dna replication.botany
TRANSCRIPT
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DNA REPLICATION
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University of the Punjab
Lahore
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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.
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DNA STRUCTURE
In 1940s various researches showed that
DNA was the genetic material.
In early 1950s structure of DNA was
determined.
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STRUCTURE OF DNA
James Watson &
Francis Crick
determined the
structure of DNA in
1953.
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STRUCTURE OF DNA
DNA is polynucleotide; nucleotides are
composed of a phosphate, a sugar and a
nitrogen containing base.
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STRUCTURE OF DNA
Sugar in DNA is deoxyribose.
Four nitrogen bases in DNA
i. Adenine
ii. Guanine
iii. Thymine
iv. cytosine
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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.
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STRUCTURE OF DNA
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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.
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REPLICATION
Replica “copy”.
DNA making copies of itself, we call it DNA
replication .
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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.
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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.
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WHERE REPLICATION OCCUR?
In prokaryotes, DNA replication occurs in the
cytoplasm.
In eukaryotes, in the nucleus.
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CLASSICAL MODELS FOR DNA
REPLICATION
Conservative
Semi conservative
Dispersive
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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.
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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.
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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.
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CLASSICAL MODELS OF DNA REPLICATION
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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.
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MESELSON AND STAHL EXPERIMENT
Hypothesis
Experimental procedure
Result
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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.
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EXPERIMENT DIAGRAM
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RESULTS
1. Light DNA
2. Heavy DNA
3. Intermediate DNA
4. Light DNA &
intermediate DNA
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RESULTS
Disproved conservative replication.
Disproved dispersive replication.
Proved that DNA replicates in
semiconservative manner.
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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.
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REQUIREMENTS OF REPLICATION
DNA template.
Free 3’-OH group.
Proteins of DNA replication
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DNA TEMPLATE
Template strand, that is to be copied.
Each old strand act as a template.
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FREE 3’-OH GROUP
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PROTEINS OF REPLICATION
1. DNA Helicases
2. DNA single-stranded binding proteins
3. DNA Gyrase
4. DNA Polymerase
5. Primase
6. DNA Ligase
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HELICASE
DNA Helicases - These proteins bind to the
double stranded DNA and stimulate the
separation of the two strands.
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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.
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DNA GYRASE
DNA Gyrase - This enzyme catalyzes the
formation of negative supercoils that is
thought to aid with the unwinding process.
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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.
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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.
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DNA LIGASE
DNA ligase- forms a covalent
phosphodiester linkage between 3'-hydroxyl
and 5'-phosphate groups.
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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.
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DIRECTION OF REPLICATION
It replicates from 3’ to 5’ of the template
strand.
From 5’ to 3’ of the newly growing strand.
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STEPS OF REPLICATION
Initiation
Elongation
Termination
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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
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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.
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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.
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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).
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LEADING STRAND
Leading strand synthesis is continuous.
From 3’ to 5’ of the template.
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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".
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LAGGING STRAND
Lagging strand synthesis is discontinuous.
Okazaki fragments.
Ligase joins discontinuous fragments.
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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).
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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.
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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.
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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.
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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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