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DNA Repilication

DNA replication is the process by which a cell makes an identical copy of its DNA before cell division. It occurs during the S phase of interphase and involves unwinding the DNA double helix, synthesizing new strands complementary to each existing strand, and rewinding the DNA into two new double helices. Key proteins involved include DNA helicase, gyrase, primase, DNA polymerase III, DNA polymerase I, and ligase. DNA polymerase builds new strands in the 5'-3' direction using the existing strands as templates while correcting any errors through proofreading.

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0% found this document useful (0 votes)
189 views57 pages

DNA Repilication

DNA replication is the process by which a cell makes an identical copy of its DNA before cell division. It occurs during the S phase of interphase and involves unwinding the DNA double helix, synthesizing new strands complementary to each existing strand, and rewinding the DNA into two new double helices. Key proteins involved include DNA helicase, gyrase, primase, DNA polymerase III, DNA polymerase I, and ligase. DNA polymerase builds new strands in the 5'-3' direction using the existing strands as templates while correcting any errors through proofreading.

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JeevikaGoyal
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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DNA Replication

The Cell Cycle

Two Main Stages


1. Interphase

Growth 1, Synthesis, Growth 2
2.Cell Division

Mitosis, Cytokinesis
THE CELL CYCLE
Telophase
Anaphase
Metaphase Cytokinesis
Prophase
G1
G2
S
INTERPHASE
Period between cell divisions
Cell undergoes growth, replication of DNA,
obtaining energy, making hormones,
repairing damage, fighting disease.
Prophase
Metaphase
Anaphase
Telophase
S Phase
• How does DNA replicate?
BACK TO INTERPHASE
DNA REPLICATION
•Produces two identical copies of the
chromosome during S phase of
interphase
•Catalyzed by many enzymes

Semi –conservative
Replication
Structure of DNA

The structure of DNA allows it to be easily


replicated
H bonds between base pairs can be broken
(unzipped) and each strand acts as a
template for adding new nucleotides
Meselson & Stahl’s
semiconservative experiment
E. coli bacteria were grown in an 15N growth
medium (heavy isotope of N). The 15
represents the atomic mass of the isotope.
Why N? Nitrogenous bases!
Over time and numerous replications all
(most) of the nitrogens within the bacteria
will be of the heavy 15N isotope.

Some of the bacteria grown in the 15N growth medium are
then placed in a 14N (normal and lighter isotope).

Any new made nucleotides will incorporate 14N isotopes.

After one round of replication some of the bacteria are
lysed and the DNA is isolated. We will call it Sample F1.

Some of the bacteria were allowed to complete 2
replications in the 14N medium before they are lysed and
the DNA is isolated. We will call this Sample F2.

Some of the bacteria were allowed to complete 3 or more
replications in the 14N medium before they are lysed and
the DNA is isolated. We will call this Sample F3.

Some of the bacteria were grown in the 15N medium are
lysed and the DNA is isolated. We will call this Sample F.

Remember centrifugation? More dense particles will travel
further.

The F1 sample when centrifuged gave only one band, with
a density midway between what would have been the
heavy (H) and the light (L - normal).

The F2 sample when centrifuged gave two bands, one with
density midway between H and L, and a band at the light.

The F3 sample when centrifuged gave the same 2 bands as
found when the F2 was spun. There was just more of the
light band.
F
Let’s rule out the Conservative
Heavy
model – The heavy band would
remain in subsequent replications
Light
as it is only used as a framework
for new stands to be formed.

Let’s rule out the Dispersive model –
After each replication parts of the heavy
parental are distributed to the next gen
while integrating more and more new
light nucleotides. This would result in a
single band that gets progressively lighter
after each replication.

Therefore the semiconservative model holds TRUE as the
parent strands act as templates for new strands to form.
This is proven by the F3 sample as the midway band will
remain present. Remember, the midway band represents
the original heavy parent strand combined with a light
strand.
DNA Replication Process
STEPS

1) Separating the DNA strands


2) Building complementary strands
3) Linking of Nitrogenous Bases
1. Separating DNA Strands
Initiated at sites along DNA called
Replication Origins – made of
specific nucleotide sequences
Enzymes and other proteins work
together to unwind and stabilize the
double helix
THE PLAYERS
•DNA helicase
•DNA gyrase
•SSBs
DNA Helicase
Recognizes specific nucleotide
sequence (origin of replication)
Unwinds double helix by breaking H
bonds between complementary base
pairs
Opens up replication bubble
DNA Gyrase
Relieves the tension produced
by the unwinding of DNA
Single-stranded Binding Proteins

SSBs
Keep separated DNA strands apart
by blocking hydrogen bonding
Keep the templates (single DNA
strands) straight
Replication Fork
The junction where the DNA strands
are still joined
2. Building complementary strands

Replication begins in two directions


from the origin(s) as a region of DNA is
unwound
Replication proceeds towards the
replication fork on one strand, and away
from the fork on the other strand
Because there may be
more than one origin of
replication in eukaryotes,
more than one replication
fork may exist
Link-replication animation:

Comp
THE PLAYERS

•DNA polymerase
•primase
•DNA ligase
DNA polymerase III
Takes free nucleotides found within
the cell and adds them in the 5’ to
3’ direction
The parent strand is used as a
template
Leading strand
The daughter strand that grows
continuously towards the replication
fork as the double helix unwinds
Occurs quickly
Requires a single RNA primer
Lagging strand
Built in short segments (in the 5’ to 3’
direction) away from the replication
fork
Requires many RNA primers
Lagging Strand Details
The 3’ to 5’ parent strand is a problem
for DNA polymerase since it must
synthesize in the 5’ to 3’ direction
Primase
Builds RNA primers (used to initiate DNA replication)

Okazaki Fragments
◻ Short fragments of DNA built by
DNA polymerase off of the RNA
primers
DNA polymerase I
Removes the RNA primers once they
have been used and replaces them with
the appropriate DNA sequence
DNA ligase
◻ Joins the Okazaki fragments into one
strand by the creation of
phosphodiester bonds
Link-leading vs lagging animation:

Leading Lagging
3. Linking Nitrogenous Bases
Nitrogen bases of nucleotides of
opposite strands (parent and daughter)
form new H bonds
Once bonds are formed, DNA twists
automatically into a double helix
QUALITY CONTROL


DNA polymerase acts as a proof-
reader by checking the newly
synthesized strand for any incorrectly
inserted bases
•If a mistake is found, act as an
exonuclease – & cut out the mistake
Summary
DNA helicase breaks H bonds and unwinds helix
Single-stranded binding proteins stabilize unwound DNA
DNA gyrase relieves tension
DNA is synthesized in a 5’ to 3’ direction by DNA
polymerase III from an RNA primer
DNA polymerase III builds in short segments called
Okazaki fragments
DNA polymerase I removes RNA primers and replaces
them with the appropriate nucleotide
DNA ligase joins fragments by completing the backbone
Animations
DNA Rep

DNA Rep2
Your Homework
Summarize Comparing DNA Replication in Euk and Prok. Know similarities
and differences

DNA Replication

Protein / Molecule Purpose

Helicase

DNA gyrase

RNA primer

Ligase

DNA polymerase I

DNA polymerase II

DNA polymerase II

Primase
Correcting Errors during DNA
Replication

A human cell can copy all of its DNA in a couple of hours
with an error rate of 1 per billion nucleotide pairs.

The most common type of error is mispairing, such that a T
is paired with G. fig 5.20

Another error is caused by strand slippage during
replication. This can result in addition or omission of
nucleotides in the newly synthesized strand. Fig 5.21

Most polymerases have the ability to proofread. The
textbook stresses Pol II for some reason.

When an incorrect nucleotide is placed on the newly
synthesized strand, replication is stalled as the 3’ end is in
the wrong position for the next to attach.

The incorrect nucleotide is then excised (along with a few
correct nucleotides) and DNA replication can continue.

Proofreading Video

Mismatch repair is another mechanism that corrects errors
in both eukaryotes and prokaryotes that were not fixed
during proofreading. The mechanism can also detect
insertions and deletions that happens with strand slippage.

A protein complex recognizes and binds to the mismatched
base. A second complex cuts the DNA near the mismatch,
and more enzymes chop out the incorrect nucleotide and
adjacent patch of DNA.

A polymerase will then add the correct nucleotides.
Telomeres

Telomeres are long, non-coding, highly-repetitive
sequences of DNA at the ends of eukaryotic chromosomes.

They are necessary as DNA synthesis can only occur in the
5’-3’ direction.

After the final RNA primer is placed on the lagging strand,
the extension on the parent chain will not be matched with
nucleotides.

Thus each subsequent replication results in shortened
telomeres. If the telomeres where not present, then coding
regions (genes) would be shortened and most likely not
functional. This would lead to cell death.

Leading strand synthesis is normal
but look at the lagging strand.

Removal of the last RNA primer
results in a gap.

The length of the telomeres
is closely linked to the
lifespan of an organism.

Telomerase is enzyme that
can synthesize telomeric
regions that have been lost.

Telomerase activity high
during childhood when
tissues are growing rapidly.
Telomerase activity slows in
somatic cells (body) as
people age.

If we could maintain high telomerase activity we would
age much slower.

However, high telomerase activity is what allows for
cancerous tumors to grow. Cancer is unregulated cell
proliferation. High telomerase activity allows for the tumor
cells to continue to replicate as important genes would not
be lost.
Comparing DNA Replication
in Eukaryotes and Prokaryotes


Review this section on Pg 227 for your text.

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