Ch 10 Lecture (Replicon) Flashcards
replicon
A unit of the genome in which an individual act of replication occurs
Each contains an origin for initiation of replication
origin of replication
Physical site on DNA where DNA is unwound and replication is initiated
replicator
Cis-acting DNA sequences sufficient to direct the initiation of DNA replication
Origin is always part of replicator, but is often only a portion of it
terminus
A segment of DNA at which replication ends
bacterial chromosomes each contain how many origins of replication
one
Eukaryotic chromosomes contain a large number of c
unevenly spaced replicons
how many times are replicons fired during eukaryotic replication
once
semiconservative replication
Both strands of a parental duplex are separated
Each strand then acts as a template for synthesis of a complementary strand
replication fork
Point at which replication occurs
Initiated at the origin and then moves sequentially along the parental duplex
replication bubble
How a replicated region appears within unreplicated DNA when viewed by electron microscopy
Appearance does not distinguish between unidirectional and bidirectional replication
bacterial chromosomes are usually
circles that replicate bidirectionally from a single origin
the early stage of bacterial replication is sometimes called
a theta structure
oriC
The replication origin of E. coli
245 bp in size
DnaA
Bacterial replication initiator protein
Binds to DnaA boxes in oriC
Binding of DnaA to oriC is influenced by
interactions with ATP and ADP
OriC contains how many copies of the palindromic sequence GATC
11
The adenine in this palindrome is a target for methylation by
Dam methylase enzymes
where are dam methylation sites located
Dam methylation sites are scattered throughout the genome, but several overlap DnaA boxes
what does Dam do before replication begins
the adenines of the palindromic sequence are methylated on both parental strands by Dam
after replication, the DNA is considered
hemimethylated
hemimethylated origins
inhibit initiation of replication
only fully methylated origins can
initiate replication
The GATC sequences at hemimethylated oriC remain
hemimethylated following replication much longer than GATC elsewhere in genome
Hemimethylated promoter inhibits expression of
DnaA protein
therefore delays re-initiation of replication
Hemimethylation also allows cell to distinguish
parental and daughter strands from each other
Cell can determine which strand to use as a template for DNA repair
Three events must occur to initiate replication at oriC
- Protein synthesis of the DnaA protein
- Transcription of one of the two genes that flank oriC
- Synthesis of the cell wall
DnaA is the
licensing factor that must be made anew in each round of replication
opening of a transcription bubble assists
DnaA in opening the replication bubble
drugs that inhibit cell wall formation also inhibit
initiation of replication
Initiation at oriC begins with formation of a large six-membered protein complex
DnaA, DnaB helicase, DnaC, HU, gyrase, SSB
Only DnaA-ATP will bind a
fully methylated oriC
oriC must be associated with
the cell membrane for DnaA-ATP binding to occur
DnaA-ATP has an intrinsic ATPase activity that is stimulated by
ssDNA and phospholipids
DnaA-ADP is unable to
initiate replication
ssDNA is produced by
replication bubble formation
Origin of replication remains anchored to the cell membrane for
1/3 of the cell cycle
Daughter strands are unable to be
methylated while bound to membrane
Opening oriC involves
9bp and 13bp repeats within the oriC sequence
The 9bp repeats are the initial binding sites for
DnaA-ATP
DnaA contains two distinct
DNA binding sites
DnaA-ATP initially binds as
an extended multimeric form at the 9 bp repeats
Multimeric extended DnaA-ATP forms a
a helical core around which oriC is wound
steps of bacterial replication initiation
- DnaA-ATP initially binds as an extended multimeric form at the 9 bp repeats
- Multimeric extended DnaA-ATP forms a helical core around which oriC is wound
- Multimeric extended DnaA-ATP binds to the 13 bp repeats
- Multimeric extended DnaA-ATP converts to a compact form that twists open and stabilizes a ssDNA bubble
- DnaA then recruits two “pre-priming” complexes
- DnaB also catalyzes the loading of DnaG onto the DnaB-(DnaC-ATP) pre-priming complex at each fork
- Loading of DnaG onto the pre-priming complex stimulates the dissociation of ATP from DnaC-ATP
- DnaB will displace DnaA from oriC as it extends the bubble
- Gyrase is also recruited
- As helicase opens the bubble, the ssDNA is immediately stabilized by single-strand binding proteins
- Protein HU is also found at the replicon
Multimeric extended DnaA-ATP converts to a
compact form that twists open and stabilizes a ssDNA bubble
Transcription of flanking genes assists in
opening bubble
“pre-priming” complex
One hexameric DnaB bound to six DnaC-ATP monomers
One complex per replication fork in bidirectional replication
DnaB is
the helicase responsible for unzipping the replication fork along the parental duplex
DnaB cannot
initially denature dsDNA
DnaB can only extend
ssDNA that has already been opened
DnaC is the
DnaB helicase loader
DnaC serves as
a chaperone to repress DnaB activity until it is needed
DnaB also catalyzes the loading of
DnaG onto the DnaB-(DnaC-ATP) pre-priming complex at each fork
DnaG is the
primase required for initiating polymerase action on the leading and lagging strands at each fork
Loading of DnaG onto the pre-priming complex stimulates
the dissociation of ATP from DnaC-ATP
The dissociation of ATP from DnaC-ATP will cause
DnaC to be released from the pre-priming complex
The release of DnaC from the pre-priming complex will activate
DnaB
DnaB will displace
DnaA from oriC as it extends the bubble
what is gyrase
type II topoisomerase
Allows one DNA strand to rotate around the other
Reduces positive supercoiling ahead of each fork that would inhibit helicase action
As helicase opens the bubble, the ssDNA is immediately stabilized by
single strand binding proteins
protein HU
Stimulates initiation
General DNA binding protein
Thought to bend DNA and catalyze bubble formation
How does the cell prevent premature reinitiation od replication
- OriC is sequestered from Dam methylase
- The amount of DnaA available for binding to oriC depends upon competition for its binding to other sites
- Influence of Hda protein on ATPase activity of DnaA
How is OriC sequestered from Dam methylase?
The SeqA protein binds tightly to GATC palindromes in hemimethylated DNA
SeqA protein remains at the origin after initiation and inhibits Dam methylase action
SeqA prevents DnaA from fully binding to oriC and re-initiating replication
SeqA infrequently dissociates from oriC, allowing Dam to methylate new daughter strand
what prevents rebinding of SeqA
Dam methylase
More than … DnaA binding sites available outside of oriC
300
DnaA acts as
transcriptional regulator at the promoter for several genes
Control of binding many DnaA molecules at
dat locus
dat is a
a site away from oriC that has a large number of DnaA binding sites
Dat locus binds
Binds a larger number of DnaA than the origin
Hda is recruited to the origin by
DNA polymerase
Hda enhances
ATPase activity of DnaA
Exchange of DnaA-ADP for DnaA-ATP is
slow
replication proceedes at how many bp/min in bacteria and eukaryothes
eukaryotes 2000bp/min
bacteria 50kb/min
how long are the replicons on a chromosome
40-100 kb
Individual replicons are activated at characteristic times during
S phase
If all replicons fired simultaneously, mammalian genomes could be replicated in about
60 minutes
how long does replication take in somatic cells
more than 6 hours
Replicons near … are replicated first in the S phase
active genes
Heterochromatin is replicated … in the S phase
late
Most eukaryotic replicons do not have … like in bacteria
termination sequences
Replication forks continue until
they meet a fork coming from a neighboring replicon
Replication events seem to be localized to
foci
how many foci are in a nucleus
100-300
each focus contains how many replication forks
more than 300
A … is necessary for initiation of replication at each origin in eukaryotes
a licensing factor
Licensing factor is present in
the nucleus prior to replication
what happens to the licensing factor during S phase
Is removed, inactivated, or destroyed during the S phase
the licensing facor is resynthesized in
the cytoplasm
Initiation of another replication cycle becomes possible only after
nuclear membrane breaks down during M phase
functional origins in eukaryotes requires
relaxed chromatin
what is required in yeast for replication
Binding site for the origin replication complex (ORC)
A region of DNA with a high AT content
A binding site for proteins that adjust local chromatin structure and facilitate ORC binding
prereplicative complex
Complex of four components that must form in an ordered manner at each replicator in order to initiate replication
what is in the pre-rc
ORC, Cdc6, Cdt1, Mcm
ORC is … in all eukaryotes
highly concerved
when can ORC bind to origins
anytime
when in ORC activated
when a part of the pre-RC
prereplicative complex steps
- Binding of the origin recognition complex to an origin
- Cdc6 binds to the replicator only in late M phase and early G1
- Active Cdt1 is only present in G1
- Cdt1 escorts Mcm to the origin
- The pre-RC has now been formed
- Cdc6 and Cdt1 are released
- Mcm is fully loaded onto the origin
Cdc6 is deactivated and degraded as S-Cdk levels … in S phase
increase
what protein inactivates free Cdt1
geminin
Geminin is specifically degraded in
early G1
Mcm2-7 contains the eukaryotic
helicase
Activation of the helicases requires
Cdks
In the G1 phase, S-Cdk levels are low, so helicase loading is … and activation of helicase is…
allowed
not allowed
In the S, G2, and early M phases, S-Cdk levels are high, so helicase activation is…
Helicase loading is…
allowed
not allowed
Once helicases move beyond the origin, they must be … to initiate another replication event
reloaded
Cell must wait until the next … to initiate more replication events at origins
G1 phase
S-Cdk levels rise to their maxima in the
S phase
what happens when SCDK levels are at their maxima
Cdc45 and GINS bind Mcm
Mcm and ORC are phosphorylated
The helicase is activated
ORC is inactivated
The Cdc45-GINS-Mcm complex recruit additional proteins that form the active replicative helicase
The CMG helicases are loaded onto single stranded DNA and move away from the origin
ORC is displaced
Any inactive origins with bound ORC that are passed by the replication fork will be inactivated
