Download MBBS DNA Replication Lecture PPT

DNA REPLICATION.

Dr. P.C.Mishra,MD.

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DNA replication is the process of

synthesis of an identical duplicate copy

of DNA from an existing DNA molecule.

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CENTRAL DOGMA.

? REPLICATION- synthesis of daughter DNA from parental DNA.
? Transcription--synthesis of RNA using DNA as the template.
? Translation---protein synthesis using mRNA molecules as the

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template.

? Reverse transcription--synthesis of DNA using RNA as the

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template.


CENTRAL DOGMA
DNA REPLICATION.

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? A reaction in which daughter DNAs are synthesized using the

parental DNAs as the template.

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? Transferring the genetic information to the descendant

generation with a high fidelity.

? DNA replication is a series of 3'-5' phosphodiester bond

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formation catalysed by a group of enzymes.


DNA replication system.

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? Template--double stranded DNA.
? Substrate--dNTP.
? Primer------short DNA fragment with a free ?OH end.
? Enzymes---DNA dependent poymerase(DDDP) and protein

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factor.
CHARACTERISTICS OF REPLICATION.

? Semi-conservative replication.

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? Bidirectional replication.
? Semi continuous replication.
? High fidelity.
Semi -conservative replication.

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? Half of the DNA molecule is conserved in each newly double

helix, paired with a newly synthesized complementary

strand.This is called semi conservative replication.

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? SIGNIFICANCE--The genetic information is ensured to be

transferred from one generation to the next generation with a

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high fidelity.

? MESELSON AND STAHL EXPERIMENT-
? semi -conservative replication

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was proposed by Watson and Crick.

? This experiment provided the proof of semi-conservative

replication of DNA.

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Meselson ? Stahl Experiment.

? Bacteria were grown in medium containing the heavy

isotopes of nitrogen N15 when all the DNA was labeled with

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heavy nitrogen. These cells were allowed to divide in a

medium containing normal nitrogen N14. In the first

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generation , all DNA molecule were half labeled.

? In the second generation half labeled and completely

unlabeled molecule were present in equal numbers.

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? From this experiment , it was prooved that DNA replication is

semiconservative.

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DNA replication is semiconservative.

(MESELSON AND STAHL EXPERIMENT)

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? Proposed by Watson and Crick.
? Meselson and stahl experiment provided the proof of

semiconservative replication of DNA.

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SEMICONSERVATIVE REPLICATION.
BIDIRECTIONAL REPLICATION.

? Replication starts from unbinding the dsDNA at a particular

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point ( called origin), followed by synthesis on each strand .

? The parental dsDNA and two newly formed dsDNA form a Y ?

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shaped strucure called replication fork.

? Once the dsDNA is opened at the origin , two replication forks

move in opposite direction as the synthesis continue.

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BIDIRECTIONAL REPLICATION.
SEMICONTINUOUS REPLICATION

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? The daughter strands on two template strands are

synthesized differently since the replication process obeys the

principle that DNA is synthesized from that 5' end to the 3'

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end.
LEADIND STRAND.

? On the template having the 3' ? end , the daughter strand is

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synthesized continuously in the 5'- 3' direction. This strand is

referred to as the leading strand.
Okazaki Fragments.

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? Many DNA fragments are synthesized sequentially on the DNA

template strand having the 5'- end. These DNA fragments are

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called okazaki fragments.

? The daughter strand consisting of okazaki fragment is called

lagging strand.

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SEMI-CONTINUOUS REPLICATION.

? Continuous synthesis of the leading strand and discontinuous

synthesis of the lagging strand represent a unique feature of

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DNA replication . It is referred to as the semi ? continuous

replication.
ENZYMES AND PROTEIN FACTOR.

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Dna A protein

Recognise origin.

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DnaB protein

Open dsDNA

DnaC protein

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Assist DnaB binding.

DNA pol

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Elongate the DNA strand.

Dna G protein

Synthesized RNA primer.

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SSB/DNA topoisomerase

Single strand binding/release supercoil

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constrant.
DNA topoisomerase.

(prokaryotes)

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? DNA- pol I, DNA pol II, DNA pol III were identified in

experiment using mutated E. Coli cell line.

? All of them possess following biological activity..

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? 5'-3 polymerizing.
? Exonuclease.
DNA POLYMERASE.

DNA-pol I

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Proofreading, filling the gaps, repairing

DNA damaze.

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DNA-pol II

DNA repairing

DNA pol III

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Elongation process.


POLYMERASES.

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DNA ?pol of eukaryotes

? DNA ? pol alpha--initiate replication and synthesize primers.--

-DnaG, primase.

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? DNA ? pol beta--replication with low fidelity.-repairing.
? DNA-pol gamma--polymerization in mitochondria.
? DNA ?pol delta--elongation--DNA-pol III.

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? DNA-pol epsilon? proofreading and filling gap.
PRIMASE

? Also called DnaG.
? Primase is the able to synthesize primers using free NTPs as

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the substrate and the ssDNA as the template.

? Primers are short RNA fragments of a several decade of

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nucleotides long.

primase privide free 3'-OH groups to react with the alpha ?P

atom of the dNTP to form phosphodiester bond.

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PRIMOSOME COMPLEX.

? Primase , DnaB, DnaC and origin form a primosome complex

at the initiation phase.

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? HELICASE--
? Also referred as Dna B.
? Opens the double stranded DNA.
? SSB protein---

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? maintain the DNA template in the single strand

form in order to prevent ds DNA formation.

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ENZYMES AND PROTEIN.
TOPOISOMERASE

? Opening the dsDNA will create supercoil ahead of replicate

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fork.

? Supercoil constraint needs to be released by topoisomerases.
? The interconversion of topoisomers of dsDNA is catalysed by a

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topoisomerase in a three step process-

? Cleavage of one or both strand of DNA.
? Passage of a segment of DNA through the break.
? Resealing of the DNA break.

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Topo I

? It cuts a phosphoester bond on one DNA strand, rotate the

broken DNA freely around the other strand to relax the

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constraint ,and release the cut.
Topoisomerase II

? It is named gyrase in prokaryotes.

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? It cuts phosphodiester bonds on both strands of dsDNA

,release the supercoil constrant, and reforms the

phosphoester bonds.

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? DNA Ligase----
? Sealing the nick in the process of replication,

repairing, recombination and splicing.

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DNA REPLICATION.

? INITIATION-
? recognise the starting point , separate dsDNA,

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primer synthesis.

? ELONGATION?
? add dNTP to the existing strand, form

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phosphodiester bond, correct mismatch bases, extending

DNA strand,

? TERMINATION--stop the replication.

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DNA REPLICATION.
Replication of prokaryotes.

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? INITIATION--
? The replication starts at a particular point called

origin.

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? The structure of the origin is 248 bp long and AT-rich.
Formation of replication fork.

? DnaA recognise ori C.
? DnaB and DnaC join the DNA-DnaA complex , open the local

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AT-rich region, andmove on the template downstream further

to separate enough space.

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? DnaA is replaced gradually.
? SSB protein binds the complex to stabilise ssDNA.


Replication fork.

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Primer synthesis.

? Primase joins and forms a complex called primosome.
? Primase starts the synthesis of primers on the ssDNA

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template using NTP as the substrate in the 5'-3' direction at

the expense of ATP.

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DNA REPLICATION.
ELONGATION

? dNTP arev continuously connected to the primer or the

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nascent DNA chain by DNA ?pol III.

? The core enzyme catalyse the synthesis of leading and lagging

strand , respectively.

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? Chain elongation is the series formation of the

phosphodiester bonds.

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? The synthesis direction of both leading strand and okazaki

fragments are same as that of replication fork.
LAGGING STRAND SYNTHESIS.

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? Primer on okazaki fragments are digested by Rnase.
? The gaps are filled by DNA ?pol in the 5'-3' direction.
? The nick between the 5' and of one fragment and the 3' end

of the next fragment is sealed by LIGASE.

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DNA REPLICATION.
TERMINATION.

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? The replication of E.coli is bidirectional from one origin and ,

and the two replication fork must meet at one point called ter

at 32.

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? All the primers will be removed , and all the fragments will be

connected by DNA-pol I ligase.
REPLICATION IN EUKARYOTES.

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? Basic principle of eukaryotic replication are same.
? Some major points of consideration are--
? There is relationship with cell cycle and replication. it occurs

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during synthetic phase of cell cycle and is regulated.

? There are multiple sites of origin proceeding bidirectionally.
? There are several DNA polymerases in eukaryote comparable

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to E.coli polymerases.
MAMMALIAN DNA POLYMERASES.

E.COLI CELLS

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MAMMALIN

FUNCTION

I

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Gap filling, repair

II

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Proof reading

DnaG

alpha

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primase

beta

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DNA repair

gamma

Mitochondrial DNA

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synthesis

delta

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Lagging strand synthesis

III

epsilon

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Leading strand synthesis.
DNA- PROOF READING MECHANISM.

? Replication is a high fidelity(accurate process).

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? Chance of incorporation of a wrong nucleotide are very less.
? The DNA polymerase III is the main enzyme responsible for

proof reading in E.coli.