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DNA Replication:

Process of replicating cells DNA


Why it is important?
1. To repair broken cell because broken cells DNA are prone to mutation which is dangerous
2. Form a replacement because cells DNA is used in metabolism again & again

Mechanism/Pathway

1. Semi conservative
Double stranded DNA is separated, which one of the strand is used as a template to form
new daughter cell, as a result it forms 2 DNA double helix which its strand is made up of
parental and new strand
2. Conservative
Two DNA strands are separated then back together after replication has occured. One
daugther cell consist of fully from parental strands whereas the other one consist of all
newly synthesized material
3. Dipersive
Material in the two parental strands is distributed more or less randomly between two
daughter molecules (DNA in strand selang seling from old and new strand)

ENZYMES & PROTEINS USED IN DNA REPLICATION


1. SSB (Single strand binding protein) prevent unwind double strand DNA from winding back
up again
2. Helicase open the double strand DNA (create replication fork) to start replication process
3. Dna polymerase enzyme used for replciation
4. Gyrase (Topoisomerase II) to relax the supercoiling state of DNA as DNA polymerase
cannot read supercoiled DNA
5. RNA primase create RNA primers (foundation of DNA synthesis) as DNA polymerase only
can add nucleotides to existing nucleotides
6. Ligase act as glue to join okazaki fragment (fragment of laging strand after RNA primer is
removed) which has gabs and filled by DNA polymerase after replication is finished
DNA polymerase
Used to replicate DNA after primer has been formed by RNA primase
1. Prokaryotes
a. Dna Pol. 1 remove RNA primer & remove mistmatched pair (3-5 direction)
b. Dna Pol. 2 no specific function
c. Dna pol. 3 replicate DNA (5-3 direction)
NOTE: DNA pol 1 + 3 exist as holoenzyme, only work in 1 direction (5-3)
so to replicate DNA simultaneously, lagging strand has to be folded
2. Eukaryotes
a. Alpha () similar activity to DNA pol 1
b. Beta () DNA repair, filling gaps with DNA nucleotides after RNA primer is
removed
c. Gamma () DNA polymerase in mitochondria
d. Delta () replicate lagging strand
e. Epsilon () replicate leading strand & similar activity to DNA pol 1

PROCESS IN EUKARYOTES
1.
2.
3.
4.
5.
6.
7.
8.

DNA in histones is made possible to be acessed


Origin of replication is bound by origin replication complex (ORC) in many places
Helicase unwind the double strand DNA forming replication fork (replication loci)
RNA primase create RNA Primer in leading strand
DNA pol epsilon add complementary bases in the leading strand (5-3 direction)
RNA primer is removed by DNA alpha & epsillon enzymatically forming telomere
RNA pimase create RNA Primer in lagging strand
DNA pol delta add complementary bases in the lagging strand (3-5 direction) discontinously
creating series of short fragment

9. RNA primer is removed and then replaced by appropiate DNA nucleotides, however if the
RNA primer that is being removed is located at the end of the DNA it will form telomere
(hanging end)
10. Gaps between Okazaki fragment and new DNA nucleotides which replace RNA primer is
joined by ligase
11. New histones will also be produced, the offspring will have both parental histones and newly
formed histones

PROCESS IN PROKARYOTES

1. Helicase unwind double staranded DNA and SSB react with single strand DNA to prevent it
from winding back again
2. RNA primase create RNA primer in leading strand
3. DNA pol 3 add complementary bases to the leading strand (5-3 direction)
4. RNA primase create RNA primer in lagging strand
5. Lagging strand is folded as DNA pol 3 is a holoenzyme and only can work in 1 direction
6. DNA pol 3 add complementar bases to the lagging strand (3 5 direction) discontinously
creating series of short fragments
7. RNA primer is removed by DNA pol 1 forming Okazaki fragment, and then was replaced with
appropiate DNA nucleotides by DNA pol 1
8. Gaps between Okazaki fragment and new DNA nucleotides which replace RNA primer is
joined by ligase

DIFFERENCE BETWEEEN REPLICATION IN PROKARYOTES AND EUKARYOTES


Prokaryotes
Histones of DNA are being removed completely
and later is recreated for offsprings
Circular replication
DNA pol 1, 2, 3 as holoenzyme
Dna pol is holoenzyme, can only work in one way
direction so lagging strand has to be fold
After primers are removed, the gaps will be
replaced by DNA pol 1
One origin site

Eukaryotes
Histones are not removed and the offspring will
have both parent & newly formed histone
Linear replication
DNA pol alpha, beta, gamma, delta, epsilon
did not work as holoenzyme
Two way replication process, DNA pol is not
holoenzyme so folding of lagging strand is not
neede
After primers are remvoed, it will create a
hanging end as it does not have complementary
base pair (TELOMERE)
Many origin sites

TELOMERE

Is hanging end which odes not have any complementary base that is present in eukaryotic
DNA
How does it forms?
o At first, during replication RNA primase will create primers in the replicated strand in
order for replication to occur. However, after the replication process is finished, RNA
primer is removed thereby creating a gap between replicated DNA. If the RNA
primer that is being removed is located at the middle of the strand (between
replicated DNA) this gap can be filled by appropiate DNA nucleotides. However if it is
located at the end of the DNA, it cannot be filled with DNA nucleotides as there is no
foundation (existing nucleotide) for DNA pol. to work. As a result, it will form
telomere, a hanging end (strand that did not ave any complementary base).
Why it is not present in prokaryotes?
o In prokaryotes the gap, formed after RNA primers is removed, is filled by DNA pol I
which is not present in eukaryotes.
What happened to telomere during replication?
o If the DNA strand, containing telomere, is replicated again, the telomere will be cut
off. However since a too short telomere can cause death to the cell, enzyme
telomerase is used to elongate the telomere therefore prevent its shortening.

WHAT IS OKAZAKI FRAGMENT

is pieces (fragment) of lagging strand DNA after RNA primers are removed.
How does it forms?
o In replication process, lagging strand is replicated discontinously by creating series of
short fragment containing RNA primer and replicated DNA sequence.

o
o

After the replication is finished and the RNA primer is being removed, it will create
gap between the replicated DNA or Okazaki Fragment.
Then the gaps, between these fragment will later be filled in DNA polymerase and
then joined together with the help of ligase.

DNA Repair:
Process of repairing cells DNA

Rate is affected by many factors

Cannot be conducted if:


Irreversible state of dormancy (senescene)
Cell suicide (apoptosis or programmed cell death)
Unregulated cell division
Why it is Important?
1. DNA need to be repaired because it is suspectible to mutation from external factors such as
ionizing radiation, UV radiation, chemical, and thermal energy.
Stages of DNA Repair
1. Accurate selection of nucleotides
2. Immediate proofreading
3. Post replicative mismatch repair
o EX: base pair have specific gemoetry and angels, if their geometry is wrong it will be
recognized by NUCLEASE
Mechanism

Base Excision
Repair (BER)
Mismatched
Repair (MMR)
Mechanism
Double Strand
Break (DSBs)

Nucleotide
Excision Repair
(NER)
repaired by NHEJ
in mammal cell

1. Transcription
Coupled Pathway
2. Global
Genomic
Pathway

1. MISMATCHED REPAIR (MMR) correction of mistake that escape DNA polymerase


proofreading activity
o DNA is repaired by help of enzyme
In prokaryotes it is recognized from daughter strand by the presence of
mehylated bases
o 2 KINDS:
Base Excision Repair (BER)
Repair the base only
Nucleotide Excision Repair (NER)
Remove bulky lesion such as pyrimidine imers or chemically altered
nucleotide
2 Pathways:
1. Transcription coupled pathway
It is the most prefered pathway as it only repair the
broken gene that is important for to be transcripted
or translated
2. Global genomic Pathway
It is the less efficient pathway because it correct all
damage in the DNA strand regarless whether it is
important or not
2. Double Strand Break (DSBs)
o Breakage of double strand of DNA that is caused by ionizing radiation & chemicals
o Repaired in mammal cells by nonomologous end joining (NHEJ)
How? NHEJ bind to broken end and catalyze reaction to repair it

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