DNA Replication

DNA and
Replication
copyright cmassengale 1
History
of DNA
History of DNA
• Early scientists thought
protein was the cell’s
hereditary material because
it was more complex than
DNA
• Proteins were composed of
20 different amino acids in
long polypeptide
copyright cmassengale chains 3
Transformation
• Fred Griffith worked with
virulent S and nonvirulent R
strain Pneumoccocus bacteria
• He found that R strain could
become virulent when it took in
DNA from heat-killed S strain
• Study suggested that DNA was
probably the genetic material
•
Griffith Experiment
History of DNA
• Chromosomes are made
of both DNA and
protein
• Experiments on
bacteriophage viruses
by Hershey & Chase
proved that DNA was
the cell’s genetic
material
Radioactive 32 P was injected into bacteria!
Discovery of DNA
Structure
• Erwin Chargaff showed the
amounts of the four bases
on DNA ( A,T,C,G)
• In a body or somatic cell:

A = 30.3%

T = 30.3%

G = 19.5%

C = copyright
19.9% cmassengale 7
Chargaff’s Rule
• Adenine must pair with
Thymine
• Guanine must pair with
Cytosine
• The bases form weak
hydrogen bonds
T A G C
DNA Structure
• Rosalind Franklin took
diffraction x-ray
photographs of DNA
crystals
• In the 1950’s, Watson &
Crick built the first
model of DNA using
Franklin’s x-rays
Rosalind Franklin
DNA
Structure
DNA
• Two strands coiled called
a double helix
• Sides made of a pentose
sugar Deoxyribose
bonded to phosphate
(PO4) groups by
phosphodiester bonds
• Center made of nitrogen
bases bonded together
DNA Double Helix
“Rungs of ladder”
Nitrogenous
Base (A,T,G or C)
“Legs of ladder”
Phosphate &
Sugar Backbone
Helix
• Most DNA has a right-hand
twist with 10 base pairs in
a complete turn
• Left twisted DNA is called
Z-DNA or southpaw DNA
• Hot spots occur where right
and left twisted DNA meet
producing mutations
DNA
• Stands for
Deoxyribonucleic acid
• Made up of subunits
called nucleotides
• Nucleotide made of:

1. Phosphate group

2. 5-carbon sugar
3. Nitrogenous base

DNA Nucleotide
Phosphate
Group
O
5
CH2
O=P-O
O O
N
Nitrogenous base
C4 C1 (A, G, C, or T)
Sugar
(deoxyribose) copyright cmassengale 16
C3 C2
Pentose Sugar
• Carbons are numbered clockwise
1’ to 5’ 5
CH2
C4 C1
Sugar
(deoxyribose)
C3 C2
5
DNA
O 3
3
O
P 5
P
5
O
1 G C 3
2
4 4
2 1
3 5
O
P P
5
T A 3
O
5
P 3 copyright cmassengale P
18
Antiparallel Strands
• One strand of
DNA goes
from 5’ to 3’
(sugars)
• The other
strand is
opposite in
direction going
3’ to 5’
(sugars)
Nitrogenous Bases
• Double ring PURINES

Adenine (A)

Guanine (G) A or G

• Single ring PYRIMIDINES


Thymine (T)
T or C

Cytosine (C)
Base-Pairings
• Purines only pair with
Pyrimidines
• Three hydrogen bonds
required to bond Guanine
& Cytosine 3 H-bonds
•
G C
• Two hydrogen bonds are
required to bond Adenine
& Thymine
T A
Question:
• If there is 30%
Adenine,
Adenine how much
Cytosine is present?
Answer:
• There would be 20%
Cytosine
• Adenine (30%) = Thymine
(30%)
• Guanine (20%) = Cytosine
(20%)
• Therefore, 60% A-T and
40% C-G
DNA
Replication
Replication Facts
• DNA has to be copied
before a cell divides
• DNA is copied during the S
or synthesis phase of
interphase
• New cells will need identical
DNA strands
Synthesis Phase (S phase)
• S phase during interphase of the
cell cycle
• Nucleus of eukaryotes
S
DNA replication takes phase
place in the S phase.
G1 interphase G2
Mitosis
-prophase
-metaphase
-anaphase
-telophase
DNA Replication
• Begins at Origins of Replication
• Two strands open forming Replication
Forks (Y-shaped region)
• New strands grow at the forks 3’
5’ Parental DNA Molecule Replication

Fork
3’
5’
DNA Replication
• As the 2 DNA strands open at
the origin, Replication Bubbles
form
• Prokaryotes (bacteria) have a
single bubble
• Eukaryotic chromosomes have
MANY bubbles
Bubbles Bubbles
DNA Replication
• Enzyme Helicase unwinds
and separates the 2
DNA strands by
breaking the weak
hydrogen bonds
• Single-Strand Binding
Proteins attach and keep
the 2 DNA strands
separated and untwisted
DNA Replication
• Enzyme Topoisomerase attaches
to the 2 forks of the bubble to
relieve stress on the DNA
molecule as it separates
Enzyme Enzyme
DNA
DNA Replication
• Before new DNA strands can
form, there must be RNA
primers present to start the
addition of new nucleotides
• Primase is the enzyme that
synthesizes the RNA Primer
• DNA polymerase can then add
the new nucleotides
DNA Replication
• DNA polymerase can only add
nucleotides to the 3’ end of the
DNA
• This causes the NEW strand to be
5’
built in a 5’ to 3’ direction
3’
RNA
5’
DNA Polymerase
Nucleotide Primer
Direction of Replication
Remember HOW the
Carbons Are Numbered!
Phosphate
Group
O 5
CH2
O=P-O
O O
N
Nitrogenous base
C4 C1
(A, G, C, or T)
Sugar
(deoxyribose)
C3 C2
Remember the Strands are
5
O
Antiparallel 3
3
O
P 5
P
5
O
1 G C 3
2
4 4
2 1
3 5
O
P P
5
T A 3
O
5
P 3 copyright cmassengale P
36
Synthesis of the New DNA
Strands
• The Leading Strand is
synthesized as a single strand
from the point of origin
toward the opening replication
5’
fork 3’
5’
RNA
Nucleotides DNA Polymerase Primer
Synthesis of the New DNA
Strands
• The Lagging Strand is synthesized
discontinuously against overall direction of
replication
• This strand is made in MANY short segments
It is replicated from the replication fork
toward the origin
Leading Strand
5’ 3’

3’ 5’
DNA Polymerase RNA Primer
5’ 3’
3’ copyright cmassengale 5’
38
Lagging Strand
Lagging Strand Segments
• Okazaki Fragments - series of
short segments on the lagging
strand
• Must be joined together by an
DNA
Okazakienzyme
Fragment
Polymerase
RNA
Primer
5’ 3’
3’ 5’
Lagging Strand
Joining of Okazaki Fragments
• The enzyme Ligase joins the

Okazaki fragments together to
make one strand
DNA ligase
Okazaki Fragment 1 Okazaki Fragment 2
5’ 3’
3’ Lagging Strand
5’
Replication of Strands
Replication Point of Origin
Fork
Proofreading New DNA
• DNA polymerase initially makes
about 1 in 10,000 base pairing
errors
• Enzymes proofread and correct
these mistakes
• The new error rate for DNA that
has been proofread is 1 in 1
billion base pairing
copyright cmassengale errors 42
Semiconservative Model of
Replication
• Idea presented by Watson & Crick
• The two strands of the parental
molecule separate, and each acts as
a template for a new complementary
strand
• New DNA consists of 1
PARENTAL (original)DNAand 1 NEW
Template
strand of DNA
Parental DNA
New DNA
DNA Damage & Repair
• Chemicals & ultraviolet radiation
damage the DNA in our body cells
• Cells must continuously repair
DAMAGED DNA
• Excision repair occurs when any of
over 50 repair enzymes remove
damaged parts of DNA
• DNA polymerase and DNA ligase
replace and bond the new
nucleotides together
Question:
• What would be the

complementary DNA
strand for the following
DNA sequence?


DNA 5’-CGTATG-
3’ copyright cmassengale 45
Answer:

DNA 5’-CGTATG-3’

DNA 3’-GCATAC-5’
Mutability
and Repair
of DNA
Outline:
1.replication errors and their

repair
2. DNA damage
3.Repair of DNA damage
Section 1:replication
errors and their repair
Important definations:

• Transitions:a kind of the

simplest mutations which
are pyrimidine-to-
pyrimidine and purine-to-
purine substitutions
• Tranversions:the other kind
of mutation which are
pyrimidine-to-purine and
purine-to-pyrimidine
• Point mutations:mutations
that alter a single
nucleotide
• DNA microsatellites:
Mutation-prone sequence
in human genome are
repeats of simple di-, tri- or
tetranucleotide sequences,
known as DNA
microsatellites
The Nature of
Mutations
Base change substitutions
transitions transversions
 Other kinds of
mutations(which cause more
drastic changes in DNA):
• Insertions
• Deletions
• Gross rearrangements of
These mutations chromosome
might be caused by insertion by transposon
or by aberrant action of cellular recombination processes.
Some Replication Errors
Escape Proofreading
 The 3’-5’ exonuclease component of
replisome only improves the fidelity
of DNA replication by a factor of
about 100.

But, that’s not enough
 The misincorporated nucleotide
needs to be detected and replaced,
otherwise it will cause mutation.
A mutation may be introduced by misincorporation of a base
in the first round of replication.In the second round of
replication,the mutation becomes permanently incorporated
in the DNA sequence.
Mismatch Repair
Removes Errors That
Escape Proofreading
• Mismatch repair system:a
system that increases the
accuracy of DNA synthesis
by an additional two to
three orders of magnitude.
• This system faces 2
challenges:(1)rapidly find
the mismatches/mispairs
 (2) Accurately
correct the mismatch
Important parts of
mismatch repair system
MutS:a dimer of

the mismatch
repair protein
which detects
mismatches
Fuctions •of MutS:
1. MutS scans the DNA,
recognizes the mismatch
from the distortion they
cause in the DNA
backbone
Functions of MutS
2.MutS embraces the


mismatch-
containing DNA,
inducing a
pronounced kink in
the DNA and a
conformational
change in MutS
itself
•
Crystal structure of MutS
Further steps
of miamatch
repair,we
must pay
attention to
the other two
important
parts of the
mismatch
repair
system---
MutL and
MutH
How these three
parts interact
MutS-mismatch-containing

DNA complex recruits

MutL, MutL in turn
activates MutH, an
enzyme causing an
incision or nick on one
strand near the site of
the mismatch. Nicking is
followed by the specific
helicase and one of three
Then we talk
about :
 how does the E.coli
mismatch repair
system know which
of the two
mismatched
nucleotides to
(Dam)methylation
replace?
• Dam methylase:the E.coli
enzyme that methylases A
residues on both strands of
the sequence 5`-GATC-3`.

 The newly synthesized

strand is not methylated by
Dam methylase in a few
minutes after the synthesis.
a.Replication
generates
hemimethylat
Dam methylation at replication fork
ed DNA in
E.coli.
b.MutH
makes incision
in
unmethylated
daughter
strand.
Different
exonucleases
are used to
remove
single-strand
DNA between
the nick
created by
MutH and the
mismatch.
Eukaryotic cells
• In fact,eukaryotes
have multiple MutS-
like proteins with
different specificities.
• MSH proteins
MutS homologs
Section 2:DNA
Damage
There are mainly three kinds

of ways that DNA is

damaged:
• DNA undergoes damage
spontaneously from
hydrolysis and
deamination
• DNA is damaged by
Alkylation,Oxidation and
Radiation
1st kind, Hydrolysis &
Deamination
a.Deamination
that Cytosine to
Uracil
which explain
why DNA
contains T
instead of U
Answer:
 If DNA naturally
contained uracil instead
of thymine,the
deamination of cytosine
will create a natrual base
which the repair system
will not easily recognize.
2nd kind,Alkylation
Oxidation and Radiation
DNA is
subject to
attack from
Reactive
oxygen
species
(O2-,
H2O2,
OH•)
UV induces a cyclobutane
between adjacent
thymines
3rd kind,base analogs &
intercalating agents
Base analogs: similar

enough to the normal bases

to be processed by cells and
incorporated into DNA
during replication.
• But they base pair
differently, leading to
mistake during replication.
• The most mutagenic base
5-bromouracil,base anolog of
thymine,can mispair with
guanine
Intercalating
agents ØIntercalati
ng agents are
flat
molecules
containing
several
polycyclic
溴乙锭 rings that
interact with
the normal
bases in DNA
through
原黄素丫啶橙 hydrogen
bonds and
base
Section 3:Repair of DNA Damage
 There are two

consequences of DNA
damage:
• Some kinds of damage
create impediments to
replication or transcription
• Other kinds of damage
create altered bases that
cause mispairing which
results a permanent
Systems that repair damage to DNA
• A repair enzyme simply

reverses the damage
• Excision repair systems,in
which damaged nucleotide
is not repaired but
removed from DNA(more
elaborate step),composed
of base excision repair
and nucleotide excision
repair
Systems that repair damage to DNA
• Recombinational
repair,which is employed
when both strands of DNA
are damaged,also known
as double-strand break
repair.(more elaborate)
• Translesion DNA
synthesis,the last way
cells choose
Direct reversal of DNA
damage
For example,photoreactivation,which directly reverses pyrimidine dimers

Direct reversal od DNA
damage
Another example,methyltransferase( 甲基转移酶 ) directly removes the methyl group

from the O6-guanine residue
Base excision repair
systems
Base excision repair enzymes
—glycosylase recognize
and remove damaged bases
by a base-flipping
mechanism,hydrolyzing the
glycosidic bond.
DNA glycosylases are lesion-
specific.
1.The AP site is created by the hydrolysis of glycosylase bond.
2.AP endonuclease&exonuclease cut out the 5’ phosphate.
3.DNA polymerase fill in the gap.
The enzyme
The damaged base which

is filpped out
The DNA
Nucleotide excision repair systems
What is the difference

between the two kinds of
excision repair systems?
Also,how does the
nucleotide```work?
• Recognize distortions to
the shape of the DNA
double helix
• Remove a short single-
stranded segment
Once encountering a distortion UvrA
exits the complex and UvrB melts the
DNA to create a single-strand bubble
around the lesion.
Next,UvrB recruits UvrC,and UvrC
creates two incisions in different
positions on one strand.
Finally,DNA polymerase and ligase fill
in the gap.
Recombinational
repair
• This is the very essencial
way that cells repair
double-strand breaks in
DNA in which both strands
of the duplex are broken.
• We call it double-strand
break(DSB)repair
pathway,which retrieve
sequence information from
sister chromosome.
Translesion DNA
synthesis

When cells cannot repair certain lesions,there is
a fail-safe mechanism that allows the
replication machinery to bypass these sites of
damage----translesion synthesis
• Translesion synthesis is catalyzed by a
specialized class of DNA polymerases that
synthesize DNA directly across the damage
site.
• Translesion polymerase is produced by cell in
response to the DNA damage
• Translesion polymerases are expressed as part
of the SOS response pathway.
Crystal structure of
a translesion
polymerase.
Translesion
DNA
synthesis in
E. coli
Protein folding is essential to life

 Mad cow disease, or

bovine spongiform
encephalopathy (BSE), is a
fatal brain disorder that occurs
in cattle. Abnormal protein
folding is considered crucial to
the onset of the disease.

 To illustrate the concept

of protein folding we chose
villin, a protein which exists in
the stomach and intestine of
animals (including homo
sapiens).


What causes mad cow?
 In a bovine epidemic that struck the UK in 1986, 170,000
cows appeared to be mad: they drooled and staggered, were
extremely nervous, or bizarrely aggressive. They all died. As the
brains of the dead “mad” cows resembled a sponge, the disease
was called bovine spongiform encephalopathy, or BSE.

 Other examples of spongiform encephalopathy are scrapie

which develops in sheep, Creutzfeld-Jacob Disease (known as CJD)
and its variant form (known as vCJD) which develop in humans.

 Dr. Prusiner, in 1982, identified the infectious agent Humans may be infected
responsible for transmitting spongiform encephalopathy in by prions in 2 ways: 1 -
“proteinaceous infectious particles”, which he named prions.

acquiring the infection
through an infecting agent
 Prions are proteins that are found in the nerve cells of all (through diet or as the
mammals. Many abnormally-shaped prions are found in the brains
of BSE-infected cows and humans afflicted with vCJD or CJD. result of medical
 procedures such as
 The difference in normal and infectious prions may lie in the surgery, injections of
way they fold. growth hormone, corneal
 transplants, possibly blood

transfusion);
and 2 - hereditary
transmission.
Brain surface of CJD patient on autopsy
showing sponge-like appearance
How do prions fold? 
Evidence indicates that the infectious agent in transmissible

spongiform encephalopathy is a protein. Stanley Prusiner 
pioneered the study of these proteins and received the Nobel  The “kiss of death”
Prize in 1997. He has named them prion proteins (referred to as 
PrP) or simply prions. 

A person ingests an abnormally-
shaped prion from contaminated food or
other contaminated sources.
Proteins have primary structures, which is their sequence of 
amino acids, and secondary structures, which is the three  The abnormally-shaped prion
dimensional shape that one or more stretches of amino acids gets absorbed into the bloodstream and
crosses into the nervous system.
take. The most common shapes are the alpha helix and the beta 
conformation.  The abnormal prion touches a

normal prion and changes the normal
The normal protein is called PrPC (for cellular). Its secondary prion's shape into an abnormal one,
thereby destroying the normal prion's
structure is dominated by alpha helices. The abnormal, disease original function.
producing protein called PrPSc (for scrapie), has the same 
primary structure as the normal protein, but its secondary  Both abnormal prions then
structure is dominated by beta conformations. contact and change the shapes of other
normal prions in the nerve cell.

 The nerve cell tries to get rid of

the abnormal prions by clumping them
together in small sacs. Because the
nerve cells cannot digest the abnormal
prions, they accumulate in the sacs
 that grow and engorge the nerve
Examples of alpha helices cell, which eventually dies.

and beta sheets  When the cell dies, the abnormal

prions are released to infect other cells.
 Large, sponge-like holes are left
where many cells die.

Why do proteins fold?


Like all proteins, villin is formed by a unique sequences of
amino-acids. However, only knowing the sequence tells us little
about what the protein villin does and how it does it.

 In order to carry out their function (for instance as enzymes

or antibodies), proteins must take on a particular shape, also
known as a "fold." Thus, proteins are truly amazing machines:
before they do their work, they assemble themselves! This self-
assembly is called "folding."

 Villin’s function is to give structure to intestinal villi, which

are a bundle of actin filaments. Intestinal villi augment the surface
of the intestine to increase food absorption. However intestinal villi
need to be “stabilized”, to add rigidity. Villin accomplishes this goal
by folding in a certain particular way in which it attaches to actin
(another protein) filaments at specific receptor point. One and only
one way of folding is the correct way.

 Distributed dynamics simulate the complexity of the

mechanisms of protein folding, which happens extremely rapidly.
Forms determines function

Suppose you have some molten
iron. You may turn it into nails, hammers,
wrenches, etc. What makes these tools
different from each other is their form (i.e.
their shape and structure).

DNA Replication

Încărcat de

Informații document

Descriere originală:

Drepturi de autor

Formate disponibile

Partajați acest document

Partajați sau inserați document

Opțiuni de partajare

Vi se pare util acest document?

Este necorespunzător acest conținut?

Drepturi de autor:

Formate disponibile

DNA Replication

Încărcat de

Drepturi de autor:

Formate disponibile

DNA and

• Single ring PYRIMIDINES

5’ Parental DNA Molecule Replication

• The enzyme Ligase joins the

• What would be the

1.replication errors and their

• Transitions:a kind of the

2.MutS embraces the

DNA complex recruits

 The newly synthesized

of ways that DNA is

enough to the normal bases

 There are two

• A repair enzyme simply

For example,photoreactivation,which directly reverses pyrimidine dimers

Another example,methyltransferase( 甲基转移酶 ) directly removes the methyl group

The damaged base which

What is the difference

 Mad cow disease, or

 To illustrate the concept

 Other examples of spongiform encephalopathy are scrapie

Evidence indicates that the infectious agent in transmissible

PrP) or simply prions. 

conformation.  The abnormal prion touches a

 The nerve cell tries to get rid of

and beta sheets  When the cell dies, the abnormal

 In order to carry out their function (for instance as enzymes

 Villin’s function is to give structure to intestinal villi, which

 Distributed dynamics simulate the complexity of the

Forms determines function

S-ar putea să vă placă și