A molecular biology

1. Genetic Information
Transfer
1

2. DNA RNA protein
transcription translation
replication
reverse
transcription
Central dogma
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. 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 DNA
6

7. Daughter strand synthesis
• Chemical formulation:
(dNMP)n + dNTP (dNMP)n+1 + PPi
DNA strand substrate
elongated
DNA strand
• 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
OH
O
PO4
base
CH2
O
base
O
P
O
C
O
–O
CH2
1
2
4
5
1
2
3
3
4
5

9. Phosphodiester bond formation
9

10. 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
DNA replication system
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
synthesized complementary strand.
• This is called semiconservative replication
13

14. Semiconservative replication
14

15. Experiment of DNA semiconservative replication
"Heavy" DNA(15
N)
grow in 14
N
medium
The first
generation
grow in 14
N
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. 3'
5'
5'
3'
5'
3'
5'
3'
direction of
replication
Replication fork
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 
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. 5'
3'
3'
5'
bidirectional replication
fusion of bubbles
3'
5'
3'
5'
5'
3'
5'
3'
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. 5'
3'
3'
5'
5'
direction of unwinding
3'
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.
Leading strand
25

26. Semi-continuous replication
3'
5'
5'
3'
replication direction
Okazaki fragment
3'
5'
leading strand
3'
5'
3'
5'
replication fork
26

27. • 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.
Okazaki fragments
27

28. 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.
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. • The first DNA-
dependent DNA
polymerase (short for
DNA-pol I) was
discovered in 1958 by
Arthur Kornberg who
received Nobel Prize in
physiology or medicine
in 1959.
§2.1 DNA Polymerase
DNA-pol of prokaryotes
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 I
• Mainly
responsible for
proofreading
and filling the
gaps, repairing
DNA damage
33

34. 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
34

35. 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
35

36. 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
36

37. DNA-pol of eukaryotes
DNA-pol : elongation DNA-pol III
DNA-pol : initiate replication
and synthesize primers
DnaG,
primase
DNA-pol : replication with
low fidelity
DNA-pol : polymerization in
mitochondria
DNA-pol : proofreading and
filling gap
DNA-pol I
repairing
37

38. §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.
38

39. 39

40. • Primers provide free 3´-OH groups to
react with the -P atom of dNTP to
form phosphoester bonds.
• Primase, DnaB, DnaC and an origin
form a primosome complex at the
initiation phase.
40

41. §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
41

42. §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.
42

43. §2.5 Topoisomerase
• Opening the dsDNA will create
supercoil ahead of replication forks.
• The supercoil constraint needs to be
released by topoisomerases.
43

44. • 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
44

45. • Also called -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.
Topoisomerase I (topo I)
45

46. • 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.
Topoisomerase II (topo II)
46

47. 47

48. • 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.
48

49. §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
49

50. • 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.
Proofreading and correction
50

51. 3´→5´
exonuclease
activity
excise mismatched
nuleotides
5´→3´
exonuclease
activity
cut primer or
excise mutated
segment
C T T C A G G A
G A A G T C C G G C G
5' 3'
3' 5'
Exonuclease functions
51

52. Section 3
DNA Replication
Process

53. • 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
Sequential actions
53

54. • 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.
§3.1 Replication of prokaryotes
a. Initiation
54

55. • 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.
Formation of replication fork
55

56. • 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.
Primer synthesis
56

57. • dNTPs are continuously connected to
the primer or the nascent DNA chain
by DNA-pol III.
• The core enzymes (、、and  )
catalyze the synthesis of leading and
lagging strands, respectively.
• The nature of the chain elongation is
the series formation of the
phosphodiester bonds.
b. Elongation
57

58. 58

59. • 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.
Lagging strand synthesis
59

60. 3'
5'
5'
3'
RNAase
P
OH
3'
5'
5'
3'
DNA polymerase
P
3'
5'
5'
3'
dNTP
DNAligase
3'
5'
5'
3'
ATP
60

61. • 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.
c. Termination
61

62. §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.
62

63. Cell cycle
63

64. • The eukaryotic origins are shorter
than that of E. coli.
• Requires DNA-pol  (primase activity)
and DNA-pol  (polymerase activity
and helicase activity).
• Needs topoisomerase and replication
factors (RF) to assist.
Initiation
64

65. • DNA replication and nucleosome
assembling occur simultaneously.
• Overall replication speed is
compatible with that of prokaryotes.
b. Elongation
65

66. Other Replication Modes

67. §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.
67

68. Viral infection of RNA virus
68

69. Reverse transcription
Reverse transcription is a process in
which ssRNA is used as the template to
synthesize dsDNA.
69

70. 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.
70

71. 71

72. 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
72

73. 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 central
dogma. 73

74. Significance of RT
• This discovery enriches the
understanding about the cancer-
causing 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.
74