Transcript lec9 DNA replication

DNA (Deoxyribonucleic acid)
DNA structure
DNA replication
DNA repair
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DNA (Deoxyribonucleic acid)
DNA is the storage site of genetic information
Functions of DNA
1- Replication (i.e. makes copy of itself): the stored information are
transmitted from parent DNA to daughter DNA during cell division by
a process called: replication.
2- Makes all proteins that cells needed through gene expression
[(transcription and translation (protein synthesis)]
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Gene expression
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Site:
Human DNA (eukarytotic) is present in the nucleus(linear)
and mitochondria ( 1 circular).
Bacterial DNA (procaryotic) is circular (number is 1)
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Structure of DNA:
Human DNA consists of two strands. Each strand is a polymer of
deoxyoribonucleotides
(dAMP,
dGMP,
dCMP,
dTMP).
The
nucleotides are arranged in each strand linked together by 3'→5′
phosphodiester bond; a bond in which phosphate group links
between C3′ of one deoxyribose to C5′ of the next deoxyribose in the
strand.
Each DNA strand has two ends: 5′ end (the end with free
phosphate group attached to C5′ of the first pentose) and 3′ end (the
end with free OH group on C3′ of the last pentose.
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This is a tetranucleotide
written as 5`-GCTA-3` or
GCTA
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The nucleotides in the polynucleotide chain is always written from 5′
→ 3′ direction
This part of polynucleotide
is written 5`-ATC-3` or
ATC
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Base pairing:
The two strands are linked together
through hydrogen bonding formed
between purine bases in one strand
with pyrimidine bases in the other
resulting in two types of base
pairing:
_
- Adenine is always paired with
thymine by 2 hydrogen bonds
(A=T)
- Guanine is always paired with
cytosine by 3 H-bonds (G ≡ C)
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So in the double stranded DNA:
1- The content of adenine equals to that of thymine and the content of
guanine is equal to that of cytosine
2- Total purine content is equal to pyrimidine content
3- The two strands are complementary to the other i.e. each base of
one strand is matched by a complementary hydrogen bonding base on
the other strand.
4- The two strand are antiparralel (opposite in direction)
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The two strands are complementary to each other in opposite
directions as shown
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DNA double helix:
The two strands of DNA wind arround
each other forming double helix which
stabilize DNA.
Also
abundance
of
hydrogen
bonds
between bases help to stabilize DNA.
Watson and Crick model
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DNA replication (DNA synthesis):
DNAs have the ability to reproduce themselves by the process of
replication, thus ensuring the transfer of genetic information from one
generation to the next. DNA replication is the process in which
DNA is duplicated before cell division.
DNA synthesizes replicate of itself by using its own structure as a
template. Each strand of the double helix serves as a template for
constructing a complementary daughter strand. The resulting double
helix contains one parental and one daughter, and the mode of
replication is thus called: Semi-conservative
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DNA polymerases
The enzyme responsible for DNA synthesis (replication) and repair is
called DNA polymerase
In Prokaryotes (bacteria): 3 enzymes
Polymerase I replicating and repair enzyme
Polymerase II repair enzyme
Polymerase III replicating enzyme
In Eukaryotes (human) there are 5 enzymes) α, β, γ, δ and ε
(epsilon)
- Pol α and pol δ: are replicating enzymes for nuclear DNA. Pol α
synthesize lagging strand while pol δ synthesize leading strand.
- Pol γ replicate mitochondria DNA
- Pol β and pol ε are involved in DNA repair.
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Nucleases: classified as:
Exonuclease
• Cut phosphodiester bond
from the ends
• Remove single nucleotide
5' →3' exonuclease
3' → 5' exonuclease
Endonuclease
• Cut phosphodiester bond
In the middle
• Produce a nick
• Does not remove nucleotide
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Steps of prokarytic DNA replication:
1- Separation (unwinding) of double strands:
Unpairing (separation, unwinding) of the two strands of the DNA
double helix by: helicase enzyme with the formation of “replication
fork ”.
This separation is
necessary because DNA
polymerase III enzyme
that responsible for
replication use only one
single stranded DNA as a
template.
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TWO problems arise from unwinding step:
Problem 1 is Repairing or rewinding
Solution:
The separation is maintained by Single stranded DND binding
(SSB) proteins: that bind to single stranded DND and keep two
strands separated and prevent rewinding
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Problem 2: supercoils
As the two strands of the double helix are separated, a problem is
found, called “ positive supercoil or supertwist”. Accumulation
of supercoils interfere with further unwinding of the double helix.
Solving the problems of supercoils:
To solve this problem there is a group of enzyme called DNA
topoisomerases which are responsible for removing supercoils in
the helix. Two types are found: Type I and Type II
topoisomerases.
DNA gyrase enzyme: is a type II topoisomerase
bacteria and plants.
found in
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Supercoiling can be demonstrated
by tightly grasping one end of
telephone cord
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Topoisomerase I Remove supercoilingby cutting (endonuclease
activity} and sealing (ligase activity) a single strand
Topoisomerase II Remove supercoiling by cutting (endonuclease
activity} and sealing (ligase activity) a double strand
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Etoposide: is an anticancer drug. It is topoisomerase inhibitor that
prevent the action of type II topoisomerase and lead to
DNA strand to break.
Cancer cells will be affected by this drug more than healthy cells
because they divide more rapidly. Therefore This causes errors of
DNA synthesis and lead to death of cancer cells.
Quinolones (e.g. Ciprofloxacin): are antimicrobial agents that
inhibit bacterial DNA gyrase
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3- Direction of replication:
The enzyme responsible for replication is called: DNA polymerase
III which forms a complementary copy from DNA template.
DNA polymerase III read nucleotide sequence on DNA template
from 3′ → 5′ direction so the direction of synthesis of the new DNA
strand will be antiparallel i.e in 5′ → 3′ direction. Therefore DNA
polymerase III has 5′ → 3′ polymerase activity
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4-Starting synthesis of complementary strand
polymerase III:
by DNA
This need RNA primer, because DNA polymerase III can’t join the
first two nucleotides to start the new strand (can’t act de novo), it adds
the nucleotides to the existing RNA primer.
RNA primer is a short segment of RNA (8-10 nucleotides with free 3'
OH end) consisting of RNA nucleotides (AMP, GMP, CMP, UMP)
Primase: is the enzyme responsible for synthesis of RNA primer
Primase is of key importance in DNA replication because no nwonk
tuohtiw dnarts AND a fo sisehtnys eht etaitini nac sesaremylop AND
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When DNA polymerase III recognizes RNA primer, it begins to
synthesize new DNA strand using the 3' OH of RNA primer as the
acceptor of the first nucleotide. As we said before each DNA template
is read from 3′ → 5′ and each new strand is synthesized from 5′ → 3'
(5′ → 3' polymerase activity)
The 2 new strands grow in opposite direction,
one in 5′ → 3′ direction toward replication fork and called “leading
strand” which is synthesized continuously and need only one RNA
primer. The other strand called “Lagging strand” which is
synthesized also in 5′ → 3′ direction but away from replication fork
and synthesized discontinuously as small fragments of DNA called
Okazaki fragments and so need many RNA primers
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5- Proofreading of newly synthesized DNA: (3'→5’ exonuclease
activity of polymerase III)
It is highly important for the survival of an organism that the
nucleotide sequence of DNA be replicated with a few errors as
possible. Misreading of the template could result in mutation and
damage and could be lethal. To ensure correct replication, DNA
polymerase III enzyme has the function of proofreading activity i.e.
check that each nucleotide added to the new strand is correctly
matched to its complementary nucleotide on the template.
If DNA polymerase III mispairs a nucleotide (add false nucleotide
that not match with the template ). it will excise (remove) the wrong
nucleotide (exonuclease activity) using its 3'→5’ exonuclease
activity.
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So DNA polymerase III has two functions:
Polymerase function (5' → 3' polymerase activity)
Proofreading function (3' → 5' exonuclease activity)
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6- Removal of RNA primers by DNA polymerase I:
When DNA polymerase III finishes synthesis of new DNA on
lagging strand, RNA primers are removed by DNA polymerase I
(5' → 3' exonuclease activity) and the gap produced is filled by
DNA synthesized by also DNA polymerase I (5' → 3' polymerase
activity) and check the added nucleotides (proofreading) using
3'→5’ exonuclease activity.
So DNA polymerase I has 3 functions:
Removal of primers (5' → 3' exonuclease activity)
Filling gaps with new DNA (5' → 3' polymerase activity)
Proofreading (3'→5’ exonuclease activity)
7- DNA ligase: Connect (ligase) the newly synthesized DNA
(Oazaki fragments) together
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Removal of RNA primers and filling the resulting gap by
DNA polymerase I
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As a result of replication, 2 double stranded DNA is formed, each
of which contain one old and one new strand, This is called:
Semiconservative manner of replication.
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