNA replication
Separation, Base pair

4. The Chemistry of DNA replication

5. DNA Synthesis by DNA polymerase

6. Nucleotide polymerizing enzyme, first discovered in 1957
DNA Polymerase
Nucleotide polymerizing enzyme, first discovered in 1957

7. DNA replication with two forks

8. This Doesn’t Work!

9. DNA replication Fork

10. DNA Proofreading

11. Structures of DNA polymerase during polymerizing and editing
E: exonucleolytic; P: polymerization

12. Why 5’->3’?
The need for accuracy

13. Site-directed mismatch repair in eucaryotes
In procaryotes, old DNAs are usually methylated on A while newly synthesized ones are not. So Cells can distinguish old and newly synthesized DNAs and mutate mismatches on new ones.

14. DNA Proofreading RNA usually doesn’t have this. Why?
Pairing, correct nucleotide has higher affinity binding to the moving polymerase
Un-Paired nucleotide is easier to be off before covalent ligation, even after binding.
Exonucleotic proofreading
Strand directed mismatch repair

16. DNA Primer synthesis
On Lagging strand

17. DNA Replication at the Lagging strand

18. DNA Ligase

19. DNA Helicase
DNA double helix are tightly coupled. High temperature is needed to break them (95oC)

21. DNA Binding Protein
SSB: Single Strand DNA-binding Proteins, also called helix destabilizing proteins

22. SSB Proteins
DNA

23. DNA Clamping Protein

25. Cycle of DNA Polymerase/Clamping Protein loading and unloading
At the lagging strand (how about leading strand?)

26. Protein machinery for DNA replication

27. A Moving Replication

28. Structure of the Moving Complex

29. DNA winding

30. DNA topoisomerase I

31. DNA topoisomerase II

32. DNA topoisomerase II

33. Mammalian replication Fork
(eucaryote, DNA polymerase (primase) a synthesize RNA/DNA, DNA polymerase delta is the real polymerase)

34. Summary DNA replication 5’->3’ DNA proof reading
Lagging strand, back-stitching, Okazaki fragment
Proteins involved:
DNA polymerase, primase
DNA helicase and single-strand DNA-binding protein (SSB)
DNA ligase, and enzyme to degrade RNA
DNA topoisomerases

35. DNA Replication in Chromosome

36. DNA replication in Bacterial Genome

37. Initiating Proteins for DNA replication
1. Initiator protein, 2. helicase binding to initiator protein, 3. helicase loading on DNA, 4. helicase opens the DNA and binds to primase, 5. RNA primer synthesis, 6. DNA polymerase binding and DNA synthesis

38. Regulation for DNA replication
In Bacteria, hemimethylated origins are resistant to initiation, delayed methylation leads to delayed initiation at the second phase
Dam methylase

39. DNA replication in eucaryotes Multiple replication origin
50 nucleotides/second, autoradiography

40. The four standard phases of a eucaryotic cell
DNA replication occurring at S Phase (DNA synthesis phase)
G1 and G2, gap between S and M

41. Different regions of a chromosome are replicated at different times
Arrows point to the replicating regions at different times

42. Some facts about Replication in eucaryotes
Multiple replication origins occurring inclusters (20-80) (replication units)
Replication units activated at different times
Within replication units, replication origins are separated 30, ,000 pairs apart.
Replication forks form in pairs and create a replication bubbles moving in opposite directions
Different regions on the same chromosome are replicated at distinct times in S phase
Condensed Chromatin replicates late, while less condensed regions replicate earlier

43. The search and identification of DNA replication sequence
ARS: autonomously replicating sequence
Only histidine expression can help cells to survive

44. The origins of DNA replication on chromosome III of the yeast S
The origins of DNA replication on chromosome III of the yeast S. cerevisiae

45. A close look at an origin of replication in yeast
ORC: origin recognition complex
B1, B2, B3: other regions binding to required proteins

46. The replication origins of human genes are more complex
Even far distant DNA sequences could be important

47. Histone remains associated with DNA
In vitro experiments
DNAs with different sizes are replicated. Only the daughter DNAs replicated from parental DNA with histones showed histone binding

48. Addition of new histones
Chromatin assembly factors (CAFs) help to add and assemble new nucleosomes

50. Bacteria DNAs are circular, not a problem
There is a problem for eucaryote DNAs: ???
Hint: Telomere

51. Reverse transcriptase with RNA template to bind to DNA strands
Telomerase Structure
Reverse transcriptase with RNA template to bind to DNA strands

52. Telomerase and its function

53. T-loops at the end of mammalian chromosomes

54. Summary
Specific DNA sequence determine replication origin, recruiting proteins to form replication machinery. relatively complex in eucaryotes
Bacteria has single replication origin. Eucaryotes have multiple origins and less defined.
Replication forks are activated at different times in eucaryotes
Telomere and telomerase

55. DNA Repair Spontaneous DNA damage Pathways to remove DNA damage
Damage detection
The repair of Double-strand break
DNA repair enzymes

56. Spontaneous Alterations of nucleotides
Red: oxidative damage; blue: hydrolytic attack; green: uncontrolled methylation

57. Depurination and deamination

58. Thymine dimer

59. Mutation Generation passed on to daughter DNAs

60. Mutation Generation passed on to daughter DNAs

61. DNA Repair I

62. DNA Repair II

63. Recognition of unusual nucleotide
By base flipping recognized by DNA glycosylase family

64. Emergency DNA Repair for Double helix break

65. DNA Repair Summary
Spontaneous DNA damage: spontaneous alteration of bases, depurination and deamination, thymine dimer
Pathways to remove DNA damage: base excision repair, nucleotide excision repair
Damage detection: base flipping
The repair of Double-strand break: nonhomolous end joining, homologous end joining
DNA repair enzymes: heat shock proteins

66. DNA Recombination
General recombination
Site specific recombination

67. General DNA Recombination

68. Heteroduplex joint

69. General Recombination
Two homologous DNA molecules cross over
The site of exchange can occur anywhere
A strand of one DNA molecule has become base-paired to a strand of the second DNA to create heteroduplex joint
No nucleotide sequences are altered

70. The procedure of general recombination
DNA synapsis: base pairs form between complementary strands from the two DNA molecules

71. The initial step for DNA recombination
DNA Hybridization
The initial step for DNA recombination

72. RecA protein-mediated DNA synapsis
Rec A has multiple DNA binding sites, hence can hold a single strand and a double helix together
Rec A is also a DNA-dependent ATPase

73. DNA Branch Migration

74. Holiday Junction for DNA recombination
Exchange of the first single strand between two different DNA double helices is slow and difficult, then intermediate state Holiday Junction, then complete exchange

76. Holiday Junction for DNA recombination
and its resolution

77. Summary for General Recombination
General recombination allows large fraction of genetic information to move from one chromosome to another.
General recombination requires the breakage of double helices, beginning with a single strand breakage.
General recombination is facilitated by Rec A in bacteria and its homologs in eucaryotes.
Holiday junction is the intermediate state of general recombination

78. Site-specific recombination
Moves specialized nucleotide sequence (mobile genetic elements) between non-homologous sites within a genome.
Transpositional site-specific recombination
Conservative site-specific recombinatinon

79. Transpositional site-specific recombination
Modest target site selectivity and insert mobile genetic elements into many sites
Transposase enzyme cuts out mobile genetic elements and insert them into specific sites.

80. Three of the many types of mobile genetic elements found in bacteria
Transposase gene: encoding enzymes for DNA breakage and joining
Red segments: DNA sequences as recognition sites for enzymes
Yellow segments: antibiotic genes

82. Cut and Paste Transposition
DNA-only

83. The structure of the central intermediate formed by transposase (integrase)

84. Replicative Transposition

85. Retrovirus-based Transposition Retroviral-like retrotransposition

87. Reverse Transcriptase
RNA

88. Non-retroviral retrotransposition

89. Conservative Site Specific Recombination Integration vs. inversion
Notice the arrows of directions

90. Bacteriophase Lambda

91. Genetic Engineering to control Gene expression

92. Summary DNA site-specific recombination transpositional; conservative
Transposons: mobile genetic elements
Transpositional: DNA only transposons, retroviral-like retrotransposons, nonretroviral retrotransposons