DNA replication ,repair and recombination ومن أحياها
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GENETICS
الفريق الطبي
االكاديمي
DNA replication ,repair and recombination
DONE BY :
Shatha Khtoum and
Heba abu hazeem
.
GENETICS
DNA replication ,repair and recombination ومن أحياها
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In last lecture we talked about DNA polymerases activity, and
most important one DNA polymerase III which has the activity
of 5' 3' polymerase activity and the 3' 5' exonuclease activity
but doesn't have the 5' 3' exonuclease activity. However, there
is some exceptions; some DNA polymerases do not have this
3’5’ exonuclease activity, ex: HIV.
In HIV; because of do not have this 3’5’ exonuclease activity,
the HIV will transcribe RNA to DNA (polymerase and RNase H,
cooperate to convert the RNA into a double-stranded linear
DNA). Because it doesn’t have the 3’5’ exonuclease activity, so
it’ll make a lot of steps to convert RNA to DNA by reverse
transcriptase. As a result, it will evolve … *didn’t hear it clearly*
(2:00) so you can’t treat HIV by drugs because it evades drugs,
why? Because it’s not have good reading during replication. It
takes a lot of steps and each time it replicates a new genome
will produce. Very fast it’s evolve, very fast it’s change its
genome and by doing this it escapes drugs.
-DNA replication in prokaryotic or eukaryotic, it’s directed to
specific sequences in the genome not in any place but in a
specific sequences, that called “origin of replication”.
In prokaryotes, because the genome is small in relative to
eukaryotes, it’s about 5-6 million pair of base in comparative to
eukaryotes which is 3 billion pair of base prokaryotes have
origin of replication because the genome is small, but only one
t’s big of replication because i many originsthe eukaryotes have
and linear which requires a lot of origin of replication.
will recognize those specific sequences replication proteinsThe
and bind to them and start replication.
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As we know DNA replication needs template and reader (in
NA polymerase; there’s polymerase the reader is D prokaryotes
I, II, III / the principle one is DNA polymerase III, polymerase I, II
are important for editing). As you know polymerase is
important for remove the primers and fill the gaps between
okazaki fragments.
Also, there must be a catalytic action in the DNA replication to
make the phosphodiester bonds.
-What makes the phosphodiester bonds during DNA
replication? DNA polymerase.
DNA polymerase reads and do the catalysis the So,
ynthesis . And remember it reads and sphosphodiester bonds
the DNA in 5’ to 3’ direction, there is no catalytic reaction the 3’
to 5’ direction.
In prokaryotes, the number of DNA polymerase I or II is several
hundreds, while there are only 3 to 4 copies of DNA polymerase
III.
enzyme in processiveDNA polymerase III is the principal and
, it doesn’t leave the DNA strand during DNA DNA replication
, while DNA polymerase I is not processive and can replication
leave the DNA strand (it leaves once it leaves the primer).
-Why DNA polymerase I does not stay, while DNApolymerase III
stay?
DNA polymerase I has 5’3’ exonuclease while DNA polymerase
III doesn’t have 5’3’ exonuclease. So, DNA polymerase I fills
between the Okazaki fragments and jump to the other
fragments, once it finishes the RNA primer removal it leaves. If
the okazaki frgaments by 5’3’ degradeit stays, it will
exonuclease activity.
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DNA polymerase III stays on the replication fork doing the
, this clamp protein called “clamp”replication because of a
protein will grap the DNA stands and DNA polymerase to the
replication fork and prevent DNA polymerase III from leaving.
-DNA clamp in eukaryotic and prokyotic is very important. If
the cell. And this killyou inhibit the DNA clamp protein, you will
used as a strategy to kill bacteria.
-The primase: it’s a RNA polymerase. It synthesizes short RNA sequences that are complementary to a single-stranded piece of DNA (15-20 pair of base). Since the DNA polymerase can't initiate the process of polymerization, we need a primer or the DNA replication will not occur. -What is the primer? It is a small stretch of RNA (small stretch of ribonucleotide) which is complimentary to the template at specific regions.
-In addition to the fact that DNA polymerase can't initiate polymerizing the new strand, it is also need (OH) at 3' end of the primer in order to synthesis the (3'- 5' phosphodiester bond) to incorporate the new deoxy nucleotides with primer. Side Note: - Usually the primer is RNA but in some cases it could be DNA or proteins.
SSB proteins: special kind of proteins found in the fork called
.tranded binding proteinsingle s What is the importance of those proteins?
to the single strand after unwinding of the 2 bindThey will -strands.
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the single stand from degradation by nucleases. protectThey - er the repairing of the 2 strands aft preventThey will -
unwinding.
DNA replication fork:
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Helicase are very important to open the double stranded DNA. plays a major role in preventing the two strands from winding, it requires ATP in order to open the 2 strands for the DNA polymerase.
DNA polymerase III has the ability to add 6000 pair of base per min, if it less than that the replication will stop. And this is a strategy to make antibiotics in the prokaryotes to stop the replication and thus will kill the cell
-Topoisomerase: are enzymes that convert DNA molecules to different isomers.
topoisomers1.the super coiled form of while the unwinding of the two During DNA replication and
by helicase, the fixed end of the DNA molecule will strandsstop the movement of l condense forming “knots”, which wil
disadvantage. thus the DNA replication will stop helicase On the other hand, the condensed form of the DNA will be
easily packaged smaller than the relaxed form so it will be advantage. inside the nucleus
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This is also a place to find antibiotics to inhibit topoisomerases in prokaryotes.
topoisomers 2.the relaxed form of
The normal DNA molecule does not contain knots (no supercoils). although the condensed form is smaller in size than the relaxed form, both of them have the same number of nucleotides. *In the cell there are two enzymes that called topoisomerases (I and II) the function: converts the DNA molecule from one topoisomer to another topoisomer. The type II of topoisomerase is called gyrase. Topoisomerase I makes cut in one strand and the other one will evade and do the supercoiling. While the topoisomerase II does the cuts in the two strands of DNA and each of them relieve the supercoiling and return back to the relaxed form.
The relaxed B form of DNA, each pair it has 10.5 bp per turn. If this number has change whether it is less or more, well it is creating tension and supercoiling. So, this is a problem in DNA replication -the supercoiling and tension- and this problem solved by topoisomerase.
-What is the importance of DNA ligase? It joins the okazaki fragments by filling the gaps between them by making phosphodiester bonds.
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Now we will talk about application of the DNA replication
polymerase chain reaction.
Remember when you take your DNA and extend it, it’s about 3
meters in length. So, if you are looking for a single gene in this
big molecule, you will be unable to find that gene because
there is a million. So, the scientists find a method to find those
gene, it called polymerase chain reaction. In which you could
choose any piece of DNA and amplify this piece to one billion
cuts.
To identify a genetic disease, you should characterize the
specific gene that is responsible for that genetic disease. How
to amplify these genes? By polymerase chain reaction.
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In this reaction, first step you should denature the DNA, you
separate the two-double strand. Then, you use primer in order
to do the replication. Then add DNA polymerase to this
reaction. You will start with one molecule and now you have
two molecules.