Genetic Information Transfer Central dogma DNA Replication General Concepts of DNA Replication

1. Genetic Information
Transfer
1

2. Central dogma
replication
transcription
DNA
translation
RNA
protein
reverse
transcription
2

3. • Replication: synthesis of daughter
DNA from parental DNA
• Transcription: synthesis of RNA using
DNA as the template
• Translation: protein synthesis using
mRNA molecules as the template
• Reverse transcription: synthesis of
DNA using RNA as the template
3

4. DNA
Replication
4

5. Section 1
General Concepts of
DNA Replication

6. DNA replication
• A reaction in which daughter DNAs are
synthesized using the parental DNAs as
the template.
• Transferring the genetic information to the
descendant generation with a high fidelity
replication
parental DNA
daughter DNA6

7. Daughter strand synthesis
• Chemical formulation:
• The nature of DNA replication is a
series of 3´- 5´phosphodiester bond
formation catalyzed by a group of
enzymes.
7

8. The DNA backbone
• Putting the DNA
backbone together
– refer to the 3′ and 5′ ends of
the DNA
PO4
5′ CH2
4′
base
O
1′
C
3′
O
–
O P O
O
5′ CH2
2′
base
O
4′
1′
3′
OH
2′

9. Phosphodiester bond formation
9

10. DNA replication system
Template:
double stranded DNA
Substrate:
dNTP
Primer:
short RNA fragment with a
free 3´-OH end
Enzyme:
DNA-dependent DNA
polymerase (DDDP),
other enzymes,
protein factor
10

11. Characteristics of replication

Semi-conservative replication

Bidirectional replication

Semi-continuous replication

High fidelity
11

12. §1.1 Semi-Conservative Replication
12

13. Semiconservative replication
Half of the parental DNA molecule is
conserved in each new double helix,
paired with a newly synthesized
complementary strand. This is called
semiconservative replication
13

14. Semiconservative replication
14

15. Experiment of DNA semiconservative replication
"Heavy" DNA(15N)
grow in 14N
medium
The first
generation
grow in 14N
medium
The second
generation
15

16. Significance
The genetic information is ensured to be
transferred from one generation to the
next generation with a high fidelity.
16

17. §1.2 Bidirectional Replication
• Replication starts from unwinding the
dsDNA at a particular point (called
origin), followed by the synthesis on
each strand.
• The parental dsDNA and two newly
formed dsDNA form a Y-shape
structure called replication fork.
17

18. Replication fork
5'
3'
3'
5'
5'
3'
5'
direction of
replication
3'
18

19. Bidirectional replication
• Once the dsDNA is opened at the
origin, two replication forks are
formed spontaneously.
• These two replication forks move in
opposite directions as the syntheses
continue.
19

20. Bidirectional replication
20

21. Replication of prokaryotes
The replication
process starts
from the origin,
and proceeds
in two opposite
directions. It is
named θ
replication.
21

22. Replication of eukaryotes
• Chromosomes of eukaryotes have
multiple origins.
• The space between two adjacent
origins is called the replicon, a
functional unit of replication.
22

23. origins of DNA replication (every ~150 kb)
23

24. §1.3 Semi-continuous Replication
The daughter strands on two template
strands are synthesized differently since
the replication process obeys the
principle that DNA is synthesized from
the 5´ end to the 3´end.
24

25. Leading strand
On the template having the 3´- end, the
daughter strand is synthesized
continuously in the 5’-3’ direction. This
strand is referred to as the leading
strand.
3'
5'
3'
3'
direction of unwinding
5'
5'
25

26. Semi-continuous replication
26

27. Okazaki fragments
• Many DNA fragments are synthesized
sequentially on the DNA template
strand having the 5´- end. These DNA
fragments are called Okazaki
fragments. They are 1000 – 2000 nt
long for prokaryotes and 100-150 nt
long for eukaryotes.
• The daughter strand consisting of
Okazaki fragments is called the
lagging strand.
27

28. Semi-continuous replication
Continuous synthesis of the leading
strand and discontinuous synthesis of
the lagging strand represent a unique
feature of DNA replication. It is
referred to as the semi-continuous
replication.
28

29. Section 2
Enzymology
of DNA Replication

30. Enzymes and protein factors
protein
Mr
#
function
Dna A protein
50,000
1
recognize origin
Dna B protein
300,000
6
open dsDNA
Dna C protein
29,000
1
assist Dna B binding
DNA pol
Elongate the DNA
strands
Dna G protein
60,000
1
synthesize RNA primer
SSB
75,600
4
single-strand binding
DNA topoisomerase
400,000
4
release supercoil
constraint
30

31. §2.1 DNA Polymerase
DNA-pol of prokaryotes
• The first DNAdependent DNA
polymerase (short for
DNA-pol I) was
discovered in 1958 by
Arthur Kornberg who
received Nobel Prize in
physiology or medicine
in 1959.
31

32. • Later, DNA-pol II and DNA-pol III
were identified in experiments using
mutated E.coli cell line.
• All of them possess the following
biological activity.
1. 5′→3′ polymerizing
2. exonuclease
32

33. DNA-pol of E. coli
33

34. DNA-pol I
• Mainly
responsible for
proofreading
and filling the
gaps, repairing
DNA damage
34

35. Klenow fragment
N end
DNA-pol Ⅰ
C end
caroid
• small fragment (323 AA): having 5´→3´
exonuclease activity
• large fragment (604 AA): called Klenow
fragment, having DNA polymerization
and 3´→5´exonuclease activity
35

36. DNA-pol II
• Temporary functional when DNA-pol I
and DNA-pol III are not functional
• Still capable for doing synthesis on
the damaged template
• Participating in DNA repairing
36

37. DNA-pol III
• A heterodimer enzyme composed of
ten different subunits
• Having the highest polymerization
activity (105 nt/min)
• The true enzyme responsible for the
elongation process
37

38. Structure of DNA-pol III
α ： has 5´→ 3´
polymerizing activity
ε ： has 3´→ 5´
exonuclease activity
and plays a key role to
ensure the replication
fidelity.
θ: maintain
heterodimer structure
38

39. 39

40. 40

41. DNA-pol of eukaryotes
DNA-pol α: initiate replication
and synthesize primers
DNA-pol β: replication with
low fidelity
DnaG,
primase
repairing
DNA-pol γ: polymerization in
mitochondria
DNA-pol δ: elongation
DNA-pol III
DNA-pol ε: proofreading and
filling gap
DNA-pol I
41

42. §2.2 Primase
• Also called DnaG
• Primase is able to synthesize primers
using free NTPs as the substrate and
the ssDNA as the template.
• Primers are short RNA fragments of a
several decades of nucleotides long.
42

43. 43

44. • Primers provide free 3´-OH groups to
react with the α-P atom of dNTP to
form phosphoester bonds.
• Primase, DnaB, DnaC and an origin
form a primosome complex at the
initiation phase.
44

45. §2.3 Helicase
• Also referred to as DnaB.
• It opens the double strand DNA with
consuming ATP.
• The opening process with the
assistance of DnaA and DnaC
45

46. §2.4 SSB protein
• Stand for single strand DNA binding
protein
• SSB protein maintains the DNA
template in the single strand form in
order to
• prevent the dsDNA formation;
• protect the vulnerable ssDNA from
nucleases.
46

47. §2.5 Topoisomerase
• Opening the dsDNA will create
supercoil ahead of replication forks.
• The supercoil constraint needs to be
released by topoisomerases.
47

48. 48

49. • The interconversion of topoisomers
of dsDNA is catalyzed by a
topoisomerase in a three-step
process:
• Cleavage of one or both strands
of DNA
• Passage of a segment of DNA
through this break
• Resealing of the DNA break
49

50. Topoisomerase I (topo I)
• Also called ω-protein in prokaryotes.
• It cuts a phosphoester bond on one
DNA strand, rotates the broken DNA
freely around the other strand to relax
the constraint, and reseals the cut.
50

51. Topoisomerase II (topo II)
• It is named gyrase in prokaryotes.
• It cuts phosphoester bonds on both
strands of dsDNA, releases the
supercoil constraint, and reforms the
phosphoester bonds.
• It can change dsDNA into the
negative supercoil state with
consumption of ATP.
51

52. 52

53. §2.6 DNA Ligase
3'
5'
5'
3'
RNAase
3'
5'
OH
dNTP
P
DNA polymerase
3'
P
5'
ATP
3'
5'
5'
3'
5'
3'
DNA ligase
5'
3'
53

54. • Connect two adjacent ssDNA strands
by joining the 3´-OH of one DNA
strand to the 5´-P of another DNA
strand.
• Sealing the nick in the process of
replication, repairing, recombination,
and splicing.
54

55. §2.7 Replication Fidelity
• Replication based on the principle of
base pairing is crucial to the high
accuracy of the genetic information
transfer.
• Enzymes use two mechanisms to
ensure the replication fidelity.
– Proofreading and real-time correction
– Base selection
55

56. Proofreading and correction
• DNA-pol I has the function to correct
the mismatched nucleotides.
• It identifies the mismatched
nucleotide, removes it using the 3´- 5´
exonuclease activity, add a correct
base, and continues the replication.
56

57. Exonuclease functions
5´→3´
exonuclease
activity
cut primer or excise
mutated segment
5'
3'
3´→5´
exonuclease
activity
excise mismatched
nuleotides
C T T C A G G A
3'
G A A G T C C G G C G
5'
57

58. Section 3
DNA Replication
Process

59. Sequential actions
• Initiation: recognize the starting point,
separate dsDNA, primer synthesis, …
• Elongation: add dNTPs to the existing
strand, form phosphoester bonds,
correct the mismatch bases, extending
the DNA strand, …
• Termination: stop the replication
59

60. §3.1 Replication of prokaryotes
a. Initiation
• The replication starts at a particular
point called origin.
• The origin of E. coli, ori C, is at the
location of 82.
• The structure of the origin is 248 bp
long and AT-rich.
60

61. Genome of E. coli
61

62. Structure of ori C
• Three 13 bp consensus sequences
• Two pairs of anti-consensus repeats
62

63. Formation of preprimosome
63

64. Formation of replication fork
• DnaA recognizes ori C.
• DnaB and DnaC join the DNA-DnaA
complex, open the local AT-rich
region, and move on the template
downstream further to separate
enough space.
• DnaA is replaced gradually.
• SSB protein binds the complex to
stabilize ssDNA.
64

65. Primer synthesis
• Primase joins and forms a complex
called primosome.
• Primase starts the synthesis of
primers on the ssDNA template using
NTP as the substrates in the 5´- 3´
direction at the expense of ATP.
• The short RNA fragments provide free
3´-OH groups for DNA elongation.
65

66. Releasing supercoil constraint
• The supercoil constraints are
generated ahead of the replication
forks.
• Topoisomerase binds to the dsDNA
region just before the replication
forks to release the supercoil
constraint.
• The negatively supercoiled DNA
serves as a better template than the
positively supercoiled DNA.
66

67. Primosome complex
Dna B
Dna A
Dna C
primase
3'
5'
3'
DNA topomerase
5'
67

68. b. Elongation
• dNTPs are continuously connected to
the primer or the nascent DNA chain
by DNA-pol III.
• The core enzymes (α 、、 and θ )
catalyze the synthesis of leading and
lagging strands, respectively.
• The nature of the chain elongation is
the series formation of the
phosphodiester bonds.
68

69. 69

70. • The synthesis
direction of the
leading strand is
the same as that of
the replication fork.
• The synthesis
direction of the
latest Okazaki
fragment is also the
same as that of the
replication fork.
70

71. 71

72. Lagging strand synthesis
• Primers on Okazaki fragments are
digested by RNase.
• The gaps are filled by DNA-pol I in the
5´→3´direction.
• The nick between the 5´end of one
fragment and the 3´end of the next
fragment is sealed by ligase.
72

73. 3'
5'
5'
3'
RNAase
3'
5'
OH
dNTP
P
DNA polymerase
3'
P
5'
ATP
3'
5'
5'
3'
5'
3'
DNA ligase
5'
3'
73

74. c. Termination
• The replication of E. coli is
bidirectional from one origin, and the
two replication forks must meet at
one point called ter at 32.
• All the primers will be removed, and
all the fragments will be connected
by DNA-pol I and ligase.
74

75. §3.2 Replication of Eukaryotes
• DNA replication is closely related
with cell cycle.
• Multiple origins on one chromosome,
and replications are activated in a
sequential order rather than
simultaneously.
75

76. Cell cycle
76

77. Initiation
• The eukaryotic origins are shorter
than that of E. coli.
• Requires DNA-pol α (primase
activity) and DNA-pol δ (polymerase
activity and helicase activity).
• Needs topoisomerase and replication
factors (RF) to assist.
77

78. b. Elongation
• DNA replication and nucleosome
assembling occur simultaneously.
• Overall replication speed is
compatible with that of prokaryotes.
78

79. c. Termination
3'
5'
5'
3'
3'
5'
5'
3'
3'
5'
connection of discontinuous
segment
5'
3'
3'
5'
5'
3'
79

80. Telomere
• The terminal structure of eukaryotic
DNA of chromosomes is called
telomere.
• Telomere is composed of terminal
DNA sequence and protein.
• The sequence of typical telomeres is
rich in T and G.
• The telomere structure is crucial to
keep the termini of chromosomes in
the cell from becoming entangled and
sticking to each other.
80

81. Telomerase
• The eukaryotic cells use telomerase to
maintain the integrity of DNA telomere.
• The telomerase is composed of
telomerase RNA
telomerase association protein
telomerase reverse transcriptase
• It is able to synthesize DNA using RNA
as the template.
81

82. 82

83. Significance of Telomerase
• Telomerase may play important
roles is cancer cell biology and in
cell aging.
83

84. Section 4
Other Replication Modes

85. §4.1 Reverse Transcription
• The genetic information carrier of
some biological systems is ssRNA
instead of dsDNA (such as ssRNA
viruses).
• The information flow is from RNA to
DNA, opposite to the normal process.
• This special replication mode is called
reverse transcription.
85

86. Viral infection of RNA virus
86

87. Reverse transcription
Reverse transcription is a process in
which ssRNA is used as the template
to synthesize dsDNA.
87

88. Process of Reverse transcription
• Synthesis of ssDNA complementary
to ssRNA, forming a RNA-DNA
hybrid.
• Hydrolysis of ssRNA in the RNA-DNA
hybrid by RNase activity of reverse
transcriptase, leaving ssDNA.
• Synthesis of the second ssDNA using
the left ssDNA as the template,
forming a DNA-DNA duplex.
88

89. 89

90. Reverse transcriptase
Reverse transcriptase is the enzyme
for the reverse transcription. It has
activity of three kinds of enzymes:
• RNA-dependent DNA polymerase
• RNase
• DNA-dependent DNA polymerase
90

91. Significance of RT
• An important discovery in life science
and molecular biology
• RNA plays a key role just like DNA in
the genetic information transfer and
gene expression process.
• RNA could be the molecule
developed earlier than DNA in
evolution.
• RT is the supplementary to the
91

92. Significance of RT
• This discovery enriches the
understanding about the cancercausing theory of viruses. (cancer
genes in RT viruses, and HIV having
RT function)
• Reverse transcriptase has become a
extremely important tool in molecular
biology to select the target genes.
92

93. §4.2 Rolling Circle Replication
3'
5'
3'
5'
3'
5'
93

94. §4.3 D-loop Replication
94