ON DNA REPLICATION

PRESENTATION

SUBMITTED BYMANASMITA MAHARANA

ADM. NO.-08ABT/15

DNA• DNA stands for – This chemical substance is present in the nucleus of

all cells in all living organisms

– DNA controls all the chemical changes which take

place in cells

– It is the genetic material in prokaryotic and

eukaryotic cell

– Carry genetic information

Deoxyribose nucleic acid

• DNA is a very large molecule made up of a long chain of sub-units.The sub-units are called

• Each nucleotide is made up of(a)a sugar called - (b)a phosphate group - and(c)an -

Nucleotide

Deoxyribose

Organic nitrogenous basePO4

1.Purine or 2.Pyrimidine

STRUCTURE OF DNA

Deoxyribose Phosphate

Purine

Pyrimidine

The bases always pair up in the same way

Adenine forms a bond with Thymine

and Cytosine bonds with Guanine

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Adenine Thymine

Cytosine Guanine

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

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DNA – DOUBLE HELICAL STRUCTUREWATSON and CRICK- Model

DNA – DOUBLE HELICAL STRUCTURE

Three possible models were proposed for DNA replication:a. Conservative model proposed both strands of one copy

would be entirely old DNA, while the other copy would have both strands of new DNA.

b. Dispersive model was that dsDNA might fragment, replicate dsDNA, and then reassemble, creating a mosaic of old and new dsDNA regions in each new chromosome.

c. Semiconservative model is that DNA strands separate, and a complementary strand is synthesized for each, so that sibling chromatids have one old and one new strand. This model was the winner in the Meselson and Stahl experiment.

Three models for the replication of DNA

• Three experiments to confirm the semiconservative mode of replication are– Meselson & Stahl Experiment- In E.coli– Cairans Experiment- In E.coli.– Tayler’s Experiment- In Vicia faba

Messelson-Stahl Experiment• They(1958) grew E. coli in a heavy isotope of nitrogen,

15N in the form of 15NH4Cl. DNA containing 15N is more dense than DNA with normal 14N.

• Once the E. coli were labeled with heavy 15N, the researchers shifted the cells to medium containing normal 14N.

• DNA was extracted from each sample and analyzed in CsCl density gradients .

• After one replication cycle - all DNA had density intermediate between heavy and normal.

• After two replication cycles - there were two bands in the density gradient, one at the intermediate position, and one at the position for DNA containing entirely 14N.

• Does not fit conservative model, because after one generation there is a single intermediate band, rather than one with entirely 15N DNA and another with entirely 14N DNA.

The semiconservative model fits the data very well.

The Meselson-Stahl experiment

• J.Cairns in 1963 used the technique of autoradiography.• Cairns grew E.Coli bacteriawhose DNA is labelled

withheavy hydrogen-tritiated thymidine(3H-TdR). • The DNA was then carefully extracted from the bacteria

and placed on photographic emulsion for a period of time.

E.Coli DNA is a circle

DNA is replicated while maintaining the integrity of the circle. An intermediate theta structure is formed which is due to the formation of replication eye.

one or two moving Y-junctions in the circle Replication forks, which further supports the Semiconservative replication.

Cairans Experiment

•Taylor (1969) conducted his experiments with root tip cells of Vicia faba. •He treated root tips with radioactive thymidine to label the DNA.• Then root tips were grown in the normal medium. •In the 1st generation, both chromatids were labeled.•In the 2nd generation of cell division, one chromatid of each chromosome was labeled and one was normal.

This demonstrated semi- conservative mode of chromosome replication.

Taylor’s Experiment

DNA Replication • Basis for inheritance• Fundamental process occurring

in all cells for copying DNA to transfer the genetic information to daughter cells

• Each cell must replicate its DNA before division.

• Occurs during interphase of thecell Cylce• Semi conservative

Introduction

• Semi discontinuous• Leading & Lagging strandsLeading strand

Lagging strand

• The nucleotides arrive as nucleoside triphosphates• DNA base, sugar with PPP• P-P-P = energy for bonding• DNA bases arrive with their own energy source for bonding• bonded by enzyme: DNA polymerase III

continuous synthesis

• Okazaki fragments• joined by ligases

• Primer is needed • DNA polymerase can only add

nucleotides to 3 end of a growing DNA strand

• need a “starter” nucleotide to make a bond

• strand only grows 53. • Template is read in the 3-5 direction

while polymerization takes place in the 53 direction

Components of Replication

Deoxynucleotide polymerization -Processive unwinding of DNA -- Relieve torsional strain that

results from helicase-induced unwinding Initiates synthesis of RNA primers Prevent premature

reannealing of dsDNA -Seals the single strand nick between

the nascent chain and Okazaki fragments on lagging strand

DNA polymerases-

DNA ligase

Single-strand binding proteins-

RNA primase-

Topoisomerases

Helicase

STEPS OF REPLICATION1.Initiation of Replication2.Unwinding of Strands3.Formation of RNA Primer4.Synthesis of DNA on RNA Primer(Base Pairing)5.Removal of RNA Primer & Nick Sealing6.Union of Okazaki Fragments7.Termination8.Proof-reading and DNA Repair

1.Initiation of ReplicationOrigin of Replication-Prokaryotes

At the origin of replication (ori), there is an association of sequence-specific dsDNA-binding proteins with a series of direct repeat DNA sequences.

In E coli, the oriC is bound by the protein dnaA.

a complex is formed consisting of 150–250 bp of DNA and multimers of the DNA-binding protein. This leads to the local denaturation and unwinding of an adjacent A+T-rich region of DNA.

Origin of Replication -Eukaryotes The Autonomously replicating

sequences (ARS) contains a somewhat degenerate 11-bp sequence called the origin replication element (ORE).

The ORE binds a set of proteins, analogous to the dnaA protein of E coli, which is collectively called the origin recognition complex (ORC).

Functionally similar ARS or replicators have been identified in yeast cells.

The ORE is located adjacent to an approximately 80-bp A+T-rich sequence that is easy to unwind. This is called the DNA unwinding element (DUE).

2.Unwinding of Strands• Breaking of H bonds between bases of the 2 antiparallel strands.• The splitting happens in places of the chains which are rich in A-T. That is because there are only two bonds between A and T (there are 3 hydrogenbonds between C and G).

Helicase/Swivelase is the enzyme that splits the two strands. The structure that is created is known as "Replication Fork".

short, work to bind individuals strands in a DNA double stranded helix and aid the helicases in opening it up into single strands. These formations are particularly useful in stabilizing the unwound single-stranded

(a)Unwinding protein

(b)Single Strand Binding Protein-

(c)Nick Formation

1.TopoisomeraseClass I topoisomerases –cleave only one strand

Class II topoisomerases -2.Dna Gyrase-

(D)Relaxing of tension in the nicked strand

cleave both strands

A enzyme uses the energy of ATP hydrolysis to introduce negative supercoiling into DNA removing positive supercoiling generated during replication

Replication forkThe replication fork is a structure that forms within the nucleus

during DNA replication. - -unwinds a short segment of the parental duplex DNA; -initiates synthesis of an RNA molecule that is essential for priming DNA synthesis; -initiates nascent, daughter strand synthesis in 5’ to 3’ direction ; and -bind to ssDNA and prevent premature reannealing of ssDNA to dsDNA.•The polymerase III holoenzyme binds to template DNA as part of a multiprotein complex•DNA polymerases only synthesize DNA in the 5' to 3' direction, •Because the DNA strands are antiparallel , the polymerase functions asymmetrically. •On the leading (forward) strand, the DNA is synthesized continuously. •On the lagging (retrograde) strand, the DNA is synthesized in short (1–5 kb)fragments, the so-called Okazaki fragments.

(4) SSBs

(3)DNA polymerase

(2) Primase (1) DNA helicase

Formation of Replication Bubbles

• Replication occurs in both directions along the length of DNA and both strands are replicated simultaneously.• This replication process generates "replication bubbles"

3.Formation of RNA Primer• -in the

initiation point of the 3'-5' parent chain. can attract RNA nucleotides which bind to the DNA nucleotides of the 3'-5‘strand due to the H bonds between the bases.

•RNA nucleotides are the primers (starters) for the binding of DNA nucleotides.

RNA Primase

RNA Primase

4. Synthesis of DNA on RNA Primer (Base Pairing):•Enzyme responsible for base pairing isDNA Polymerase.This

enzyme will be in function if the buffer contains Mg2+ & Mn2+. •DNA Polymerase can function polymerase activity to pick up deoxyribonucleotide triphosphate[ATP, GTP, CTP and TTP].•They will be paired with the fitting nucleotide complemenatrily with that of template strand.

a)5'-3' Template: The 3'-5' proceeding daughter strand -that uses a 5'-3' template- is called leading strand because DNA

Polymerase III can "read" the template and continuously adds nucleotides (complementary to the nucleotides of the template, for example A opposite to T etc).

• (b)The 3'-5'template cannot be "read" by DNA Polymerase III. The replication of this template is complicated and the new strand is called lagging strand. In the lagging strand the RNA Primase adds more RNA Primers. DNA polymerase III reads the template and lengthens the bursts. The gap between two RNA primers is called "Okazaki Fragments".

5.Removal of RNA Primer & Nick Sealing

• Primers are removed by DNA polymerase I by replacing ribonucleotides with deoxy Ribonucleotides

• Nicks are sealed by DNA ligase• Multiple primers on the Lagging strand

while single primer on the leading strand.

6.Union of Okazaki fragments

• The discontinuous fragments of Okazaki are joined to make continuous strand.

• The union of Okazaki fragments takes place with the help of a joining enzyme called polynucleotide ligase.

• The replication may take place either in one direction(Unidirectional) or in both the directions (Bidirectional)from the point of origin.

7.Termination

• DNA replication terminates when replication forks reach specific “termination sites”.

• The two replication forks meet each other on the opposite end of the parental circular DNA .

In prokaryotes:

• This process happens when the DNA Polymerase reaches to an end of the strands & when the RNA primer is removed, it is not possible for the DNA Polymerase to seal the gap (because there is no primer).

• So, the end of the parental strand where the last primer binds isn't replicated. These ends of linear (chromosomal) DNA consists of noncoding DNA that contains repeat sequences and are called telomeres.

In eukaryote

8.Proof-reading and DNA Repair• 1000 bases/second =

lots of typos!• Enzymes like nucleases

remove the wrong nucleotides and the DNA Polymerase fills the gaps.

• DNA polymerase I – proofreads & corrects typos – repairs mismatched bases– removes abnormal bases• repairs damage

throughout life– reduces error rate from

1 in 10,000 to 1 in 100 million bases

THANK YOU