چرخه سلولي 10-12 6-8 4-5 1-2 go objectives: to know and explain about: what are needed for...
TRANSCRIPT
Objectives: To know and explain about:
What are needed for ReplicationTemplate properties Start site of replication( the
origin) Enzymes of ReplicationEnzymes of Replication Protein Factors of ReplicationProtein Factors of Replication Different Stages of ReplicationDifferent Stages of Replication Simultaneous DNA synthesis in
two forks (bidirectional) Simultaneous DNA synthesis in
two strands, (leading & lagging)
Objectives Objectives contcont..
Replication termination in E. Coli The fidelity of DNA replication How to ensure initiation of replication
occures only once per cycle ? How to control the multiple replicons
to be activated only once time in a single cell cycle
Comparison of replication in eu and pro Termination in eu Toxins and antibiotics that inhibit
replication
Replication( overall view)Replication( overall view) During cell division During cell division
WholeWhole DNA must DNA must replicatereplicate
The DNA double helix The DNA double helix unwindsunwinds
The exposed bases bind to The exposed bases bind to free floating nucleotides free floating nucleotides in the nucleoplasmin the nucleoplasm
DNA polymerase binds DNA polymerase binds the complimentary the complimentary nucleotidesnucleotides
Theory of ReplicationTheory of Replication
Bacteria grown in 14N media
Bacteria grown in 15 media
Transfer to 15 N media for one replication
Which one is correct?
Let’s check!
1- Template ( Double Stranded 1- Template ( Double Stranded DNA)DNA)
2- Building blocks ( dNTPs & NTPs)2- Building blocks ( dNTPs & NTPs) 3- Enzymes3- Enzymes 4- Energy ( Energy of dNTPs & ATP)4- Energy ( Energy of dNTPs & ATP) 5- Protein Factors5- Protein Factors
What are needed for What are needed for ReplicationReplication
The templateThe template
It is a double stranded DNAIt is a double stranded DNA It should have a specific sequence It should have a specific sequence
(origin) for replication to be started(origin) for replication to be started It is replicated in semiconservative It is replicated in semiconservative
mannermanner It is replicated bidirectionalyIt is replicated bidirectionaly
Replication: Is it bidirectional or Replication: Is it bidirectional or unidirectional / How to prove itunidirectional / How to prove it
By using HBy using H33T T with two with two different different concentratioconcentrations it was ns it was proved that proved that in most cases in most cases it is it is BidirectionalBidirectional
uUnidirectional
Bidirectional
Replication / The originReplication / The origin Replication starts from OriginReplication starts from Origin Origin has specific AT rich sequencesOrigin has specific AT rich sequences Because of the shape of the Because of the shape of the origin, replicating bacteria DNA is origin, replicating bacteria DNA is
called called tetateta form form
Initiation – establishment of replication forks
Localised melting of duplex
4 of 9 mer 3 of 13 mer
Ori
Teta form
A minimal Origin sequence is consisted of :
245bp
Enzymes of ReplicationEnzymes of Replication1- DNAPs ( DNA synthesis, gap filling)
2- RNAP =primase ( priming DNA synthesis)
3- Helicases ( opening the helix turns)
4- Topoisomerase (removing the superheix turns)
5- Ligase ( sealing the nicks between Okazaki fragments)
Function:Function:1- 51- 5’’ to 3 to 3’’DNA synthesisDNA synthesis2- 32- 3’’ to 5 to 5’’ Exonuclese activity (proof Exonuclese activity (proof reading) reading) 3- 53- 5’’ to 3 to 3’’ exonuclese activity just exonuclese activity just DNAP1 DNAP1
Structure:Structure: Is oligomeric and is composed of Is oligomeric and is composed of different subunitsdifferent subunits
Types:Types: DNAP I:DNAP I: fills gaps left by the removal fills gaps left by the removal of RNA primer and involves in DNA repair of RNA primer and involves in DNA repair Its Klenow fragment (C-large Its Klenow fragment (C-large fragment=68kDa) has fragment=68kDa) has 55’’-3-3’’ polymerase and 3 polymerase and 3‘‘-5-5’’ exonuclease activity exonuclease activity DNAP II:DNAP II: Involves in DNA repair Involves in DNA repairDNAP III:DNAP III: Chromosome replicating Chromosome replicating enzymeenzyme
22 - -Enzymes/a: DNA Enzymes/a: DNA polyerasespolyerases
DNA polymerase III holoenzyme is DNA polymerase III holoenzyme is very complex,with many polypepetidesvery complex,with many polypepetides
1- major subunits ,core
2-minor subunits
dsDNA β subunite
ε 3 5 exo….. 250-1000n/sα Polymerase activityDimerization of core enzyme ז
DNA polymerase I IIIII
5‘ 3‘
3‘ 5‘
5‘ 3‘ polymerase
Exonuclease( Proof reading)exonuclease
+ + +
+ + +
+ - -
TNO( nt/min) 600 30 9000
Mass (kDa) 103 90 900
Numbers/ cell 400 10-20???
Bioactivity 1 0.05 15
Gene pol C*
pol A pol B
DNA Polymerases in Bacteria
•DNAPs can not initiate DNA DNAPs can not initiate DNA synthesis synthesis (no de novo synthesis)(no de novo synthesis) and there must be small pre-and there must be small pre-existing primers existing primers
The enzyme responsible for The enzyme responsible for primer synthesis is called primer synthesis is called PrimasePrimase•It synthesize the primer from It synthesize the primer from 55’’ to 3 to 3’’•It has It has not exonuclease activitynot exonuclease activity•It is part of It is part of primosomeprimosome
22 - -Enzymes/b: Primase( Dna Enzymes/b: Primase( Dna G)G)
22 - -Enzymes/c: Enzymes/c: TopoisomerasesTopoisomerases•Enzymes which relieve stress on the DNA by allowing free rotation around a single or double stranded DNA
•There are two classes: 1 & 2
•Class 2 in bacteria is called gyrase.
•Gyrase reduces two turn of DNA each time ( convert +ve superhelix into –ve)
22 - -Enzymes/d: HelicasesEnzymes/d: Helicases
•There are different types of helicases :
• Dna B
• Other Helicases (rep protein)
Enzyme which catalyze the unwinding and separation ( bearking H-bonds) of the parental double helix by using ATP
Ligases seal nicks in DNAThe energy is provided by ATP or NAD
Phage--ATP
E.coli--NAD
*T4 DNA ligases can join two blunt DS DNA
22 - -Enzymes/e: LigaseEnzymes/e: Ligase
Eukaryote--ATP
Dna ADna A direct primosome to the direct primosome to the origin origin
Dna BDna B( Helicase activity) ( Helicase activity) Dna C & CDna C & C’’ are co-activator for are co-activator for
primaseprimase Dna GDna G( primase)( primase) SSBPSSBP binds to each ss DNA, keep binds to each ss DNA, keep
the separated strands apartthe separated strands apart Tus Tus recognize the recognize the terter sequence for sequence for
termination of replication termination of replication
Protein Factors of Protein Factors of ReplicationReplication
Different StagesDifferent Stages
Recognition Recognition of the Ori of the Ori
sequence by Dna Asequence by Dna A Initiation Initiation by primosomeby primosome
Elongation Elongation by replisomeby replisome
Termination Termination at ter sequence at ter sequence by tus proteinby tus protein
Initiation of replication at Ori C
Initiation of replication at oriC
• DnaA binds and begins to melt double helix
• Helicase (DnaB) continues to separate strands
1- Recognition of the origin by Dna A molecules2- Twisting of DNA around them result in a short unwinded DNA( loop) 3- Progressive unwinding of the DNA by Dna B and eye loop formation4-Assembly of primosome at replication origin5- Synthesis of the primer by primase followed by DNAP activity
Recognition & Initiation of replication
Different ways of Priming reaction in replication
1-Primase:RNA polymerasePrimer ~10 bases 2-Nicked
DNA
3‘-OH
3-Terminal protein:Ser OH
2- Elongation: 5’ to 3’ PDE bond formation
Replisome, a protein complex,associate with particular DNA structure to unwind the DNA and synthesis daughter strands.
Pyrophosphate hydrolysisis necessary for irreversibility of this reaction
The two strands are run in opposite direction The DNA synthesis is only from5’ to 3’The two strands are synthesized SimultaneouslyBut how ? One strand is made continuously (leading strand) and The other is made discontinuously (lagging strand, Okazaki fragments)
Simultaneous synthesis of two strands of DNA
OKAZAKI FRAGMENTOKAZAKI FRAGMENTIts structure:A short sequence(10 nt) of RNA primer
+ about 100 nt of DNA
How it is made:Primer by primase and DNA by DNAP III
How it is joined to the other fragments:First the primer of the previous Okazaki fragment is hydrolyzed and replace by DNAP I, then the two ends of DNA is linked by ligase
Priming
Extension
Removal of primer
Gap filling
Nick ligation
Discontinuous Replication Stages1- Primer synthesis by primase
2- DNA synthesis by DNAP III
3- Removal of primer by DNAP I
4- DNA synthesis by DNAP I
5- Sealing the nick between two Okazaki fragments by ligase
Nick translation: is referred to these last three stages
How the two strands are synthesize in same How the two strands are synthesize in same directiondirection??
E. coli replication
Synthesis of the two strands in same Synthesis of the two strands in same directiondirection
3- Termination of repli-cation in E.Coli
1- Ter sequences is aconsensus sequences with 23 bases 2- Tus protein is a 36 kDa protein that binds to the ter site3- Contra helicase activity of the Ter- Tus complex4- DNA replication stop5- Separation of strand by topoisomerase
Replication of circular DNA inE. coli (3.10):
1. Two replication forks result in a theta-like () structure.
2. As strands separate, positive supercoils form elsewhere in the molecule.
3. Topoisomerases relieve tensions in the supercoils, allowing the DNA to continue to separate.
The fidelity of DNA replication
Control at two different stages:
1- Presynthetic control: Control at the incoming base
2 -Proofreading:
Substitution Determined by 3’ to 5’ exonuclease activities
Replication in EukaryotesReplication in EukaryotesThe differences between Pro and EuThe differences between Pro and Eu
The Replication rate in eukaryotes could be low at least because:
1- DNA is so long2- There are many physical barriers
BUT
How to overcome this problem?
Having hundreds of origins
There are hundreds of Origin in There are hundreds of Origin in eukaryotic genome, each is called eukaryotic genome, each is called ARSARS
ARS (Autonomous replicating sequence)ARS (Autonomous replicating sequence)
The licensing factor in Yeast
ORC: origin recognition complex, bind to A and B1 in ARS
Cdc6, a highly unstable protein (half-life < 5 min), synthesisonly in the G1 phase
Cdc6, allow Mcm bind to complex
Replication initiation--Cdc6-Mcm are displaced
DNA polymerase δ
replicasePrimingreplication
repair
location
α β γ ε
Nu Nu NuNuMito
repairfunction replication
Different DNAPs In Eukaryotic
5‘-3’ plolymerization
3‘-5’ exonuclease
+ + + + +
- - + + +5’ -3‘exonuclease +- - - -
Different StagesDifferent Stages
Recognition Recognition of the ARS of the ARS
sequence by ORCsequence by ORC Initiation Initiation by DNAP by DNAP αα
Elongation Elongation by replisomeby replisome
Termination Termination by telomeraseby telomerase
Toxins and antibiotics that inhibit replication
Mitomycine: : makes cross link makes cross link between the two strands of DNA, between the two strands of DNA, preventing them of being preventing them of being templatetemplate
Nalidixic acidNalidixic acid: : Prevents gyrase Prevents gyrase activityactivity