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Eukaryotic DNA Replication Chromosomesare denselyTheaccuracyaccuracyof.
Fertilised Egg CharacteristicsCharacteristicsof Budding yeast replication origins map within such ARSelements on both chromosomal and plasmid DNA .
ARS elements comprise a short 11 bp A element or ARSconsensus sequence 5 A T TTTA T C A G TTT A T 3 plus flanking regions of 100 200 bp B elements thatenhance origin function B3 B2 B1 ACS.
Which proteins bind to and defineeukaryotic replication origins ReplicationReplication originsorigins in.
in metazoansmetazoans somatic somatic cells The structure of replication origins inhigher eukaryotes is unclear .
Small extrachromosomal DNA sequencesreplicate poorly even when carrying 10kb genomic DNA known to act as originswhen in the chromosome Replication initiates at specific.
regions at a characteristic time in Sphase Both place and timing may changewith cell type Replication forks can potentiallyinitiate at a number of different sites.
The Origin Origin Number Number ParadoxGenome 4 Mb 4 x 106 bpFork rate approx 800 bp sec.
Replication time approx 40 minutes 2 400 secsAmount replicated by 2 forks in 40 mins 2 x 2400 x 800 3 840 000 bp 4 Mb EukaryotesGenome 20 Mb yeast up to 6 000 Mb human .
Fork rate 10 bp sec frog 50 bp sec mammal Amount replicated by 2 forks in 8 hr human cells 2 x 50 x28 800 2 880 000 3 Mb a 2 000 fold deficit 46 chromosomes human cells with one origin perchromosome at least 92 replication forks gives approx 140.
Mb replicated in 8 hours still a 40 fold deficit e solution eukaryotes replicate their chromosomes from multiple replication oriElectron micrograph showing an approx 300 kbstretch of replicating chromosomal DNA from the.
yeast S cerevisiae Replication forks areindicated by an arrow Petes Newlon Byers Fangman 1974 Cold Spring Harb Symp Quant Biol 38 9 16 ofreplication.
replicationoriginsoriginsusingfibreautoradiographyautoradiographyPulse label proliferating cells with 3H thymidine for 5 min pulse.
Dilute label to 1 5 activity for further 5 min pulse II Isolate DNA and spread on a photographic plateexpose for 6 monthsdevelop and examine grains under microscopeheavy labelling.
light labellingInterpretation Before pulse I End of pulse I End of pulse II .
ChromosomeChromosome regionsregions replicatereplicate atat different.
different timesa Pulse cells at different BrdU BrdUtimes with BrdU for 1 BrdUb Chase collect BrdUchromosomes BrdU BrdU.
c Stain with anti BrdUDuration BrdU BrdUantibodies 8 2 1 BrdU BrdUG1 S G2 M BrdU BrdU BrdU.
9 hr late S mid S early S2 hr chase5 hr chase9 hr chase BrdU pulse OrganizationOrganization of.
of replicationreplication duringduring SS phaseTypical somatic cellG1 template DNA.
early firing originsS late firing originsG2 duplicated DNA The global pattern of origin usage can also change egearly embryonic versus somatic cells .
Drosophila somatic cell transcriptionally active S phase 10 hours 600 mins mean origin spacing Early Drosophila embryo transcriptionally quiescent S phase 3 4 mins mean origin spacing 7 9kbEarly Drosophila embryo.
near synchronousS initiationmany origins o allow sections of the genome to replicate faster llow different sections of the genome to replicate at different time.
revent problems if origins do not initiate with 100 probability s origins are used to lower the probability of a lethal double stalstalled forkreplication completed byother fork of pair.
double stall no way of replicating intervening DNA Rate of progression of replication forks is fairlyconstant for a given organism Forks generally stop only when they encounter anoppositely.
movingChromosome forkreplication is regulated mainly throughcontrol of the initiation of new replication forksFor example .
by regulating the number and spacing of origins that fireeg during development by regulating the time during S phase at which differentorigins are activatedFacts IIII.
In somatic mammalian cells most inter origin distances replicon sizes are between 30 300 kb ie would take 5 50 min to replicate completely Some adjacent origins origin clusters typically 2 5 origins initiate synchronously.
Different origins origin clusters initiate atdifferent times during S phaseTypical mammalian cell replicates 6 000 Mb in 8 hr 6x 109 28 800 bp sec ie 200 000 bp secFor fork rate of 50 bp sec 200 000 50.
4 000 forks active at any given time in S phase Restoration of chromatin afterreplicationThe principle chromatinassembly reactions during.
DNA replication Reaction a parental nucleosomesare partially disrupted duringDNA replication and thehistones are directly.
transferred to the replicatedDNA reassembling intonucleosomes Reaction b the assembly of newnucleosomes from newly.
synthesized and solublehistones is mediated by achromatin assembly factor InitiationInitiation of.
replicationreplicationSV40 T antigen binds anddistorts the viralRP A replication.
protein A binds tothe single strandedDNA polymerase a primaseputs down an RNA primerand extends it with DNA .
RF C displaces pol a primase and loads PCNAto establish theleading strand Trykkfeil Cdt1 ikke Ctd1.
Mcm2 7Mcm2 7 mini chromosome maintenance proteins wereoriginally identified in yeast because as mutantsaffecting replication origin usage ractionation showed them to be a key component of Licensing Factor .
Highly conserved throughout eukaryotes archaea alsopossess an Mcm2 7 homologueThey are loaded onto DNA in anaphase and are removedfrom chromatin during S phase hey form a hexameric ring capable of encircling double stranded DNA.
Whether a cell has only one chromosome as in prokaryotes or hasmany chromosomes as in eukaryotes the entire genome must bereplicated precisely once of every cell division Two general principles 1 Initiation of DNA replication commitsthe cell to a further division Replication is controlled at the stage of.
initiation Once replication has started it continues until the entiregenome has been duplicated 2 If replication proceeds theconsequent division cannot be permitted to occur until thereplication event has been completed the unit of DNA in which an individual act of replication occurs .
site at which replication is initiated site at which replication stops A genome in a prokaryotic cell constitutes a singlereplicon thus the units of replication and segregation A plasmid is an autonomous circular DNA genome that.
constitutes a separate replicon may show single copycontrol or under multicopy control Any DNA moleculethat contains an origin can be replicated autonomously in Each eukaryotic chromosome contains a large number ofreplicons each must be activated no more than once in.
each cell cycle The DNA of mitochondria and chloroplasts may beregulated more like plasmids that exist in multiple copiesper bacterium Replicons Can Be Linear or Circular.
Key Concepts A replicated region appears as an eye within nonreplicated A replication fork is initiated at the origin and then movessequentially along DNA Replication is unidirectional when a single replication fork is.
created at an origin Replication is bidirectional when an origin creates tworeplication forks that move in opposite directions Figure 15 1 Replicated DNA is seen as a replication eyeflanked by nonreplicated DNA .
Figure 15 2 Replicons may beunidirectional or bidirectional depending on whether one ortwo replication forks are formedat the origin .
Figure 15 3 A replicatin eye forms a structure in circular DNA Origins Can Be Mapped by Autoradiography and ElectrophoresisKey Concepts Replication fork movement can be detected by autoradiography usingradioactive pulses .
Replication forks create Y shaped structures that change theelectrophoretic migration of DNA fragments Figure 15 5 Different densities of radioactive labeling can be used todistinguish unidirectional and bidirectional replication Figure 15 6 The position of the origin and the number of replicating forks.
determine the shape of a replicating restriction fragment which can befollowed by its electrophoretic path solid line The dashed line showsthe path for a linear DNA 15 4 Does Methylation at the Origin Regulate Initiation Key Concepts.
oriC contains eleven GATC CTAG repeats that are methylated onadenine on both strands Replication generates hemimethylated DNA which cannot initiatereplication There is a 13 minute delay before the GATC CTAG repeats are.
remethylated What feature of a bacterial or plasmid origin ensures that it isused to initiate replication only once per cycle Some sequences that are used for this purpose are included in theorigin oriC contains eleven copies of the sequence GATC CTAG .
which is a target for methylation at the N6 position of adenine bythe Dam methylase Figure 15 7 If the plasmid is methylated it undergoes a single round ofreplication and then the hemimethylated products accumulate Figure 15 8 Hemimethylated origins cannot initiate again until the.
Dam methylase has converted them into fully methylated origins Figure 15 7 Replication of methylated DNA gives hemimethylated DNA which maintains its state at GATC sites until the Dam methylase restoresthe fully methylated condition Figure 15 8 Only fully methylated origins can initiate replication .
hemimethylated daughter origins cannot be used again until they havebeen restored to the fully methylated state Origins May Be Sequestered after ReplicationKey Concepts SeqA binds to hemimethylated DNA and is required for delaying.
rereplication SeqA may interact with DnaA As the origins are hemimethylated they bind to the cell membraneand may be unavailable to methylases The nature of the connection between the origin and the membrane.
is still unclear Figure 15 9 A membrane bound inhibitor binds to hemimethylated DNAat the origin and may function by preventing the binding of DnaA It isreleased when the DNA is remethylated Each Eukaryotic Chromosome Contains Many Replicons.
Key Concepts Eukaryotic replicons are 40 to 100 kb in length A chromosome is divided into many replicons Individual replicons are activated at characteristic times during S Regional activation patterns suggest that replicons near one another.
are activated at the same time S phase usually lasts a few hours in a higher eukaryotic cell Individual replicons in eukaryotic genomes are relatively small typically 40 kb in yeast or fly and 100 kb in animal cells The rateof replication is 2000 bp min which is much slower than the.
50 000 bp min of bacterial replication fork movement A mammalian genome could be replicated in 1 hour if all repliconsfunctioned simultaneously S phase actually lasts for 6 hours in atypical somatic cell implying that no more than 15 of thereplicons are likely to be active at any given moment .
Visualization of replicating forks by labeling with DNA precursorsidentifies 100 to 300 foci instead of uniform staining each focusshown in Figure 15 11 probably contains 300 replication forks Figure 15 11 Replication forks are organized into foci in the nucleus Cells were labeled with BrdU The leftmost panel was stained with.
propidium iodide to identify bulk DNA The right panel was stained usingan antibody to BrdU to identify replicating DNA 15 7 Replication Origins Can Be Isolated in YeastKey Concepts Origins in S cerevisiae are short A T rich sequences that.
have an essential 11 bp sequence The ORC is a complex of six proteins that binds to an ARS Any segment of DNA that has an origin should be able to replicate so although plasmids are rare in eukaryotes it may be possible toconstruct them by suitable manipulation in vivo This has been.
accomplished in yeast although not in higher eukaryotes The discovery of yeast origins resulted from the observation thatsome yeast DNA fragments when circularized are able totransform defective cells very efficiently These fragments cansurvive in the cell in the unintegrated autonomous state that is .
as self replicating plasmids This segment is called as ARS for autonomously replicatingsequence ARS elements are derived from origins of replication Replication origins in metazoans (somatic cells) • The structure of replication origins in higher eukaryotes is unclear. • Small extrachromosomal DNA sequences replicate poorly, even when carrying >10 kb genomic DNA known to act as origins when in the chromosome. • Replication initiates at specific regions at a characteristic time in S phase.

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