DNA repair systems are critical for correcting damage to DNA that occurs naturally. There are multiple DNA repair mechanisms that have been discovered, including direct reversal of damage, mismatch repair, recombinational repair, and excision repair. Excision repair involves removing damaged sections of DNA. It is a conserved process in prokaryotes and eukaryotes that utilizes DNA glycosylases to recognize and remove damaged bases, AP endonucleases to excise areas around the damaged bases, DNA polymerase to fill in the gaps, and DNA ligase to seal the gaps.

1. 1 Assignment
July 4, 2017
DNA Repair System Mechanism
History
As we know that ,the discovery of DNA was in the 1950s by James Watson and Francis Crick
but actually the first DNA was identified by Johann Friedrich Miescher in 1860s that was a swiss
chemist.After the discovery of DNA there were many problems including DNA damage so
scientists identified the DNA repair system mechanism in middle of 1930s by Alexander
Hollander that was the director of biology and he spend his career. He was the the director in
Oak Ridge National Laboratory.Later there were many scientists who contribute too much to
Repair the DNA including Richard B Setlow, Jane Setlow,William Kimball and R C Von
Borstel.After that there were three important scientists who discovered the new ways to repair or
fix the damage of DNA and got the noble prize in 2015 for discovery of the DNA repair named
as Tomas Lindahl,Paul Modrich and Aziz Sancar.They are searching on it from the last 40
years.Multiple DNA repair mechanism were also discovered later.
What is DNA damage?
DNA damage is a change in the chemistry of structure of DNA which can occur in strand of
DNA and can also break the strand of DNA or the double strand breaks .The Primary structure of
dna effected by the majority of dna damage .The dna damage can be also in the form of oxidative
and hydrolytic.
DNA damage can be repaired by DNA repair mechanisms.
What is DNA repair?
DNA repair is a biochemical term so the ,DNA repair is a method in which include collection of
different processes by which cell identifies and than fix damage of DNA that encodes of its
genome.it is a constantly active process and it responds as soon as possible to the damage which
occur in the structure of dna.DNA repair is a multistep process in some cases including the dna
structure irregularity,removal of abnormal dna and the synthesis of normal dna.
Mutation are not good so the systems of Dna repair are very necessary for the survival of all
living organism.In many living cells there are different mechanisms or systems of dna repair that
can fix the different change or dna damge.
This system of dna repair can be divided into two catagories.
 To repair the bases which are damaged
 To repair wrong pairing of bases during the replication
DNA repair rate depend on the following factors.

2. 2 Assignment
July 4, 2017
 Age of the cell
 Type of the cell
 Environment of the cell
There are many different mechanism in the DNA repair system.
 Reversal of damage
 Mismatch repair
 Recombination repair
 Excision repair
 Then ,Restriction-modification systems
1-Reversal of damage
This is the mechanism of dna repair in which simply involve the reversal of double helix dna
damage.These are the processes which require only one single protein that first able to recognize
the damage of dna then catalyse reversal of damage to the dna and then restore the deoxyribo
nucleic acid into the its original state.Direct reversal of the dna damage is a one repair
mechanism which are used to restore the damage of dna so this is called the energy efficient
method.In this the area which is damaged is repaired directly .
So there are many kinds of covalent changes in the bases of dna directly reversed in this
mechanism.This phenomena occurs because of specific enzymatic system then recognize the
change in base and breaks the different bonds and then remove the adduct and base will back to
its real structure.
In the 1940s the first repair mechanism was independently discover the enzymatic
photoreactivation( EPR).
Photoreactivation :-
It is a process which is light- dependent and this process is used by bacteria which helps to
reverse the dimers of pyrimidine that are formed by the uv radiation.There is a most important
and specific enzyme photolyase which attach or binds to the dimer of pyrimidine and then
photolyase catalyzes the second other photochemical reaction and it uses visible light and then
breaks cyclobutane ring structure and then reform 2 adjacent thymidylates in the dna .Energy
from the visible light used in the bonds breakage that are between the pyrimidines then there is
no release of uv radiation so the result is the real dna structure is return. The important
photolyase have two main subunits that are packed or encoded by the phrA,phr B genes present
in the E.Coli.

3. 3 Assignment
July 4, 2017
Removal of methyl groups:-
It is another important example of reversal of damage.so if enzyme that is O6-methylguanine
methyltransferase which is encoded by the gene that is adagene in the E.coli so it first recognizes
the O6 methylguanine in the dna which is duplexe after it removes the methyl group and then
transfer it into an aminoacid of enzyme and the activation of methylated enzyme will no longer
so in the end it is known as the suicide mechanism for enzyme.
Recombinational DNA repair:
What is recombination:
The rearrangement of genetic materials,especially by cross over in
chromosome or by the artificial joining of segment of DNA from different organisms.
What is DNA repair:
DNA repair is a condition in which cells corrects the DNA damaged molecule
in the genome .
Recombinational DNA repair:
Recombinational DNA repair is critical for the maintain of uv
radiation damaged cells ,that accouts for about 50% of the survival of uv irradiated e.coli.it is
complex process that requires two DNA duplex,and the exchange of the strand of DNA from one
DNA duplex and the other strand of DNA from the other DNA duplex ,and it produce mutations.
Nucleotide excision repair that is first indicatation has various mechanism it is only the
mechanism by which cell repair damage to their DNA was result that deficiency of nutrientrs in
bacterial cell repair a genetic recombinantion sensitive uv radiation as show smillar level of
survival after it,then uv reditation and other the single nutrients the stronger is double mutant
from the most basic principle of radiation biology and genetic theses data give information that
,these two system ,i.e,coded by the urvA nad the resA gene ,function largerly independent to
each other and they are of equal importance to the survival of uv irradiated cells of e.coli .
Third step of DNA repair which is the repair of DNA double strand breaks ,metabolically that is
produced after uv irradiation.
Recombinational repairs of double stranded breaks:

4. 4 Assignment
July 4, 2017
 A double strand break in one of the two homologous is converted to a double strand gap
by the action of exonucleases .strand with 3 end are degraded less than those with 5
end,producing 3 single strand extensions .
 An exposed 3 end pair with its complement in the intact homolog.The other strand of the
duplex is displaced.
 The invading 3 end is extended by DNA polymerase plus branch migration,eventually
generating a DNA molecules with two crossovers called hollyday intermedaiates.
 further DNA replication replaces the DNA missing freom the site of original double
stranded break.
 cleavage of the Holliday intermediated by specialized nucleases generate either of the
two recombinational product .
There are two ways to cleave the Holliday intermediated :
 T he DNA flanking the region containing the hybrid is not recombined .
 the flanking region is recombined.
Mismatch repair mechanism
During the process of DNA replication and recombination , there is erroneous deletion , insertion
and mis-incorporation of nucleotides occurs and all these errors are recognized and corrected
through the DNA mismatch repair mechanism.
DNA mismatches that are produced during the process of replication are corrected through the
mismatch repair mechanism. Hence mutations are controlled to become permanent in the
dividing cells. The errors that are produced in the replication process which are known as
replication associated errors reduced by the MMR, So spontaneous mutation rate is increased
when there is defect in MMR system. In human cells , MMR inactivation belongs to hereditary
and sporadic human cancers and programmed cell death that is the response of certain DNA
damages and cell cycle arrest need MMR system mechanism. Thus MMR play a vital role in
preventing both mutagenesis in the short term and tumorigenesis (the production of tumors) in
the long term.
This mechanism is strand specific it means during synthesis of DNA errors will be occur in the
newly synthesized dna strand which is known as daughter strand.
Hemimethylation
Before starting repair mechanism , the machinery will identify the newly synthesized strand that
is formed in front of the template(parental) strand. And the strand is distinguished through the

5. 5 Assignment
July 4, 2017
concept of methylation or non-methylation. The parental strand is methylated and the daughter
strand is not methylated. This process is called hemimethylation in which one strand is
methylated and one is non methylated. It occurs in gram negative bacteria while the exact
mechanism is not clear syill in other prokaryotes and eukaryotes.
DNA mismatch repair in E.Coli:
There are specific sequences at the N6 position of adenine where DNA is methylated in the
E.Coli and the sequence is dGATC. From replicating DNA, transiently unmethylated daughter
strand comes due to the presence of hemimethylated seguences. Through this mechanism hemi-
methylated Dgatc sites determine the specificity of stsnr of repair.
There are different proteins used in the MMR of E.Coli including
dam methylase (DNA adenine methylase)
In the GATC sequence, methylates adenine is used to create 6-methyladenine.During replication,
the newly synthesized strand is temporarily unmethylated. To attain methylation of newly
synthesized strand dam methylase is used and hence methylation of hemi-methylated DNA
occurs quickly.In cells that lacks dam methylase activity possess fully unmethylated DNA.
MutH
It is used to recognize the hemimethylated Dgatc sequences and belongs to the family of type 2
restriction endonucleases.
Unmethylated strand at just 5'to the G in GATC sequence cleaves through this endonuclease.
Hence there remains a 3'-OH and 5'-P at the cleavage site. For activation of latent endonuclease
activity , there is need of MutL and MutS.
MutL
It is used To join the MutS and MutH use ATP hydrolysis and also for stimulation of MutH
endonuclease activity and also binds to and loads helicase 2.
Adds to complex of MutS at mismatch in ATP dependent (but not hydrolysis dependent) step.
It works as "molecular matchmaker".
MutS
Except C-C, it binds to all mismatches like insertion or deletion. Heteroduplexes having four
extra nucleoties are repaired weakly but on the other hand larger heterologies not appear to
recongnized.

6. 6 Assignment
July 4, 2017
 Helicase II
It can also called as the mutU/uvrD gene product. It perform function with MutS and MutL to
load on at the endonucleolytic cleavage site ("nick").
the incised strand unwinds to get the sensitivity to the appropriate single-strand exonuclease
activity that will be specific.
 Exonuclease VII
Hydrolyzes single-stranded DNA in the 5'-to-3' direction.
 RecJ
Hydrolyzes single-stranded DNA in the 5'-to-3' direction.
 Exonuclease I
Hydrolyzes single-stranded DNA in the 3'-to-5' direction.
 DNA polymerase III holoenzyme
The replicative DNA polymerase in E. coli.
 SSB
single strand binding protein.
 DNA ligase
Uses NAD+ to form phosphodiester bonds at "nicks".
Steps in the MMR system
 MutS Homodimer binds to the mismatch
 By the combine action of Muts, MutL, MutH and ATP mismatch cleavage occurs.
 Generation of nick at hemi-methylated dGATC by MutH
 Filling of gap through DNA polymerase
 Sealing of gap through DNA ligase
Key features of MMR mechanism.
 Repair is strand specific.
 Repair is bi-directional(5′→3′ or 3′→5′)

7. 7 Assignment
July 4, 2017
 MMR has broad substrate specificity including base-base mismatches and small ID
mispairs
So there is need of MutH, MutS, and MutL.because throughout evolution this mechanism is
highly conserved and E.Coli MMR mechanism behave as excellent model for MMR in
eukaryotic cells.
MMR in human cells
There is strong similarities between human MMR and prototypical E.Coli MMR including
bidirectionality, substrate specificity and nick directed strand specificity.the working of hemi-
methylation for strand discrimination is not conserved from e.coli to human MMR, but hemi-
methylated Dgatc site is MutH dependant nicking and because in humans the nick directed is in
vivo. So there is assumed that both systems distinguish daughter and template strand by using
strand specific nick.
There are some proteins used in the human MMR, identified on their homology to E.Coli MMR
proteins.
 homologs of MutS, MutL, EXO1
 single-strand DNA-binding protein RPA 27
 proliferating cellular nuclear antigen (PCNA)

8. 8 Assignment
July 4, 2017
 DNA polymerase δ (pol δ) and DNA ligase
The human MutS and MutL homologues are heterodimers while these are homodimers in E.Coli.
hMSH2 heterodimerizes with hMSH6 or hMSH3 to form hMutSα or hMutSβ, respectively, both
of which are ATPases that play a critical role in mismatch recognition and initiation of repair
Excision Repair Mechanism
Introduction:
It is a General Process in which we can remove damage DNA backbone and the bases from the
genome. In this mechanism usually for the DNA Pol I initiation 3’OH provided by ss nick/gap.
The ss nick /gap also provides DNA ligase seals nick. Excision Repair is conserved throughout
evolution, it is found in all organisms including eukaryotes and prokaryotes organisms
Three step process:
1. Error is recognized and enzymatically clipped out by a nuclease which cleaves the
phosphodiester bonds (operate the uvr gene products in this step).
2. DNA Polymerase I fills in the gap by inserting the appropriate nucleotides.

9. 9 Assignment
July 4, 2017
3. DNA Ligase seals the gap.
Types of excision repair
 Base excision repair: Cleave the base, then excise next to the apurinic or
apyrimidinic site, and DNA Pol I start the repair procedure.
 Nucleotide excision repair: Remove out a segment of DNA from a
damaged base.
Base excision repair (BER)
In genetics and biochemistry, base excision repair (BER) is a cellular mechanism which repairs
damaged DNA during the cell cycle. It is involved primarily for removing small, non-helix
distorting base lesions in the genome of the organisms. The related nucleotide excision repair
pathway repairs bulky helix-distorting lesions. Correction of damage nitrogenous bases due to
the spontaneous hydrolysis of DNA bases and the hydrolysis of DNA bases which have the
agents that chemically alter them.
The enzymes known as DNA-N-glycosylases involves in the BER. These enzymes can identify a
single damaged base and cut the bond between it and the de oxy ribose sugar in the DNA.Using
Pol adding to 3’ ends then ligase attaching to 5’ end, cleave one base, excises more around it,
and replaces with many new bases. , this repair mechanism can eliminate damaged bases such as,
3-methyladenine, Uracil, Thymine dimers and 7-methylguanine. Which are depends on the
species.
DNA polymerase and DNA ligase
The removal of 39 or 59 dirty ends BER enzymes creates a small gap in DNA, which is then
ready to be filled by a DNA polymerase. DNA polymer is the major source of BER associated
repair system in nuclear extract and Pol2, the catalytic subunit of Pol e, binds with high affinity
to a BER intermediate in vitro.

10. 10 Assignment
July 4, 2017
Base Repair Mechanism…
Proteins involved in base excision repair:
DNA glycosylases: DNA glycosylases are responsible for initial recognition of the lesion. They
flip the damaged base out of the double helix and remove the N glycosidic bond of the damaged
base by the leaving an AP site.
The AP endonucleases: Remove an AP site from the 3' hydroxyl adjacent to a 5' deoxyribose
phosphate (dRP).
Nucleotide excision repair (NER)
It is a different and more flexible damage repair mechanism present in living organisms. Which
recognizes damaged fragments of DNA based on their abnormal chemistry, their irregular
structure as well as, and then excises or replaces them.

11. 11 Assignment
July 4, 2017
Recognizes bulky lesions which can inhibit or stop the DNA replication, lesions produced by
carcinogens for example UV pyrimidine photo dimers. It is a Common distortion in helix.
Incision on both sides of lesion. Cut the Short patch of DNA and repaired by repolymerization
and ligation. Replication blocking lesions such as UV photo dimers can be repaired by NER but
pose a serious problem if they are in ssDNA.
In all organisms, NER involves the following steps:
 Damage recognition
 Binding of a multi-protein complex at the damaged site
 Double incision of the damaged strand several nucleotides away from the
damaged site, on both the 5' and 3' sides
 Removal of the damage-containing oligonucleotide from between the two nicks
 Filling in of the resulting gap by a DNA polymerase
 Ligation
NER Mechanism…..
NER and human genetic diseases:
We know that genes encoding several of NER proteins in the human. The first identified in
genetic studies of the human DNA repair disease, Xeroderma pigmentosum (XP) which is due to

12. 12 Assignment
July 4, 2017
the mutations in any of 7 genes (XPA, XPG) and cause the disease. Two other human genetic
disordered are involve in this defects with XP and related to NER. These two additional diseases
are Cockaynes syndrome (CS) and trichothiodystrophy (TTD). All 3 diseases are different in
term of clinical manifestations.
Patients suffering from XP have
 severe light sensitivity
 frequent neurological defects
 severe pigmentation irregularities
 early onset of skin cancer at high incidence
 elevated frequency of other forms of cancer
Cockayne's syndrome:
CS cells are deficient in transcription-coupled NER but are proficient in global genome NER.
The patients Shows the characteristics
 light sensitivity in some cases
 neurological abnormalities
 premature aging of some tissues
 facial and limb abnormalities
 dwarfism
 early death due to neurodegeneration
Trichothiodystrophy patient’s display
 light sensitivity in some cases
 premature aging of some tissues
 facial abnormalities
 short stature
 ichthyosis (fish-like scales on the skin)
 sulfur deficient brittle hair
Restriction modification system
Introduction:
In restriction modification system, restriction enzymes and one or two modification enzymes
protect the DNA of cell from foreign DNA . Such as DNA from bacteriophage. It was observed

13. 13 Assignment
July 4, 2017
50 years ago that some strains of bacteria are less susceptible to the infections caused by
bacteriophage than others.
Restriction modification enzyme restricts the entry of foreign DNA in the bacterial cell.it
converts the foreign DNA into fragments with the help of restriction enzymes. Bacterial DNA is
methylated by methyltransferase while the foreign DNA is not methylated so it is recognized.
More than 25% bacterial strains have one restriction enzyme modification system. Some strains
contain six R-E systems. Genes for restriction enzyme modification system can be present on
chromosomes, on a prophage or on a plasmid. Many of their genes are sequenced. It activates a
kind of immune system in bacteria that has characteristics of immune system in eukaryotes
R-M Gene Expression and Organization
Expression of those genes that are present in RM system is tightly regulated. A genome that
contains RM system should be protected from its restriction enzymes during tough times such as
starvation. RM system has organized genes.
Types of restriction enzyme modification system:
There are four types of restriction modification system.
 Type I
 Type II
 Type III
 Type IV
Type I
Type 1 restriction modification systems are the complex systems of all. Less than 20 different
type 1 restriction modification system are found. These are present in E.coli salmonella and
freundii. They contain modification and restriction functions in one multisubunit enzyme. It
contain thee polypeptides in total. M (modification) R (restriction and S (specificity). These
polypeptides will form a complex that methylates and cleaves the DNA.. The subunit S is for
thee specificity of both functions. The bands resulted from cleavage can not easily visualized by
gel electrophoresis because DNA is cleaved at a distance from recognition site.
Type II R-M Systems
Over 2600 type II R-M Systems are known. These are the simplest systems. Endonuclease and
methyl transferase work independently because two separate proteins encode them. they do not
work as complex. Methyltransferase work as homodimer and monodimer. In monodimer one
strand of the duplex is methylated. When endonuclease works as homodimer it cleaves the both
strands. It cleaves the DNA at recognition sequence or close to the recognition sequence and
produce fragments.

14. 14 Assignment
July 4, 2017
Type III R-M systems:
Type III systems that have been found are few in number. They form complex of cleavage and
modification by the help of proteins M and R. M protein can methylate itself in Type III systems
methylation occur at one strands. Methyl transferase and endonucleases are catalyzed by single
enzyme. When one or two recognition sites are present in the same orientation then the Type III
restriction activity is absent. If they occur 25-30 bp away from the DNA sequence cleavage
occur. There will be no cleavage if one or both strands of DNA are not methylated. M subunit
work as methyltransferase independently. It requires methylation of one strand and AdoMet at
one time. Methylation does not depend on restriction site orientation and number
Type IV R-M systems
Type IV systems are not said to be a true restriction modification system. They do not contain
methylase.
Applications:
We can clone restriction modification system into the plasmids. When plasmid starts to replicate.
The plasmid DNA will be methylated and protected.
Restriction Fragment Length Polymorphisms: restriction enzymes are used to identify mutations
In Neisseria meningitides has type 2 R.E systems that are used in natural genetic transformation
system. At first R.M systems focused on protected bacteria from invader bacteriophages now it is
said that it restricts DNA that is introduced by natural transformation.
RM systems also act as selfish genetic material that ensures their survival in cell.