سلولي سلولي چرخه چرخه

10-12

6-8

4-5

1-2 Go

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

DNADNA

DNADNA

DNA overall viewDNA overall view

1’

2’3’

4’

5’

(see, no oxygen at 2’…)Deoxyribose

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

The Permission

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 polymeraseDNA polymerase

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

Initiation of Replication in Initiation of Replication in EukaryotesEukaryotes

Elongation Nucleosome Elongation Nucleosome problemproblem

End Replication ProblemEnd Replication Problem

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

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 3.19 Synthesis of telomeric DNA by telomerase