1. DNA Replication  The basic rules for DNA replication
 DNA Polymerases
 Initiation of replication
 DNA synthesis at the replication fork
 Termination of replication
 Regulation of re-initiation
 Other modes of DNA replication

2.  Initiation of replication
 Common features of replication origins
 Common events of initiation
 Priming

3.  The replicon model of replication initiation:
(proposed by F. Jacob, S. Brenner and J. Cuzin, 1963)
All the DNA replicated from a particular
origin as a replicon.
Binding of the initiator to the replicator
stimulates initiation of replication.

4. Replicator
Initiator
Replicator: the entire set of cis-acting DNA sequences that is
sufficient to direct the initiation of DNA replication
(*the origin of replication is part of replicator)
Initiator: the DNA-binding protein that specifically recognizes
a DNA element in the replicator and activates the
initiation of replication

5. Priming and DNA synthesis
Easily melted region
Initiator binding site
Replicator
(Origin)
DNA binding
Initiator
DNA unwinding
The functions of initiator
Protein recruitment
Priming and DNA synthesis

6. E. coli oriC DnaA DnaB/DnaC DNA binding DNA unwinding
Protein recruitment
DnaB/DnaC
Priming and DNA synthesis

7. oriC : the origin of replication in E. coli
Figure 14.26
Easily melted
(AT rich)
Initiator (DnaA) binding site

8. oriC: L, M, R repeats (13 bp) 1~4 repeats (9 bp) GATCTNTTNTTTT
Consensus sequence
Consensus sequence
GATCTNTTNTTTT
CTAGANAANAAAA
TTATNCANA
AATANGTNT

9. Origin recognition complex (ORC)
The replicator (origin) of S. cerevisiae:
ARS (Autonomously replicating sequence)
~ 150 bp
Origin recognition complex (ORC)
(Initiation complex)
ACS: ARS consensus sequence
圖引用自：Cooper, G. M. (1997) The cell: a molecular approach. ASM Press. Fig. 5.17

10. Mutations in B elements reduce origin function.
Figure 13.20
Mutation in ARS
Mutations in B elements reduce origin function.
Mutations in core consensus abolish origin function.

11. E. coli oriC DnaA DnaB/DnaC DNA binding DNA unwinding
Protein recruitment
DnaB/DnaC
Priming and DNA synthesis

12. DnaA.ATP
DNA helicase
(DnaB)
DNA helicase
Loader (DnaB)
Figure 14.27
Watson, J. D. et al. (2004) Molecular Biology of the gene. 5th ed. CSHL Press. Fig

13. (histone like protein)
DnaAATP
HU + ATP
(histone like protein)
DNA bending
Super-helical tension
Strand separation
Binding of DnaA, plus the basic proteins, bends the DNA rather sharply and creates negative super-helical tension. In turn this tension causes DNA unwinding in the 13 bp regions.

14. DNA helicase
(DnaB)
DNA helicase
Loader (DnaB)

15. Figure 14.10

16. bind to the ssDNA, preventing it
Two types of function are needed to convert dsDNA to the single-stranded state:
Helicases separate the strands of
DNA, usually using the hydrolysis of
ATP to provide the necessay energy
2. Single-strand binding proteins
bind to the ssDNA, preventing it
from reforming the duplex state

17. Single-strand binding proteins
DNA helicase
(DnaB)
DNA helicase
Loader (DnaB)
Single-strand binding proteins
Primase

18. For DNA replication, a primase is required to catalyze the synthesis of RNA primer.
Primase in E. coli:
 An RNA polymerase
 Synthesizing short stretches of RNA
 Encoded by the dnaG gene

19. Figure 14.14

20. Protein required to initiate replication at the E. coli origin:
Summary
Protein required to initiate replication at the E. coli origin:
 Protein
 Function
DnaA protein
Recognizes origin sequence; open duplex
at specific sites in origin
DnaB protein (helicase)
Unwinds DNA
DnaC protein
Required for DnaB binding at origin HU
DNA bending protein; stimulates initiation
Primase (DnaG protein)
Synthesizes RNA primers
Single-strand DNA-binding protein (SSB)
Binds single-strand DNA
DNA gyrase
(DNA topoisomerase)
Relieves torsional strain generated by DNA
unwinding
Dam methylase
Methylates 5’-GATC-3’ sequences at oriC
The timing of replication initiation is affected by DNA methylation and interactions with the bacterial plasma membrane. The oriC region of E.coli is highly enriched in GATC sequences, containing 11 of them in its 245 bp.( See below for regulation of re-initiation).

21. DNA Replication  The basic rules for DNA replication
 DNA Polymerases
 Initiation of replication
 DNA synthesis at the replication fork
 Termination of replication
 Regulation of re-initiation
 Other modes of DNA replication

22.  DNA synthesis at the replication fork
 Proteins at the replication forks
Coordinating synthesis of the lagging
and leading strands
 in E. coli
 in eukaryotic cells

23.  DNA replication is semidiscontinuous.
Figure 14.9
Okazaki
fragments
nt in prokaryotes
nt in eukaryotes

24. DNA replicase (DNA polymerase)
Proteins required at the replication forks:
Topoisomerase
Helicase
Primase
SSB
DNA replicase
(DNA polymerase)
Primosome
Primer removal enzyme
DNA ligase
*SSB: Single-strand binding proteins

25. Different replicase units are required to synthesize the leading and lagging strands.
In E. coli both units contain the same catalytic
subunit of DNA Pol III.
In other organisms, different catalytic subunits
may be required for each strand.

26. The helicase creating the replication fork is connected to two
DNA polymerase
catalytic subunits.
Each polymerase
catalytic subunit is
held on DNA by a
sliding clamp.
Figure 14.19

27. synthesizes the lagging strand dissociates at the end of Okazaki
The polymerase that
synthesizes the lagging
strand dissociates at
the end of Okazaki
fragment and then
reassociates with a
primer in the single-
stranded template loop
to synthesize the next
fragment.
The polymerase that
synthesizes the
leading strand moves
continuously.
Figure 14.19

28. In E. coli:
DnaB
DNA Pol III
holoenzyme
DnaG

29. E. coli DNA Polymerase III holoenzymeg d d’ e q a t b c
Based on Figure 14.17

30. a a t t c y g d d’ Clamp loader b Sliding clamp b Sliding clamp e e q
proofreading e e
polymerization
polymerization a a q q
Core enzyme t t
Core enzyme dimerization

31. y d’ c y d’ c g d g d b y d’ c g d b b y d’ Pi c g d b b Clamp loader
ATP
ATP c y g d d’ b b b
Sliding
clamp
ADP c y g d d’ b b
ATP c y g d d’ Pi
ATP
hydrolysis b b

32. Processivity
Core enzyme: 10 ~ 15
Holoenzyme: >500000
b converts Pol III from a distributive enzyme to a highly processive enzyme.
Figure 14.18

33. a a t t y d’ c b g d b e e q q DnaB (helicase)
Leading strand synthesis
Lagging strand synthesis e e a a q q t t
t subunits maintain dimeric structure of Pol III and interact with DnaB
DnaB
(helicase)

34. DnaB (helicase) is responsible for forward movement at the replication
fork.
Each catalytic core
of polymerase III
synthesizes a
daughter strand.
Figure 14.20

35. What happens to the loop when the Okazaki fragment is completed?
Figure 14.21

36. 1 4 2 3 Initiation of Okazaki fragment Reassociation of b clamp
5. Reassociation of core 1 4
Initiation of Okazaki fragment
Reassociation of b clamp 2 3
Termination of Okazaki fragment
Dissociation of core and b clamp
Figure 14.20

37. Each Okazaki fragment is synthesized as a discrete unit.
Lagging strand
Primase synthesizes RNA primer.
Leading strand
DNA Pol III extends primer into Okazaki fragment.
Next Okazaki fragment is synthesized.

38. Okazaki fragments are linked together.3’ 5’ 3’ 5’
RNA primer
DNA Pol I uses nick translation to replace RNA primer with DNA.
Ligase seals the nick.
Figure 14.22

39.  5’3’ Exonuclease activity of DNA Pol I:
Figure 14.5

40. Mechanism of the DNA ligase reactionP O O-
Ribose
Adenine R
AMP
NAD+ (R = NMN)
or ATP (R = PPi)
Ligase
NH3 + +
Adenylylation of
DNA ligase 1
Ligase
NH2 + P O O-
Ribose
Adenine
+ NMN (or PPi)

41. 2 * 3 Activation of 5’ phosphate in nick O + Ligase NH2 P O Ribose
Figure 14.23 O 2 +
Ligase
NH2 P O
Ribose
Adenine O-
Activation of 5’ phosphate in nick
Ligase
NH3 + * 3

42. Adenine
Ribose O O- 3 P O O O-
The 3’-hydroxyl group attacks the phosphate and displaces AMP, producing a phosphodiester bond. P O O HO ●● 3’

43. Eukaryotic cell
Pold/e
Pold
PCNA
Pola/primase
(RFC)

44.  Eukaryotes have many DNA polymerases.
Figure 14.24

45. a + - d e Eukaryotic DNA polymerases for replication in nucleus: DNA
Primase
activity
Processivity
Proof-reading
Function a +
moderate -
Primer
synthesis d
High
Leading/lagging?
strand e
lagging
strand synthesis

46. DNA polymerase a (Pola/primase):
2 subunits: Pol a
DNA synthesis
2 subunits: primase
RNA synthesis 3’ 5’ 5’ OH 3’
RNA
DNA (iDNA)
~ 10 bp
20-30 bp

47. DNA polymerase switching during eukaryotic DNA replication:
DNA Pol a/ primase
RNA primer synthesis by primase a P
Watson, J. D. et al. (2004) Molecular Biology of the gene. 5th ed. CSHL Press. Fig
RNA
DNA synthesis by Pol a
iDNA

48. iDNA
Sliding
clamp *
Pola/primase
DNA Pol e (or d)

49. *
R-FC binds to the 3’ end of iDNA and displaces pol a/primase
R-FC attracts PCNA
PCNA binds pol d or e
RF-C: Clamp loader
PCNA: Sliding clamp

50. PCNA Proliferating cell nuclear antigen ： (trimer)
圖引用自：Voet, D., Voet, J. G. and Pratt, C.W. (1999) Fundamentals of Biochemistry. John Wiley & Sons, Inc. Fig. 24-1

51. DNA topoisomerases are also required!
Summary
Proteins required at the replication forks:
Figure 14.25
DNA topoisomerases are also required!

53. There are two ways to think of the relative motion of the DNA and replication machinery:
The replication machinery moves along
the DNA.
(similar to a train moving along its track)
2. The DNA moves while the replication
machinery is static.
(similar to film moving into a movie
projector)

54. The two replisomes of E. coli are linked together and tethered to one point on the bacterial inner membrane.
Pol III holoenzyme
Pol III holoenzyme
Helicases
(double-hexamers)
圖引用自：Nelson, D. L. and Cox, M. M. (2005) Lehninger Principles of Biochemistry. 4th Ed., Worth Publishers. Fig a

55. Cell elongates as replication continues
Origin
Cells divide
Replication begins
Chromosome
Terminator
Replisomes
Chromosomes
separate
Origins separate
Cell elongates as replication continues
圖引用自：Nelson, D. L. and Cox, M. M. (2005) Lehninger Principles of Biochemistry. 4th Ed., Worth Publishers. Fig a