DNA replication, repair and recombinationDNA replication, repair and recombination اهايحأ نمو Page 6 Nucleotide Excision Repair Our genes are always insulted/ mutated/ destroyed

DNA replication, repair and recombination ومن أحياها

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DNA replication, repair and

recombination

DONE BY :Shatha Khtoum


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Prokaryotic vs Eukaryotic DNA Replication:

Location

Here we have a comparison between prokaryotic and eukaryotic DNA

replication, principally they are the same with some differences.

We all know that there is no nucleus in the prokaryotic, so the replication

takes place in the cytoplasm where the genetic material actually located.

However, in eukaryotic there is a nucleus where the genetic material found, so

the replication occurs in the nucleus, as well as the transcription (but the

translation occurs in the ribosomes outside the nucleus).

Origin of the replication

The DNA replication starts at specific sites (specific sequence of DNA) called

the origin of replication. In prokaryotic, there is only one site or one origin of

replication because it’s small genome. The same happens in eukaryotic, but

the number on origin of replication is many many thousands of origins of

replication because the size of our genome is much bigger than the size of

prokaryotic.so we can see thousands of origins of replication in the eukaryotic

while only one in the prokaryotic.

The initiation

These specific DNA sequences (origin of replocation) is recognized by specific

proteins (DnaA and DnaB in prokaryotic \ Origin Recognition Complex in

eukaryotic). So, the initiation carried out by these proteins.


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Topoisomerases

In both we have topoisomerases in prokaryotic as well as in eukaryotic in

order to relive the supercoiling during DNA replication when tension has

occurred while opening the two strands. Now if you didn’t relive the

supercoiling by topoisomerases in both prokaryotic and eukaryotic the DNA

replication will stop. And this is one of the biochemical strategies to treat

cancer; we inhibit the cancer cell topoisomerase and this will stop the

replication of cancer cells.

The replication in prokaryotic is very rapid while in eukaryotic is slow. It

depends on the time or the life cycle of replication in each species. It’s about

24 hours in prokaryotes while in eukaryotes it’s 25 hours!

We will take about another important thing, about DNA replication in

eukaryotic. What applied on prokaryotic like initiation and polymerization it

applied on eukaryotic as well. In eukaryotic you need two double strands to

open, you need one primer for the leading strand and many primers for the

lagging strand, you need templates for each base, you need DNA polymerases,

you need helicases and topoisomerases, you need clamping proteins for the

DNA polymerase. So, it’s the same things but the naming is different in

prokaryotic (you not supposed to remember the names of them but you have

to remember it replicates as the prokaryotic).

So, the problems that will happen in eukaryotic is the same that will happen in

prokaryotic, except one problem that hasn’t found in prokaryotic while it’s

found in prokaryotic. It’s very dangerous problem in DNA regulation of

eukaryotic system; it confers termination of replication of eukaryotic DNA.

In order to introduce this problem to you, you have to know something called

Telomeres and Telomerases.

They are repeated sequences of DNA at the end of linear chromosome. In

prokaryotic we don’t have telomeres because we don’t have linear


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chromosomes; it’s circular chromosome. And these telomeres are found to

protect the ends of DNA linear chromosomes from different nucleases. These

telomeres are short sequences repeated different of times at the ends of

linear chromosome, they are protective agents for the chromosomes.

• Telomeres and telomerase activity (it will be easier if you listen to the record first (15:00), you

will see some differences that because I wrote those below in my way, but it’s easy) - The free ends of linear DNA molecules introduce several complications that must be resolved by special enzymes. - In particular, complete replication of DNA ends is difficult because DNA polymerases act only in the 5’ - to- 3’ direction. - The lagging strand would have an incomplete 5’ end after the removal of the RNA primer. - Each round of replication would further shorten the chromosome. - The ends of chromosomes are called telomeres. - Telomeric DNA contains hundreds of tandem repeats of a 6-nucleotide sequence. - One of the strands is G rich at the 3’ end, and it is slightly longer than the another strand.

Telomeres

And this picture

shows you the

telomeres under the

microscope using

fluorescence

antibodies.


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- In humans, the repeating G-rich sequence is AGGGTT and it is generated by an enzyme termed telomerase. - Telomerase is a specialized reverse transcriptase that carries its own RNA template. - The RNA template is used to extend the 3’ overhang. - The single-stranded region at the very end of the structure has been proposed to loop back to form a DNA duplex with another part of the repeated sequence. Such structures would nicely mask and protect the end of the chromosome.

Another biochemical strategy used to kill cancer cells; cancer cells have

enzymes called telomerases and those telomerases extend the chromosomes

of cancer cells which lead them to replicate uncontrolled and the cancer will

stay there. While in our somatic cells this telomerase in old age is partly

inactive. So, if this problem wasn’t solved and each cycle of DNA replication,

the chromosomes are shrinkage and you’ll die. But it solved by using

telomeres and telomerases.


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Cancer cells have active telomerases while other cell have in active

telomerases, and because of this one of the reasons of aging is the inactivation

of telomerases. With age; our telomeres will become shorter and shorter then

the cells are killed.

Do you remember Dolly the sheep? Dolly after it has cloned from somatic

cells, it didn’t survive. Dolly died after 2 years. Why? Because it was cloned

from somatic cells which telomeres were short and the telomerases were

inactive.

- The stem cells have an active telomerase.

Telomerase Action


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Nucleotide Excision Repair

Our genes are always insulted/ mutated/ destroyed by radiation, chemical,

pollutions and by diet. So, if you are subjected to X-ray or to UV your genes

will be disturb and mutations will happen. if you are exposed to chemicals, to

pollution (smoke pollution from cars and from smoking cigarettes) they will

produce radicals that are highly harmful to our DNA. Our genes are insulted to

these agents every minute, and they are cause mutations to our genes.

Fortunately; we have a system in out cells to repair these mutations.

One of many these factors will cause what called DNA Adduct. This DNA

adduct, it’s an abnormal structure that changes or makes some covalent bonds

between the neighboring nucleotides. So, the whole structure of DNA is

different from the normal. Because of these covalent bonds there are a bulk

region or structure, it’s not smooth. Our repair system can recognize this bulk

and goes to the bulking area and start working on it and repair what happen to

this region.

Bulky DNA Adduct


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Nucleotide Excision Repair

what you see here is normal DNA subjected to UV light, the UV light will cause

any two thymines which are neighbor to each other on the same strand to

covalently bonded to each other (thymine dimers) and it’s an example of an

adduct. Fortunately; there is an enzyme called photolyase will deactivated and

rebind to the bulking and break the covalent bond between the thymine in the

same strand.

Some people when they want to relax on the beach, they expose their bodies

to sun, they expose their bodies to UV light. If those people have deficiency in

their repair system of Nucleotide Excision Repair, there is high prevalence to

have thymine dimers and the repair system is unable to repair these thymine

dimers and their skin will be highly sensitive to sun and highly exposed to skin

cancer.


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Nucleotide Excision Repair: it removes many nucleotides include the region

of lesion and resynthesize the removes piece.

(Thymine dimers)

This is the thymine dimer, there is a bulking, this bulking was recognized by

specific proteins that you are not supposed to memorize their names. After

recognition, they will make incision in this region, and they will remove the

whole region where the lesion -thymine dimer- located. Then by using DNA

polymerases from 5’ to 3’ they will polymerize and fill this gap by nucleotides,

and finally DNA ligase will make the phosphodiester bond.


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Base Excision Repair

In this case, what is removed isn’t nucleotide; but only one base.

Ex: if a cytosine in your DNA was deaminated, this cytosine will become an

uracil. But uracil in our DNA is an odd nitrogenous base, it must be removed.

There are some enzymes that will take this uracil and remove it from the

Thymine Dimer

Repair

Partial Strand

Removal

Cytosine Deamination

Cytosine Uracil


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twisted DNA, by using specific enzymes. And those enzymes will bind to uracil

and remove it.

Base Excision Repair

Flipping out of

base from helix


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DNA Mismatch Repair

Now in this case, during DNA replication if there is any mistake, there are

enzymes that responsible for correcting these mistakes and remove it. But

sometimes, some mistakes are still there and the proofreading system doesn’t

discover them and they will pass to dormant cells. It’s very dangerous because

it’ll pass to the next generation and there will be a lot of mutations.

So here, we have the DNA Mismatch repair system.

Mismatch

This system must recognize which strand contains the mutation. The old strand is methylated and the new strand has not been methylated yet. Therefore, it will act on the non-methylated strand.


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“Trust in dreams, for in them is hidden the gate to eternity”

Documents

DNA replication, repair and recombinationDNA replication, repair and recombination اهايحأ نمو Page 6 Nucleotide Excision Repair Our genes are always insulted/ mutated/ destroyed