Molecular Genetics PCB4522 Spring 2004 Lecture 5Replicationpart D Dr' Eva CzarneckaVerner

1
Molecular GeneticsPCB4522 Spring 2004Lecture
5-Replication-part DDr. Eva Czarnecka-Verner

• Course web page
• http//PCB4522.IFAS.UFL.EDU

--Or go to Microbiology Cell Science home page
and look under course material.
2
Replication of E. coli chromosomeGenes VIII,
Chapter 14
3
Replication requires DNA Polymerase III
In E. coli 1. Single type of catalytic subunit
(dnaE) used in replication of both strands 2.
Active replicase is a dimer each half (enzyme
unit) contains dnaE subunit other proteins In
B. subtilis 1. Two different catalytic
subunits a) Pol C (homolog of E. coli dnaE)
synthesizes the leading strand b) dnaEBS
synthesizes the lagging strand In eukaryotes 1.
The same overall structure of DNA pol III 2.
Different enzyme units synthesize leading and
lagging strands 3. Not clear whether the same or
different catalytic subunits used
4
Replication requires DNA Polymerase III

• DNA polymerase III holoenzyme 900 kDa complex
• a) a catalytic core a subunit (dnaE)
• b) a 3-5 proofreading e subunit (dnaQ)
• c) a q subunit that stimulates exonuclease
• d) a dimerization component t that links two
  cores
• e) a processivity component b that keeps
  polymerase on DNA (clamp)
• f) a clamp loader g that places the processivity
  subunit b on DNA (complex of 5 proteins)

5
Forms of DNA polymerase III from biochemical
studies
Core
25 kD proofreading
e
a
q
10 kD
130 kD
structural-holds together
catalytic
6
Forms of DNA polymerase III from biochemical
studies
gd complex makes b clamp bind to primed template
Pol III
Asymmetric Only one gd complex gd complex adds a
pair of b dimers
c
d
y
Clamp loader
d
32 kD
g
52 kD
e
q
a
t
t
7
Assembly stages for Pol III holoenzyme
gd is the clamp loader
b dimer gd recognize primer-template
ATP
ADP P
b dimer clamps on DNA
Increased processivity
8
Assembly stages for Pol III holoenzyme
QAsymmetric only one clamp loader- why?
Lagging strand
Leading strand
b
c
d
y
b
e
b
b
d
g
b
b
q
a
e
q
a
t
t
t
ADifferent abilities to dissociate from DNA
t forms dimeric structure
holoenzyme
Q Which 2 subunits are encoded by the same
DNA? A tau (t) and gamma (g) use different
reading frames of the same DNA.
9
? subunits of DNA pol III (head to tail dimer)
b dimer makes holoenzyme highly processive
b dimer bound to DNA but slides along
Ice-skating
12 a-helices/6-fold symmetry
b dimer is ring shaped assembly or removal
requires energy (gd)
Interactions with DNA via water molecules
The clamp

• Fig. 14.18, Genes VIII by B. Lewin

10
Replication of E. coli chromosome
3
dnaB helicase
Pol III
b
5
1
11
Replication of E. coli chromosome
3
5
g
2
12
Replication of E. coli chromosome
3
5
3
13
Replication of E. coli chromosome
3
DnaG
5
g
RNA primer
4
14
Replication of E. coli chromosome
3
5
DnaG
The template for a lagging strand is pulled
through creating a loop in DNA
5
15
Replication of E. coli chromosome
3
5
The template for a lagging strand is pulled
through creating a loop in DNA
6
16
Replication of E. coli chromosome
2nd Core Pol III
3
b
5
The loop is released
7
1st Core Pol III
17
Replication of E. coli chromosome
3
g
5
New b clamp present on DNA
Core Pol III
8
18
Organization of the oriC Replication Fork
DNA is pulled through the primosome
ATPAMP
DnaB
b
Pol III core
DnaB/DnaC
DnaG primase stimulated by DnaB
Pol III core
b
t
Note the loading of DnaB helicase by DnaC only
occurs at the origin.
SSB
5 end of Okazaki fragment
3
5
19

• What is responsible for recognizing the sites for
  initiating synthesis of Okazaki fragments?

• Dual properties of dnaB helicase
• Propels the replication fork
• Interacts with dnaG primase at a correct site

20
Semidiscontinuous replication
The lagging strand fragments are known as
Okazaki fragments. Usually 1,000 to 2,000
bases in length.
5
5-CTG-3
GAppp-5
3
1
2
lagging strand
RNA primer (11-12 bases)
(RNA polymerasednaG primase)
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OriC Primosome- directed synthesis
Schematic of one side of the replication fork
DnaB
DnaG primase stimulated by DnaB
Related fact At oriC, the primosome consists of
DnaB and DnaG.
3
5
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DnaB
Role of DnaB 1.) propels the replication fork
through its helicase activity. 2.) required to
activate primase (DnaG).
DnaG primase stimulated by DnaB
3
5
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OriC Primosome- directed synthesis
t binds dnaB to attach pol III core to
replication fork.

• Speed of DNA synthesis increased 10x
• Prevent leading strand from falling off
  (increased processivity)

Role of Pol III 1.) Synthesis of leading
strand. 2.) synthesis of lagging strand by
extending the RNA primer. Displaces primase. 3.)
pulls the lagging strand template through the
holoenzyme.
DnaG (primase)
3
5
24

• oriC replication fork

25
Eukaryotic DNA pol a/primase
DNA Pol ?(I) primase complex- bifunctional
Heterotetrameric phosphoprotein
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Eukaryotic DNA Replication

• 1. DNA pol ? (I) / primase
• a) initiates synthesis of lagging and leading
  strands.
• b) RNA (10 b)-iDNA (20-30 b) primer.
• 2. DNA pol ? (III)
• a) elongates leading strand continuously
• b) highly processive (interacts with RF-C
  PCNA)
• c) can dimerize- may also elongate the lagging
  strand
• 3. DNA pol e
• a) may be involved in lagging strand synthesis
• b) other functions
• 4. Replication factor C (RF-C)
• a) clamp loader binds to 3 end of iDNA
  loads PCNA b) ATPase activity used to open PCNA
  ring

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Eukaryotic DNA Replication

• 5. PCNA (proliferating cell nuclear antigen)
• a) tethers DNA pol d to the template
• b) acts as processivity factor for strand (like
  b clamp) elongation
• d) trimer forms a ring that surrounds DNA
• 6. Replication factor RF-A
• a) single strand binding protein
• 7. Topoisomerases I II maintains DNA winding
• 8. Exonuclease MF1
• a) removes RNA primers
• 9. T antigen helicase T antigen loading
  helicase
• DNA ligase I
• a) seals the nicks

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Eukaryotic DNA Replication
Notes
Eukaryotic replication fork contains one complex
of DNA pola/primase two other pol complexes
either 2x ds or one d one d/e

• In mammalian systems (DNA pol has no 5-3 exo
  activity)
• Okazaki fragments removed by
• RNAse HI (specific for RNA-DNA hybrid)
  endonuclease cuts
• b) FEN1 exonuclease removes the RNA (5-3)

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Similar functions at bacterial and mammalian
replication forks
Function
E. coli
HeLa/SV40
helicase
DnaB
T antigen
loading helicase
DnaC
T antigen
single strand
SSB
RF-A
priming
DnaG
Pol a(I)/primase
sliding clamp
b
PCNA
clamp loading
gd
RF-C
catalysis
Pol III core
Pol d(III)
holoenzyme dimerization
t
???
RNA removal
Pol I
MF 1 exonuclease
ligation
ligase
ligase 1
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• Origins
• 1. ColE1 (RNA II acts as primer)
• 2. FC174 replicative form
• 3. oriC

hairpin
FC174
ColE1 plasmid
()
RNA II
primosome
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Creating replication forks at oriC

• 1. the strands melt at the origin over a short
  distance.
• 2. DNA is unwound.
• 3. first nucleotides synthesized into RNA primer.
  Occurs only once for leading strand-many times
  for lagging strand.

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Creating replication forks at oriC

• 1. Initiation at oriC starts with complex
  formation of 6 proteins
• a) DnaA, DnaB, DnaC, HU, Gyrase and SSB

2. DnaA uniquely involved in initiation
3. DnaB/C engineof initiation at origin
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Minimal oriC
DnaA binding
Region of melting
L
M
R
1
2
3
4
13-mers
9-mers
245 bp
GATCTNTTNTTTT
TTATNCANA
Note GATC is Dam methylation site 11 copies of
GATC in oriC
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Creating replication forks at oriC

• 1. Binding of DnaA to four 9 bp sites at on right
  side of the origin.
• 2. 2-4 DnaA monomers form a tetramer and DNA
  melts at the three 13 bp sites on the left side.
• 3. DnaB/DnaC joins the complex to form
  bidirectional replication forks.

35
Minimal oriC
2-4 monomers bind cooperatively
DnaA protein
(4) 9-mer sites
ATPAMP
DNA strands melted at (3) 13-mer sites
Central core
DnaB binds displaces DnaA from 13 bp repeats
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Minimal oriC
Q Does DnaA act as the titrator that measures
number of origins vs. cell mass?
A Mutations DnaA-replication asynchronous
overproduction of DnaA-initiation starts at
reduced cell mass
ATPAMP
37
Creating replication forks at an origin

• Other proteins are involved in replication
• 1. Gyrase acts as a swivel allowing one strand
  to rotate around the other.
• 2. SSB stabilizes single stranded DNA as it
  forms
• 3. HU (HU1/HU2) general DNA (double stranded
  single stranded) binding protein. Bends DNA
  structural role? Similar to histones. No
  cooperativity in binding. Causes DNA to bend and
  fold into structure that leads to open complex
  formation resembles beaded chromatin

38
Creating replication forks at an origin

• ATP required in replication
• 1. For helicase to unwind the strands
• 2. For gyrase to swivel strands
• 3. For primase to initiate
• 4. For DNA pol III to be activated

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Methylation state of DNA may regulate replication
Me
Active origin
(13 min. delay)
Active origin
Single round replication
Me
(N6)
Dam methylase
methylated DNA
Me
methylated DNA
Inactive origin
Me
hemimethylated DNA accumulates
40
Methylation state of DNA may regulate replication
Membrane-bound inhibitor- competes with DnaA for
oriC
Me
GATCnnnnnnnnnnn
hemimethylated DNA
CTAGnnnnnnnnnnn
Inactive origin
Dam methylase (delayed)
13 min. at oriC vs lt1.5 min for GATC elsewhere
in the genome
DnaA protein
Me
GATCnnnnnnnnnnn
dnaA promoter repressed also has delayed
methylation reduced level of DnaA protein
CTAGnnnnnnnnnnn
Me
Active origin
Inhibitor released DnaA can initiate
41
Methylation state of DNA may regulate replication
1. SeqA inhibitor binds to hemimethylated DNA
what delays re-replication 2. SeqA may interact
with DnaA 3. Hemimethylated origins bind to cell
membrane- inaccessible to methylases 4.
Methylated origins do not bind to membranes 5.
No clear connection between the origin and
membrane
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The end of lecture 5