Replication

G C

A T

T A

1 nm

G C
3.4 nm
C G

A T

C G

T A

T A

A T

A T

G C
0.34 nm
A T

Figure 16.7a, c (a) Key features of DNA structure (c) Space-filling model
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 Quick Review
• DNA DNA is what?
• DNA RNA is what?
• RNA Protein is what?
• DNA Protein is what?

• Today we will focus on REPLICATION

– THINK! Where would this occur?

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 DNA replication
• The parent molecule unwinds, and two new
daughter strands are built based on base-
pairing rules

T
A T A T A A T A T A T
C G C G C G C G C G C G
T A T A T A T A T A T A
A T A T A T A T A T A T
G C G C G C C G C G C
G

(a) The parent molecule has two
complementary strands of DNA.
Each base is paired by hydrogen
bonding with its specific partner,
A with T and G with C.
(b) The first step in replication is
separation of the two DNA
strands.
(c) Each parental strand now
serves as a template that
determines the order of
nucleotides along a new,
complementary strand.
(d) The nucleotides are connected
to form the sugar-phosphate
backbones of the new strands.
Each “daughter” DNA
molecule consists of one parental
strand and one new strand.

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 DNA Replication is “Semi-conservative”

• Each 2-stranded
daughter molecule is
only half new
• One original strand was
used as a template to
make the new strand

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 DNA Replication
• The copying of DNA is remarkable in its speed and accuracy
• Involves unwinding the double helix and synthesizing two new
strands.
• More than a dozen enzymes and other proteins participate in
DNA replication
• The replication of a DNA molecule begins at special sites
called origins of replication, where the two strands are
separated

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 Origins of Replication
• A eukaryotic chromosome may have hundreds or
even thousands of replication origins

Origin of replication Parental (template) strand

0.25 µm
Daughter (new) strand

1 Replication begins at specific sites

where the two parental strands
separate and form replication
bubbles. Bubble Replication fork

2 The bubbles expand laterally, as

DNA replication proceeds in both
directions.

3 Eventually, the replication

bubbles fuse, and synthesis of
the daughter strands is
complete. Two daughter DNA molecules

(a) In eukaryotes, DNA replication begins at many sites along the giant (b) In this micrograph, three replication
DNA molecule of each chromosome. bubbles are visible along the DNA of
a cultured Chinese hamster cell (TEM).
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 Origins of Replication
• A prokaryotic DNA strand has 1 replication origin site
• This is because their DNA is CIRCULARso no
chromosomes per say….

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 Introduction to Major Players
• Helicase • dNTPs
• ssb/ssbps • Leading strand
• Topoisomerase/Gyrase • Lagging strand
• RNA Primase II • Okasaki fragments
• RNA Primer
• DNA Polymerase III
• DNA Polymerase I
• Ligase
• Nucleases
• DNA Polymerase II
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 Enzymes in DNA replication

RNA
Helicase unwinds Binding proteins Primase adds
parental double helix stabilize separate short primer
strands to template strand

DNA polymerase III DNA polymerase I Ligase joins Okazaki

binds nucleotides (Exonuclease) removes fragments and seals
to form new strands RNA primer and inserts other nicks in sugar-
the correct bases phosphate backbone 9
10
 Mechanism of DNA Replication
• DNA must be unwound at the origin site through helicase
• Single-stranded binding proteins (ssb/ssbps) stabilize new single strands
• Topoisomerase/gyrase prevents supercoiling upstream

Topoisomerase/
gyrase

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 Mechanism of DNA Replication
• DNA replication is catalyzed by DNA polymerase III which needs an RNA
primer
• RNA primase synthesizes primer on DNA strand
• DNA polymerase adds nucleotides to the 3’ end of the growing strand but
can’t just ‘start’. It MUST have a 3’OH from the RNA primer to start

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 Mechanism of DNA Replication
• Nucleotides are added by complementary base pairing with the template
strand
• The substrates, deoxyribonucleoside triphosphates, are hydrolyzed as
added, releasing energy for DNA synthesis.
• SO MUCH ATP!

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 The Mechanism of DNA Replication
• DNA synthesis on the leading strand is continuous
• The lagging strand faces opposite since the strand is antiparallel, but
DNA Poly. III still can only build in the 5’3’ direction
• Therefore, DNA synthesis on the lagging strand is discontinuous
• DNA is added as short fragments (Okasaki fragments) that are
subsequently ligated together (glued) by ligase

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15
DNA polymerase I degrades the
RNA primer and replaces it with
DNA

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 The Mechanism of DNA Replication
• Many proteins assist in DNA replication

• DNA helicases unwind the double helix, the

template strands are stabilized by other
proteins

• Single-stranded DNA binding proteins make

the template available

• RNA primase catalyzes the synthesis of short

RNA primers, to which nucleotides are added.

• DNA polymerase III extends the strand in the

5’-to-3’ direction

• DNA polymerase I degrades the RNA primer

and replaces it with DNA

• DNA ligase joins the DNA fragments into a

continuous daughter strand
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 Replication
3’
3’ 5’
5’
3’

5’ 3’
5’

Helicase protein binds to DNA sequences called

origins and unwinds DNA strands.
Binding proteins prevent single strands from rewinding.
Primase protein makes a short segment of RNA
complementary to the DNA, a primer.

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 Replication
Overall direction 3’
of replication
3’ 5’
5’

3’

5’ 3’
5’

DNA polymerase enzyme adds DNA nucleotides

to the RNA primer.

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 Replication
Overall direction
3’
of replication
3’ 5’

5’
3’

5’ 3’
5’

DNA polymerase enzyme adds DNA nucleotides

to the RNA primer.
DNA polymerase proofreads bases added and
replaces incorrect nucleotides.
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 Replication
Overall direction
3’
of replication
3’ 5’

5’
3’

5’ 3’
5’

Leading strand synthesis continues in a

5’ to 3’ direction.

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 Replication
Overall direction
3’
of replication
3’ 5’

5’
Okazaki fragment
3’

5’ 3’ 5’ 3’
5’

Leading strand synthesis continues in a

5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
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 Replication
Overall direction
3’
of replication
3’ 5’

5’
Okazaki fragment
3’

5’ 3’ 5’ 3’
5’

Leading strand synthesis continues in a

5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
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 Replication

3’
3’ 5’

5’
3’
5’ 3’ 5’ 3’5’ 3’
5’

Leading strand synthesis continues in a

5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.

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 Replication

3’
3’ 5’

5’
3’
5’ 3’5’ 3’5’ 3’
5’

Leading strand synthesis continues in a

5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.

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 Replication

3’
3’ 5’

5’
3’
5’ 3’5’ 3’5’ 3’
5’

Exonuclease activity of DNA polymerase I removes RNA

primers.

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 Replication

3’
3’

5’
3’
5’ 3’5’ 3’
5’

Polymerase activity of DNA polymerase I fills the gaps.

Ligase forms bonds between sugar-phosphate backbone.

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Replication Fork Overview

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 Proofreading
• DNA must be faithfully replicated…but
mistakes occur
– DNA polymerase III (DNA pol) inserts the wrong
nucleotide base in 1/10,000 bases
• DNA pol II has a proofreading capability and can correct
errors
– Mismatch repair: ‘wrong’ inserted base can be
removed
– Excision repair: DNA may be damaged by
chemicals, radiation, etc. (aka mutagens)
Mechanism to cut out and replace with correct
bases

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 Mutations

• A mismatching of base pairs, can occur at a

rate of 1 per 10,000 bases.
• DNA polymerases proofreads and repairs
accidental mismatched pairs.
• Chances of a mutation occurring at any one
gene is over 1 in 100,000
• Because the human genome is so large,
even at this rate, mutations add up. Each of
us probably inherited 3-4 mutations with
EACH replication!

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 Telomeres and Telomerases
• Telomeres are the ends of chromosomes
– Repeated sequences of Nonsense DNA (called
UTR -untranslated region)
• When replication happens at the end,
there is an overhang
– Due to DNA Pol.III
only being able to
build in the 3’ end
• Telomerase helps to
extend one end so the
overhang is lesser, but
it still exists and part will
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get cut off to make it even
 Dying of Old Age
• Telomeres and the overhang during replication is why we die
of old age
• The overhang gets cut off so the strands are even
• Gradually, we replace our cells (replication) and so the
telomere region gets shorter with every replication
• Eventually we cut into real genes because there is no more
telomeres

Prokaryotes
don’t have this
problem.
Why?

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 How has technology changed DNA?
Genetic Engineering: Technology used to manipulate an
organism’s DNA by inserting the DNA of another organism.
Transgenic Organism: Organism that is genetically
engineered by inserting a gene from another organism.
– Trans=across, genic=genes so means taking genes across
species so example: we like to make things glow

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 How to create Transgenic
• Plasmid-circular DNA
• CUT and then PASTE. That’s it. Just with enzymes.
This is how we produce
Insulin for diabetics. We
put the human insulin
gene in bacteria or pigs,
make them produce it,
then extract and refine

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 You should now be able to
• Name the location of replication
• Write out the key terms and players and
define their function
• Draw and label a replication fork/bubble
• Describe the difference between prokaryotic
and eukaryotic replication
• Describe why we die of old age and what
proteins we have to try to mitigate this

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