DNA REPLICATION

University of the Punjab

Lahore

DNA STRUCTURE

In early 1900s scientist knew that

chromosomes are made up of DNA and

proteins containing genetic information.

However, they didn’t know whether protein or

DNA was actual genetic material.

DNA STRUCTURE

In 1940s various researches showed that

DNA was the genetic material.

In early 1950s structure of DNA was

determined.

STRUCTURE OF DNA

James Watson &

Francis Crick

determined the

structure of DNA in

1953.

STRUCTURE OF DNA

DNA is polynucleotide; nucleotides are

composed of a phosphate, a sugar and a

nitrogen containing base.

STRUCTURE OF DNA

Sugar in DNA is deoxyribose.

Four nitrogen bases in DNA

i. Adenine

ii. Guanine

iii. Thymine

iv. cytosine

STRUCTURE OF DNA

Watson and Crick showed that DNA is

double helix in which

A is paired with T

G is paired with C

This is called complementary base pairing

because a purine is always paired with

pyrimidine.

STRUCTURE OF DNA

DOUBLE HELIX

Each side of the double helix runs in

opposite (anti-parallel) directions.

The beauty of this structure is that it can

unzip down the middle and each side can

serve as a pattern or template for the other

side.

REPLICATION

Replica “copy”.

DNA making copies of itself, we call it DNA

replication .

WHY DNA REPLICATE ITSELF?

To reproduce, a cell must copy and transmit

its genetic information (DNA) to all of its

progeny. To do so, DNA replicates.

DNA carries information for making all of the

cell’s protein.

REPLICATION IN DIFFERENT CELLS

Different types of cells replicated their DNA at different rates.

Hair cells, finger nails, bone marrow cells. constantly devide.

Cells of brain, heart and muscles. cells go through several rounds of cell division and stop.

Skin cells and liver cells. stop dividing, but can be induced to divide to repair injury.

WHERE REPLICATION OCCUR?

In prokaryotes, DNA replication occurs in the

cytoplasm.

In eukaryotes, in the nucleus.

CLASSICAL MODELS FOR DNA

REPLICATION

Conservative

Semi conservative

Dispersive

CONSERVATIVE MODEL

Conservative Model

In this model the two parental DNA strands

are back together after replication has

occurred. That is, one daughter molecule

contains both parental DNA strands, and the

other daughter molecule contains DNA

strands of all newly-synthesized material.

SEMI CONSERVATIVE MODEL

Semi conservative Model

In this model the two parental DNA strands

separate and each of those strands then

serves as a template for the synthesis of a

new DNA strand. The result is two DNA

double helices, both of which consist of one

parental and one new strand.

DISPERSIVE MODEL

Dispersive Model

In this model the parental double helix is broken

into double-stranded DNA segments that, as for

the Conservative Model, act as templates for

the synthesis of new double helix molecules.

The segments then reassemble into complete

DNA double helices, each with parental and

progeny DNA segments interspersed.

CLASSICAL MODELS OF DNA REPLICATION

MESELSON AND STAHL EXPERIMENT

Nobody knew for sure how DNA replication

really worked until two scientists named

Matthew Meselson and Franklin Stahl

devised an ingenious experiment in 1958.

Show that DNA follows semi conservative

model to replicate itself.

MESELSON AND STAHL EXPERIMENT

Hypothesis

Experimental procedure

Result

HYPOTHESIS

Three hypotheses had been previously

proposed for the method of replication of

DNA.

Semiconservative hypothesis, proposed

by Watson and Crick.

Conservative hypothesis proposed that the

entire DNA molecule acted as a template.

Dispersive hypothesis is exemplified by a

model proposed by Max Delbruck.

EXPERIMENT DIAGRAM

RESULTS

1. Light DNA

2. Heavy DNA

3. Intermediate DNA

4. Light DNA &

intermediate DNA

RESULTS

Disproved conservative replication.

Disproved dispersive replication.

Proved that DNA replicates in

semiconservative manner.

SEMI CONSERVATIVE REPLICATION

Semiconservative replication describes the

mechanism by which DNA is replicated in all

known cells. This mechanism of replication

was one of three models originally proposed

for DNA replication.

REQUIREMENTS OF REPLICATION

DNA template.

Free 3’-OH group.

Proteins of DNA replication

DNA TEMPLATE

Template strand, that is to be copied.

Each old strand act as a template.

FREE 3’-OH GROUP

PROTEINS OF REPLICATION

1. DNA Helicases

2. DNA single-stranded binding proteins

3. DNA Gyrase

4. DNA Polymerase

5. Primase

6. DNA Ligase

HELICASE

DNA Helicases - These proteins bind to the

double stranded DNA and stimulate the

separation of the two strands.

DNA SINGLE-STRANDED BINDING

PROTEINS

DNA single-stranded binding proteins -

These proteins bind to the DNA as a tetramer

and stabilize the single-stranded structure

that is generated by the action of the

helicases. Replication is 100 times faster

when these proteins are attached to the

single-stranded DNA.

DNA GYRASE

DNA Gyrase - This enzyme catalyzes the

formation of negative supercoils that is

thought to aid with the unwinding process.

DNA POLYMERASE

DNA Polymerase - DNA Polymerase I (Pol I)

was the first enzyme discovered with

polymerase activity, and it is the best

characterized enzyme. The DNA

polymerases travel up the DNA molecule

from an initiation site which is a region along

the DNA that the enzyme complex can

recognize.

adds 5' C to 3' C in a phosphodiester linkage.

PRIMASE

Primase - The requirement for a free 3'

hydroxyl group is fulfilled by the RNA primers

that are synthesized at the initiation sites by

these enzymes.

DNA LIGASE

DNA ligase- forms a covalent

phosphodiester linkage between 3'-hydroxyl

and 5'-phosphate groups.

DNA POLYMERASE FUNCTION

Requires an RNA or DNA primer (RNA primer in

eukaryotes).

Reads DNA template in a 3'-->5- direction only

Synthesizes new strand in 5'-->3' direction only -

adds 5' phosphate to 3' hydroxyl group.

DIRECTION OF REPLICATION

It replicates from 3’ to 5’ of the template

strand.

From 5’ to 3’ of the newly growing strand.

STEPS OF REPLICATION

Initiation

Elongation

Termination

INITIATION

1. The first major step for the DNA Replication to take place is the breaking of hydrogen bonds between bases of the two antiparallel strands.

2. Helicase is the enzyme that splits the two strands

INITIATION

1. One of the most important steps of DNA Replication is the binding of RNA Primase in the initiation point of the 3'-5' parent chain.

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

ELONGATION

RNA primase lays down primers.

Replication starts at primer and lays down

nucleotides 5’ to 3’.

Leading strand goes continuously, lagging

strand goes discontinuously.

ELONGATION

The elongation process is different for the 5'-3' and 3'-5' template.

a)5'-3' Template: The 3'-5' proceeding daughter strand -that uses a 5'-3' template- is called leading strand because DNA Polymerase ä can "read" the template and continuously adds nucleotides (complementary to the nucleotides of the template, for example Adenine opposite to Thymine etc).

LEADING STRAND

Leading strand synthesis is continuous.

From 3’ to 5’ of the template.

ELONGATION

5'-3'Template: The 5'-3' template cannot be "read" by DNA Polymerase ä. 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 å reads the template and lengthens the bursts. The gap between two RNA primers is called "Okazaki Fragments".

LAGGING STRAND

Lagging strand synthesis is discontinuous.

Okazaki fragments.

Ligase joins discontinuous fragments.

ELONGATION

In the lagging strand the DNA Pol I -exonuclease- reads the fragments and removes the RNA Primers. The gaps are closed with the action of DNA Polymerase (adds complementary nucleotides to the gaps) and DNA Ligase (adds phosphate in the remaining gaps of the phosphate - sugar backbone).

TERMINATION

The last step of DNA Replication is

the Termination.

RNA primer is removed. Replaced with DNA

nucleotides.

DNA ligase joins okazaki fragments with

phosphodiester bonds.

Helicase rewinds DNA together.

TERMINATION

This process happens when the DNA

Polymerase reaches to an end of the

strands.

These ends of linear (chromosomal) DNA

consists of noncoding DNA that contains

repeat sequences and are called telomeres.

A part of the telomere is removed in every

cycle of DNA Replication.

TERMINATION

The DNA Replication is not completed before a mechanism of repair fixes possible errors caused during the replication. Enzymes like nucleases remove the wrong nucleotides and the DNA Polymerase fills the gaps.

TERMINATION

Protein which binds to this sequence to

physically stop DNA replication proceeding.

This is named the DNA replication terminus

site-binding protein or in other words, Ter-

protein.

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