Molecular Genetics PCB4522 Spring 2004 Lecture 3 Replication part B Dr' Eva CzarneckaVerner

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Molecular GeneticsPCB4522 Spring 2004Lecture
3- Replication- part BDr. Eva Czarnecka-Verner

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

--Or go to Microbiology Cell Science home page
and look under course material.
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The Replication/Replicon part BChapter 12
(Genes VII)
Chapter 13 (Genes VIII)
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Plasmid incompatibility is connected to the
regulation of copy number and segregation
The Replicon/Replication

• 1. Compatibility group a set of plasmids that
  are unable to coexist in the same bacterial cell.
  
• a) Both plasmids have the same type of origin.
  
• b) Copy number is controlled repressor (RNA
  I) that measures the concentration of origins
• 2. ColE1 plasmid as a negative control model for
  copy number and incompatibility system. (ca. 20
  copies per cell). Selfish plasmids with
  territorial rights.

.
.
Fig. 13.39 Genes VIII
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Plasmid incompatibility is connected with copy
number
1. Primer RNA 555 bases, starts upstream of the
ori and extends into the ori. a. cleaved by
RNase H (cuts RNADNA hybrids) b. 3 OH of RNA
serves as primer to initiate DNA synthesis. c.
only cut by RNase H if it is not duplexed with
antisense RNA (RNA I)
Fig. 13.40 Genes VIII
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Replication of ColE1 plasmid DNA-multicopy
control Replication starts with transcription
RNA II is required for priming DNA synthesis at
the origin. Positive regulation.
-555
-20
P-II
P-I
origin
P-I and P-II are promoters for the regulatory
transcripts.
Anti-sense RNA I (108 b) acts as a negative
regulator
RNA I is complementary to the 5-terminal region
of RNA primer II
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Replication of ColE1 plasmid DNA-multicopy control
-555
-20
-265
origin
P-II
3
P-I
RNA-loop structure is required to stabilize the
RNADNA persistent hybrid .
RNaseH creates the 3-terminus if loops are
present.
RNA II
5
ds stem
ss loop
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Replication of ColE1 plasmid DNA-multicopy control
-555
-20
-265
origin
P-II
3
P-I
RNA II serves as primer for DNA synthesis for
replication.
RNA II
Replicase
5
8
Replication of ColE1 plasmid DNA-multicopy control
-555
-20
-265
origin
P-II
P-I
RNA I
RNA II
RNA I hybridizes to the 5 terminal region of RNA
II and disrupts the RNA loops.
9
Replication of ColE1 plasmid DNA-multicopy control
-555
-20
P-II
origin
P-I
3
No replication but transcription continues
RNA I
5
RNA II
Without loops, RNA II is not able to form a
persistent hybrid at the origin.
DNA synthesis can not proceed without a stable
primer
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Plasmid incompatibility is connected with copy
number

• 1. RNA I antisense inhibitory transcript forms
  duplex with Primer RNA II.
• 2. The Primer RNARNA I duplex molecule is not
  cut at ori by RNase H, and the persistent hybrid
  at the origin is not formed.
• DNA synthesis is not initiated.

Fig. 13.40, G. VIII
Fig. 13.42, G. VIII
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New incompatibility group generated by mutations
Fig. 13.43, G. VIII
4. Mutations in Primer RNA/RNA I interaction
region may result in formation of a new
compatibility group. RNARNA duplex can not be
formed between the mutated RNA primer and the
original RNA I. The mutated and original
replicons no longer regulate each other.
Original mutant plasmids behave as members of
different compatibility groups.
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Plasmid incompatibility is connected with copy
number
Q How does RNA I negative regulator counts the
copy number?
A P-I promoter regulates the expression level of
RNA I regulatory transcript. 1) At low
levels of RNA I- replication occurs. 2) At
higher levels of RNA I- replication shut down.

13
Plasmid incompatibility is connected with copy
number
Q How many levels of regulation?
A Positive- Primer RNA II enhances replication
by providing 3OH end B Negative- RNA I
repressor shuts down replication but allows
continued transcription of RNA II C Negative-
Rom protein helps to shut down replication
because it enhances RNA primer/RNA I duplex
formation
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Origins of yeast replicons ARS (autonomously
replicating sequence)

• about 50 bp in length and very AT-rich.
• 2. contains two domains.
• a. A domain (14 bp) 11 bp core ARS consensus.
• Critical for ARS function
• Binding site for the origin recognition complex
  (ORC) which contains 6 proteins.
• b. B Domain mutations reduce ori function
• 1. several B elementsimperfect copies of the
  core ARS consensus (9/11).
• 2. transcription factor binding sites.

Fig. 13.10, G. VIII
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The yeast ARS
The ORC (origin recognition complex).
ORC
B2
B1
B3
A domain (core consensus)
B domains
imperfect consensus
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The yeast ARS
Origin recognition complex 1. 6 proteins
400,000 Dalton (400kDa) 2. must bind ATP before
it can bind to the ARS core consensus.
ORC
ATP
A domain (core consensus)
B1
Origin can function effectively with functional A
domain and any two B elements
ORC binds to A and B1 domains. Events at A and
B1 critical for initiation
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The yeast ARS
Fig. 14.40, G. VIII
ORC
ORC bound to ori through cell cycle (A-B1
protected against DNase hypersensitive site
in B1)
Early G1
Late G1
S phase DNA synthesis initiated
G2
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Licensing factor consists of MCM proteins Genes
VII, Chapter 13 Genes VIII, Chapter 14

• MCM2-7 proteins form a 6-protein ring complex
• ORC hydrolizes ATP and loads MCM onto DNA
• ORC identifies origin of replication for Cdc6
  MCM
• Cdc6 MCM control initiation and licensing
• . to replicate or not.to replicate?That is
  the question.
• MCM needed for initiation elongation
• a) may contribute to helicase activity to
  unwind DNA

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Licensing factor controls eukaryotic replication
Genes VII, Chapter 13 Genes VIII, Chapter 14,

• The eukaryotic genome tens of thousands of
  replicons. Each origin activated only once in a
  single cell division.
• A rate limiting factor involved. Used up by the
  replication event at each origin. Must be
  renewed after cell division to allow further
  replication at that origin.
• 3. Experimental System Xenopus eggs- can
  replicate DNA in a nucleus injected into an egg
  (devoid of original nucleus) without any new gene
  expression. Material with DNA can be in the form
  of a sperm nucleus or an interphase nucleus.

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Xenopus eggs as system to study eukaryotic DNA
replication
(replication with heavy DNA precursors LL, HL,
HH)
nucleus
Inject new nucleus
Cytoplasm
Cytoplasm
Remove nucleus
Egg cell
One round of DNA synthesis
Second round of DNA synthesis
Block protein synthesis
New protein synthesis leads to permeabilization
of nuclear membrane
No further DNA synthesis
Licensing factor must enter nucleus, but can not
pass the nuclear membrane.
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Licensing factor controls eukaryotic replication
One DNA replication licensing factor inactivated
nucleus
Cytoplasm
Licensing factor
Cell division breakdown of nuclear membrane
Licensing factor can not enter the nucleus.
nucleus
Cytoplasm
new licensing factor enters the nucleus
Licensing factor ensures that only single round
of DNA replication occurs.
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Components of the licensing factor

• In yeast MCM2, 3, 5 protein complex. Enters
  nucleus only during mitosis.
• In animal cells MCM2, 3,5 complex remains in the
  nucleus through the cell cycle. MCM3 bound to
  DNA before replication but released after it.
  Other component of LF (able to enter only at
  mitosis) may be necessary for MCM2, 3, 5 to
  associate with DNA (cdc6).
• Ubiquitination system mutations allow
  re-replication. Licensing factor degraded after
  start of replication.

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Mitochondrial origins

• 1. Purple sulphur bacteria able to handle oxygen
  gave beginning to eukaryotic mitochondria
• 2. Mitochondria are not sexually transmitted
• We all have maternal mitochondria
• How is mitochondrial dsDNA replicated?
• a) use different ori to initiate replication of
  each DNA strand
• b) replication of H-strand starts first in a
  D loop (new L strand produced)
• c) replication of L-strand starts (new H-strand
  produced ) when ori on the old L strand is
  exposed by the replication fork movement (RNA
  primer- then DNA)

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D loops maintain mitochondrial origins Genes VII,
chapter 12 Genes VIII, Chapter 13.7

• Single D loop (displacement loop), opening of
  500-600 bp in mammalian mitochondria.
• 2. Tetrahymena mitochondrial DNA has 6 D loops
  at a time (many origins), and plant chloroplasts
  have 2.
• 2. The new short strand is unstable, degraded and
  re-synthesized to keep the loop open.
• 3. Starts by synthesis of short RNA primer which
  may be extended by DNA pol (this is normal for
  all DNA synthesis)

Fig. 13.11, G. VIII
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D loops maintain mitochondrial origins
Replication of mammalian mitochondrial DNA has
separate origins for each strand
Origin on H strand
Origin on L strand
H strand
L strand
D loop displaced L strand is single stranded
H
L

• RNA primer synthesized by RNA polymerase L
  strand displaced
• 3 end cut by a 3-strand -specific endonuclese at
  specific sites
• 3 OH extended into DNA by DNA polymerase

Fig. 13.11, G. VIII
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Replication of mammalian mitochondrial DNA has
separate origins for each strand
L
H
H
H
L
L
Partially replicated
Duplex circle
H
Duplex circle
L
L
Fig. 13.11, G. VIII
Gaps in new strands are sealed.
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D loops may be maintained at mitochondrial origins
Fig. 13.11, G. VIII
The displaced strand of the D-loop remains
single stranded until the origin on the L strand
is reached. This second origin initiates H
strand synthesis. This illustrates the
principle that an origin may be used to initiate
a single strand only in the case of D loops and
rolling circle (PhiX174) modes of
replication. An origin can be a sequence of
DNA that initiates DNA synthesis using one strand
as a template
Re-defined origin of replication
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The problem of linear replications
Since the new strand is always synthesized in
the 5 to 3 direction, it is easy to finish the
new strand by running off the end of the
template, but how does the polymerase initiate at
the 3 end of the template strand?
DNA pol usually binds region that surrounds
origin.
3
Fig. 13.12, G. VIII
5
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The problem of linear replications
Solutions 1. convert linear to circular or
multimeric molecules (rolling circles)
examples lambda (?) phage is circular and T4
phage multimeric.
Fig. 13.18, G. VIII
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The problem of linear replications
Solutions 2. create unusual structure at the
end. example hairpin so there is no free end
linear mitochondrial DNA of Paramecium crosslinks
the ends.
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The problem of linear replications

• Solutions
• end may be variable.
• example
• Eukaryotic chromosomes have short sequence
  repeats at the termini.
• A separate mechanism adds or removes these
  repeats. It is not necessary to replicate to the
  end as long as some of the repeats are copied.

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The problem of linear replications

• Solutions
• protein intervenes.
• Often used by viral nucleic acids that have
  proteins that are covalently linked to the 5
  terminal base.
• adenovirus DNA (80 kDa)
• phage ?29 DNA
• poliovirus RNA (22 amino acids)

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Strand displacement
Fig. 13.13-15, G. VIII
Linear template Adenovirus 1. bottom strand is
used as template and the top strand is
displaced. 2. top strand forms terminal duplex
before initiating DNA synthesis.
5
Free single strand
3
5
5
3
3
Duplex origin formed by base pairing Replicated
independently
5
5
3
3
5
3
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Strand displacement Linear template Adenovirus
1. Terminal protein-dCTP/DNA polymerase complex
binds to 5 end of the top strand of adenovirus
DNA 2. 3 OH of dCTP serves as a primer for
DNA synthesis 3. New strand is covalently
linked to the initiating dCTP 4. Old TP is
displaced by the new TP for each new replication
cycle
5
5
5
3
3
Fig. 13.13-15, G. VIII
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Strand displacement Linear template Adenovirus
1. Terminal protein-dCTP binds to 5 end of the
top strand of adenovirus DNA between 9 18
nucleotide 2. Host protein, nuclear factor I,
essential for the initiation binds between 17
48 nucleotide 3. Initiation complex forms
between positions 9 and 48 at a fixed distance
from the actual DNA end
NF1
5
3
Fig. 13.13-15, G. VIII
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The end of lecture 3
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FC174 phage
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There are two types of DNA replication in E.
coli 1- FC174 phage each strand synthesized
separately (unidirectional replication
fork) a) synthesis of the (-) strand to form
the double-stranded RF form serves as a
model for lagging strand synthesis. b)
synthesis of the () strand to form
single-strands for packaging into phage
particles servers as a model for leading
strand sysnthesis. 2- OriC origin of bacterial
chromosomal replication both strands
synthesized at the same time (bidirectional
replication fork)
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??174 phage as a simple model for replication
Rolling circle replication
Lagging strand synthesis
()
()
strand
Replicative form (RF) ds plasmid
()
strand packaged to form virion
Rolling circle replication is a model for leading
strand synthesis.