replication

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I- Introduction :

During the Cell Cycle, Rejoinder cell contents and then divides into two giving :

  • New organization in unicellular beings
  • Maintenance of the’integrity of the’organism in multicellular beings (growth, renewal of lost cells by natural or programmed death (apoptosis)).

Duplicating P DNA results in the formation of two identical daughter molecules with one another and the parent molecule.

The precise mechanism of this duplication is called « Replication of the’ADN »

II- basic mechanisms of replication :

– These mechanisms are similar in prokaryotes and eucaiyotes.

  • The replication is done in the direction 5′ => 3′, complementarily, according to the rules of’base pairing : A=T / G = C.
  • It is antiparallel fashion (the complementary matrix strand is copied in the direction 3′ => 5′).

Replication is semi-conservative :

The parent compound gives one of its strands to each daughter molecule, which is complemented by a newly synthesized chain.

Meselson and Staid Experience 1958 :

Hypotheses :

Experience Meselson and Stahl itself :

To highlight the semi character- conservative of the replication of the’DNA of bacteria grown for several generations in a medium containing nitrogen molecules 14N are subcultured on a medium containing nitrogenous molecules 14N. Fractions are collected at regular intervals. L’DNA is extracted, placed in a cesium chloride solution and centrifuged 24. The position of DNA is identified by measuring optical density beyond.

– at t = Q : a single band corresponding to the DNA construction 15N

– After two generations in the medium containing l4N : appearance’a band having a density intermediate between that of heavy DNA1 *N and that of the light DNA l4N (hybrid DNA), which means that DNA molecules are formed from’original heavy unbnn and d’unbnn light neo synthesized.

– After three generations in the medium containing l4N : appearance of two bands, a corresponding hybrid (heavy, lightweight), l’other corresponding to molecules made up of two light bnn.

■ The DNA replication starts at one or more sites (s) called (s)ongine(s) replication (OR) then s’expands in the form of a bubble (s) replication. Each bubble has two replication forks that move apart’from each other. There are thus two replication systems that evolve in opposite directions. Replication is called bidirectional

NB :
There is only one replication ongin in prokaryotes, while there are several eukaryotic

Replication is semibatch :

The synthesis of the new bnn d’DNA always runs in the 5 ’to 3’ direction:

– the bnn read in the direction of the replication fork gives rise to continuous strand (bit early or advanced)

– the bnn read in the opposite direction of the fork gives rise to the discontinuous strand (late or secondary strand) synthesized in the form of small fragments.

Each replication fork is thus asymmetrical

■ Several enzymes are required for replication (helicase, primase, DNA polymerase … )

III- Replication in prokaryotes (E. coli) :

A- The different proteins involved :

  • Dna the protein A (initiation factor replication): binds to the origin of replication and allows replication initiation
  • helicases (DNA or B) : place the double helix by breaking the hydrogen bonds present between the nitrogen bases of the two strands of DNA P, with ATP consumption.
  • The SSB protein (pour single stranded binding protein) : have a high affinity for single stranded Y DNA and prevents it to retract when migrating replicative forks.
  • La primase : is a DNA dependent RNA polymerase which synthesizes primer (first egg), RNA primers consist of a nucleotide lOaine.


■ Topoisomerases : Decrease significantly the winding rate of DNA. They are 2 types :

– Topoisomérases de type I
Bind to DNA → cut 1 → single strand sequence of DNA repair →

– Type II topoisomerases intersect 2 strands

E-Coli type II isomerase is called l’ADN-gyrase.

  • Les ADN ligases: catalyzes the formation of phosphodiester made friends his, but is unable to move the nucleotides. It liaises Okazaki fragments
  • The Tus protein (Terminus substance use): reconnaitle termination site and terminates the withdrawal cation
  • Les ADN polymerases

– The enzyme catalyzes the formation of a phosphodiester bond between the free 3'-OH group of the last nucleotide beyond growing chain and the 5 'phosphate group of the nucleotide to be incorporated: the choice of nucleotide integrate respects pairing rules bases with the template strand (In paired with T and G with C paired).

– DNA polymerase needs the following compounds to synthesize a chain of’ADN :

– The four deoxyribonucleosides 5′-triphosphate (dATP, dGTP, dTTP, dCTP)

– a primer (primer) as acceptor of dNMP incorporated into the new strand.

– A matrix d’ADN simple brin.

– The Mg 2+.

at E. coli exists 3 DNA polymerases

* ADN polymérase I a 3 functions :

  • A polymerization function 5′ => 3′ for the replacement of primers’RNA by a strand d’DNA and gap filling during l repair’ADN.
  • An exonuclease function 5’=> 3’which will remove the primers’ARN
  • An exonuclease function 3’=> 5′ will eliminate evil paired nucleotides and progress in replacing them with correct nucleotide. This self-correcting activity reduces the error rate.

DNA polymerase I lacking the exonuclease function 5’=> 3Is called Klenow fragment

* DNA polymerase II : mainly involved in repairing the’DNA damaged.

* DNA polymerase III has 2 functions :

  • A polymerase function d’addition of dNMP to’end 3’OH d’a nucleotide chain. C’is this enzyme that works at the replication forks, is the répllcase
  • An exonuclease function 3’=>5′ as for DNA polymerase I

B- The steps of replication :

Replication takes place in three stages :

  • Initiation
  • Elongation
  • Termination

1- Replication initiation :

  • The replication starts at a precise and unique area of ​​the bacterial circular chromosome called Ori C replication origin
  • Ori = Sequences 300 Bp approximately formed of repetitive sequences. Specifically recognized by initiation proteins (DnaA)

  • Opening of the double helix making appear drafts of 2 replication forks
  • Fixing the Dna protein B (helicase).
  • forming two replication forks that move in opposite directions from each other on the chromosome.
  • Attaching SSBs (Single-Strand-binding protein) on single DNA strands to prevent their re-association.
  • Formation of primosome at each fork after adding the Dna G = RNA-dependent DNA polymerase ^ primase) pre-initiation complex (A dna, DnaB, SSB)
  • Synthesis of RNA primers (primer) thirty pair of nucleotides in length.

2- elongation :

– The replication fork moves along the template strand which is gradually denatured by helicases and son strands are synthesized by the replicase (DNA polymerase Ht)

– The replication fork moves in the 5'-3 'on one strand and 3'-5' on the other

– DNA polymerases will incorporate nucleotide at a 3'-OH end free (synthesis in the direction 54-3’)

On the leading strand :

  • The synthesis takes place continuously by elongation of the primer in the 5'-3 'as the parental duplex place.
  • SSB proteins are driven at progressively the template strand

On the lagging strand :

– A single stranded DNA sequence must be exposed

– A segment is synthesized in the reverse direction (relative to the movement of the fork). A series of these fragments (fragments d'Okasaki) from 1000 at 2000 Pb is synthesized each from 5 'to 3' (synthesis discontinuous).

– They are then connected to each other to give rise to a delayed intact strand.

– The template strand of the discontinuous strand must be wound to allow the DNA polymerase to replicate and move in the same direction as the fork.

On the leading strand :

– 1 single initiation event at the origin

On the lagging strand :

– A series of introductory events (1 par fragment d'Okasaki) each initiated by a primer

  • Removal and replacement of RNA primers by DNA polymerase I.
  • The DNA fragments formed are linked by a DNA ligase

3- Termination :

  • The termination occurs at the meeting of the two forks.
  • It is done at a TER sequence located opposite the origin of replication recognized by the Tus protein, The Tus-Ter complex blocks forks, ending the replication
  • When replication of a circular chromosome is complete, the 2 molecules obtained are joined together, like the links of a chain (concaténées).
  • Separation and ligation is done by topoisomerase IV (topoisomérase de type 2)


IV- Differences between eukaryotic and prokaryotic :

eukaryotes Procaryotes
replication origin multiple unique
Recognition of the replication origin T antigen A dna
DNA double strand separation of single-stranded DNA (helicase activity) T antigen Dna 6
Stabilizing proteins’ADN simple brin RP-A (Replication protein A) SSB
RNA primer Yes
– synthesized by polymerase alpha, followed by alpha and delta, or epsilon
– degraded by RNAse H -1 ET FEN 1
– Delta replaced by pol
Yes
– synthesized by primase (dNA G)
– followed by DNA pol III
– degraded, replaced by l’ADN pol I
DNA polymerases alpha :
– polymerase
– primase
– no d’exonuclease activity 3′->5′
delta :
– exonuclease activity 3′- >5′
– no primase
epsilon :
– exonuclease activity 3′- >5′
– no primase
beta :
-repair short fragments of’ADN
gamma :
– replica l’ADN mitochondrial
– exonuclease activity 3′- >5’
ADN pol III :
– polymerase
– exonuclease activity 3′- >5′
ADN pol I :
-exonuclease activity 5′- >3’
– polymerase
– exonuclease activity 3′- >5'these 2 past activities form Klenow
Fragments d’Okazaki 300 Pb 1000 at 2000 Pb
ADN ligase I, I II III ADN ligase

V- The replication in eukaryotes :

The DNA replication in eukaryotes takes place during the S phase of the cell cycle.

A- Initiation :

– Replication begins at replication origins. The T antigen recognizes this initiation site, it fixed and causes the opening of the DNA double helix by its helicase activity ; In the RP-protein binds to the single-stranded DNA regions to prevent their reassociation.

– L'DNA polymerase α (Pol a) synthesized RNA DNA primer of each continuous strand and each strand batch, through its primase activity, followed by a DNA fragment 20 at 30 nucleotides thanks to its DNA polymerase activity.

B- Elongation :

– The RF-C protein (replication factor C) recognizes the template-primer complex and recruits the PCNA protein (Proliferative Cell Nuclear Antigen) which causes the replacement of DNA polymerase α by DNA polymerase δ or DNA polymerase ε, the latter shows the synthesis as T antigen helicase function as continuously and that the topoisomerase I relaxes the voltage to the replication fork.

  • The Brim continued east synthétisé without interruption by the DNA polymerase δ or the DNA polymerase ε (DNA pol δ participe également à la réparation de ADN.
  • Lorsque sur le Brim discontinu the DNA polymerase δ (you ε) approaches the RNA-DNA primer synthesized previously, the latter is removed by RNase H1 (an endonuclease that detaches the sequence of ribonucleotides except that related to the first deoxynucleotide that is removed by exonuclease FEN-1).

– The DNA polymerase δ in the engagé synthèse du fragment of Okasaki "bouche est" by the ear from the amorce

– DNA ligase I ligation finally the 5'-P of the sequence of the primer of the Okazaki fragment upstream to the 3 'OH end of the Okazaki fragment downstream and so on.

C- Termination :

– When synthesis 2 copies is completed, topoisomerase 11 décatène the 2 chromosomes.

WE- Mitochondrial DNA replication :

the replication of the’Circular mitochondrial DNA is not limited to the S phase of the cell cycle.
It uses two replication origins and involves an intermediate structure 3 strands
The DNA polymerase is responsible for Gamma LADN mitochondrial replication whose
Replication is independent of T nuclear DNA.

the replication of the’Mitochondrial DNA is controlled by nuclear genes and involves
Many protein factors.

references

  1. Christian Moussard. Biochemistry and Molecular Biology .de Boeck 2th print 2011, ISBN :978-2- 8041-6229-0.
  2. Neddjma Ameziane ,Mark Bogard,Jerome Lamoril. Molecular Biology principle in clinical biology .Elsevier p50-60.ISBN 2-84299-685-2.
  3. Jack J.Pasternak. molecular genetic humaine.de boeck PL02-l 15.ISBN 2-7445-0147-6.
  4. Internet

Course Dr S. hannachi – Faculty of Constantine