Chapter 2

Safeguard Systems of DNA

The microbial cells are equipped with different enzymes which help them to override
mutations that may occur due to errors in DNA replication during cell division, exposure to
mutagens etc. Some enzymes also help to protect their genetic material genetic from invading
pathogens like bacteriophages, prions etc. They can be collectively referred as the safeguard
systems of DNA.

2.1. Restriction enzymes: Significance, Roles and features of Type I, Type II

and Type III restriction enzymes.
Restriction enzymes belongs to the group of enzyme category known as nucleases.
Nucleases

 Nucleases are enzymes that degrade nucleic acids.

 Ribonucleases (RNases) attack RNA and Deoxyribonucleases (DNases) attack DNA.
 Most nucleases are specific, but the degree of specificity may vary.
 Some nucleases will only attack single stranded nucleic acids, others will only attack
double stranded nucleic acids and a few will attack either kind.
 Depending on the site of cleavage, nucleases are of 2 types:

1. Exonucleases: They attack at the end of nucleic acid molecules and usually remove just a
single nucleotide or sometimes a short oligonucleotide. Any particular exonuclease
attacks either the 3’end or the 5’end not both.
Eg: Exonuclease Bal31, Ecoli exonuclease III, Lambda exonuclease.

2. Endonucleases: They can recognize specific base sequence within nucleic acid molecule
and cleave internal phosphodiester bonds. Some endonucleases are non specific but
others, especially restriction enzymes are extremely specific and will only cut DNA after
binding to specific recognition sequences.
E.g.: EcoRI, HindIII, BamHI

Restriction enzymes
 A restriction enzyme, also called restriction endonuclease is a protein that recognizes a
specific, short nucleotide sequence and cuts the DNA only at that specific site, which is
known as restriction site or target site.
 They have the capacity to recognize specific base sequences on DNA and to cut each strand
at a given place. Hence they are also called as molecular scissors.
 More than 400 restriction enzymes have been isolated from the bacteria that manufacture
them.
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  In living bacteria, restriction enzyme function to defend the cell against invading viral
bacteriophages.
 Restriction sites in the viral genome are cleaved by the bacterium’s restriction
enzymes, fragmenting and destroying DNA of invading bacteriophages before it can
incorporate into the host's genome and take over the cell.
 A bacterium is immune to it’s own restriction enzymes, even if it has the target
sequences ordinarily targeted by them.
 This is because the bacterial restriction sites are highly methylated, making them
unrecognizable to the restriction enzymes.

History
 In 1970, the first restriction endonuclease enzyme HindIII was isolated.
 For the subsequent discovery and characterization of numerous restriction
endonucleases, in 1978, Daniel Nathans, Werner Arber and Hamilton O Smith were
awarded the Nobel Prize for Physiology and Medicine.
 Since then restriction enzymes have been used as an essential tool in recombinant DNA
technology.

Nomenclature of RE
 Restriction enzymes are named based on the organism in which they were discovered.
 For example, the enzyme HindIII was isolated from the bacterium Haemophilus influenzae,
strain Rd.
 The first three letters (Hin) of the name are italicized because they abbreviate the genus
(H) and species (in) names of the organism. The fourth letter (d) typically comes from the
bacterial strain designation.
 Typically, the Roman numerals (I, II, III) etc. indicates the order in which restriction
enzymes were discovered in a particular strain.

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Restriction site
 Each restriction enzyme identifies a specific sequence of nucleotides (between four and
eight bases) and makes cut in both strands of the double stranded DNA.
 Many of the restriction sites are palindromic in nature i.e. reads same forward and
backward.
 These palindromes could be mirror like (e.g. GTAATG) or inverted repeats (e.g. GGATCC
being complimentary to CCTAGG).
 Inverted repeat palindromes are more common with greater biological importance than
mirror like palindromes.
Nature of cleavage by restriction endonucleases
 The nature of cleavage produced by restriction endonucleases is of considerable
importance.
 They cut the DNA molecules in two ways.
1. Blunt ends-
 Many restriction endonucleases cleave both strands of DNA simply at
the same point within the recognition sequence
 As a result of this type of cleavage, the DNA fragments with blunt ends
are generated
 It may be also referred to as protruding ends
 PvuII, HaeIII, AluI are examples
2. Sticky ends-
 In the other style of cleavage by restriction endonuclease, the two
strands of DNA are cut at two different points.
 Such cuts are termed as staggered cuts ie. One strand of the double helix
extends a few bases beyond the other strand.
 Such ends are called sticky or cohesive ends.

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The following table shows sticky and blunt ends generated in the restriction sites by various
enzymes:

Mechanism of cleavage of restriction enzymes

 When a restriction endonuclease recognises a particular sequence, it snips through the
DNA molecule by catalysing the hydrolysis of the bond between adjacent nucleotides.
 To cut the DNA, all restriction enzymes make two incisions, once through each sugar
phosphate backbone of the DNA double helix.

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Classification of restriction endonucleases
There are three major classes of restriction endonucleases based on the types of
sequences recognised, the nature of the cut made in the DNA and the enzyme structure
 Type I-

o These enzymes have both restriction and modification activities. Restriction depends
upon the methylation status of the target DNA.
o Cleavage occurs approximately 1000 bp away from the recognition site.
o The recognition site is asymmetrical and is composed of two specific portions in which
one portion contain 3–4 nucleotides while another portion contain 4–5 nucleotides and
both the parts are separated by a non-specific spacer of about 6–8 nucleotides.
o They require S-adenosyl methionine (SAM), ATP, and magnesium ions (Mg2+) for
activity.
o These enzymes are composed of mainly three subunits, a specificity subunit that
determines the DNA recognition site, a restriction subunit, and a modification subunit.
o Ex: EcoK, EcoB

 Type II-

o Type II restriction enzymes are the kind used for most molecular biology applications
such as gene cloning, DNA fragmentation and analysis.
o They cleave DNA at fixed positions with respect to their recognition sequences.
o Restriction and modification are mediated by separate enzymes so it is possible to cleave
DNA in the absence of modification. Although the two enzymes recognize the same target
sequence, they can be purified separately from each other.
o These enzymes are used to recognize rotationally symmetrical sequence which is often
referred as palindromic sequence.
o These palindromic binding site may either be interrupted (e.g. BstEII recognizes the
sequence 5 ́-GGTNACC-3 ́, where N can be any nucleotide) or continuous (e.g. KpnI
recognizes the sequence 5 ́-GGTACC-3 ́).
o They require only Mg2+ as a cofactor and ATP is not needed for their activity.
o Over 3500 type II enzymes have been characterized, recognizing over 350 different DNA
sequences.
o They vary widely in size, amino acid sequence, domain organization and subunit
composition, cofactor requirements and modes of action.
o Some examples are EcoRI, PvuII, HindIII.
o They are loosely grouped into subtypes based on their enzymatic properties.
o The four most important subtypes are Type IIP, IIS, IIC and IIT.
o Type IIP enzymes account for over 90% of the enzymes used in molecular biology.
o They recognize symmetric or palindromic sequences 4-8 base pairs in length and
generally cleave within that sequence.
o The subunit composition of type IIP enzymes depends on the length of the enzymes
recognition sequences.

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 o Enzymes that recognize short, 4bp sequence act as monomers, comprising single protein
chains, while enzymes that recognize longer, 6-8bp, sequence typically act as
homodimers comprising two identical protein chains.
o Still other type II enzymes act as dimers of diners or homotetramers.
o These later bind to and cleave 2 or more recognition sequence at once.
o Upon cleavage, some type IIP enzymes leave single stranded overhangs, while others
leave blunt ends.
o In contrast to type IIP enzymes, in which the amino acids that catalyse cleavage and those
that recognize the DNA are integrated into a single protein domain, in the larger type IIS
enzymes, those amino acids are partitioned into two separate domains, linked by a short
polypeptide connected.
o Due to the separation, the catalytic domain is positioned to one side of, and several base
pairs away from the sequence bound by the recognition domain, causing cleavage to be
shifted to one side of the sequence.
o Type IIS enzymes generally bind to DNA as monomers and recognize asymmetric
sequence but cleave as dimers.
o In Type IIC enzymes, restriction and modification activities are combined into a
composite enzyme with three domains: one for cleavage, one for methylation and a third
for sequence recognition.
o Type IIC enzymes also cleave outside of their recognition sequence.
o Type IIT enzymes, in contrast to the previous three subtypes, use two different catalytic
sites for cleavage, each specific for our particular DNA strand.
o Disruption of either catalytic site results in the creation of a DNA-nicking enzyme that
cleaves only one DNA strand.
The steps involved in DNA binding and cleavage by a type II restriction endonuclease:
 These enzymes have nonspecific contact
with DNA and initially bind to DNA as
dimmers.
 The target site is then located by a
combination of linear diffusion or “sliding”
of the enzyme along the DNA over short
distances, and hopping/jumping over
longer distances.
 Once the target restriction site is located,
the recognition process (coupling) triggers
large conformational changes of the
enzyme and the DNA, which leads to
activation of the catalytic centre.
 Catalysis results in hydrolysis of
phosphodiester bond and product release.

 Type III-

o Type III restriction enzymes are infrequently used in molecular biology, as they have
few relevant applications.
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 o Therefore, they are less well characterized than type II enzymes.
o While the mechanism of type III restriction modification system is uncertain, the
enzymes themselves consists of two proteins that function as a single protein complex:
the methyl transferases or the M protein, which contains the specificity domain and the
restriction endonuclease or the R protein.
o Type III enzymes recognize a 5-6 bases, non palindromic sequence and require two
inversely oriented recognition sites for cleavage.
o Once bound, the slides along the DNA and upon encountering a second complex, cleaves
downstream of the recognition sites, typically 25-28 bases away.
o Mg+2 ions, ATP are needed for DNA cleavage and process of cleavage is stimulated by
SAM.
o EcoP15I is currently the only type III restriction enzyme commercially available.

Applications of restriction enzymes

 The most interesting use of a restriction enzyme is in a living bacteria, where the they
function to defend the cell against invading viral bacteriophages.
 Restriction sites in the viral genome are cleaved by the bacterium’s restriction enzymes,
fragmenting and destroying DNA of invading bacteriophages before it can incorporate
into the host's genome and take over the cell.
 A bacterium is immune to it’s own restriction enzymes, even if it has the target sequences
ordinarily targeted by them.
 This is because the bacterial restriction sites are highly methylated, making them
unrecognizable to the restriction enzymes.
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In addition, there are various applications related to genetic engineering where DNA is cleaved
by using these restriction enzymes.
Some of the applications include-
o Gene cloning and protein expression- Restriction enzymes in combination with DNA
ligase help in insertion of genes into plasmid vectors during gene cloning and protein
expression. For this, both plasmid DNA containing multiple cloning sites and gene insert
are treated with the same restriction enzymes and then glued together with the help of
DNA ligase. Restriction enzymes are also used after cloning to confirm that insertion of
gene has taken place correctly.

o DNA mapping- A DNA map generated by restriction digestion can be used to find the
relative positions of the genes. The restriction digestion generates different lengths of
DNA which gives a specific pattern of bands after gel electrophoresis. This can be used
for DNA fingerprinting.

o Restriction fragment length polymorphism (RFLP)- Restriction enzymes are used to

digest genomic DNA for gene analysis using Southern blot. With the help of this
technique, researchers can find copy number of a gene present in the genome of one
individual as well as number of gene mutations (i.e., polymorphisms) within a
population. This is called RFLP.

o Studying epigenetic modifications- The sensitivity of restriction endonucleases

toward methylated bases has been used to map modified bases within genomes.

o Preparation of DNA libraries- Restriction enzymes are used in SAGE (serial analysis of
gene expression) for identification and quantification of a large number of mRNA
transcripts in cancer research to diagnose mutations and study gene expression.

2.2. Modification: Enzymes and Significance

DNA modification refers to changes or alterations occurring in DNA molecules during:

o Replication
o Transcription
o Repair
o Genetic recombination
o Epigenetic regulations

DNA Modifying enzymes

The enzymes that effect change in the DNA chemical constitution or topology are generally
referred to as DNA modifying enzymes. Broadly they can be grouped into two categories based
upon the nature of modification performed:

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 I. Composition modifiers: These are the enzymes that change the chemical constitution and
require net gain or loss of covalent bonds. Major composition modifiers include enzyme
classes such as Nucleases, Methyltransferases (Methylases and Demethylases),
Phosphatases, Kinases, Polymerases and Ligases.

II. Topology modifiers: They change the topology of the DNA molecule and does not result in
net gain or loss of a covalent bond. These enzymes are primarily involved in DNA replication.
They are generally classified into two classes based upon the number of DNA strands cut by
the enzyme in single reaction, i.e., Topoisomerase type I (single strand cut and seal) and
Topoisomerase type II (double strand cut and seal).

Important DNA modifying enzymes are:

1. Nucleases
 This class of enzymes break the phosphodiester bonds of the DNA backbone.
 They produce single and double stranded breaks in their target molecule.
 In living organisms, they are essential machinery for many aspects of DNA repair. Defects
in certain nucleases can cause genetic instability.
 There are two primary classes of nucleases:

I. Exonucleases: They attack at the end of nucleic acid and usually remove just a single
nucleotide or sometimes a short oligonucleotide. Any particular exonuclease either
the 3’ end or 5’end not both, e.x. Bal31, exonucleaseIII

II. Endonucleases: Enzymes belonging to this class of nucleases degrade/makes cut(s)

at internal site. A number of different specificity endonucleases exist. Some examples
are
i. S1 nucleases: They cleaves only single-stranded DNA, including single-stranded
(ss) nicks in double-stranded (ds) molecules.

ii. Restriction endonucleases: They function to

defend the cell from invading viral
bacteriophages. They mostly cleave dsDNA, but
only at a limited number of sequence specific
sites. This subcategory of endonucleases
recognizes and/or cleaves DNA at specific
sequences (recognition sequences or restriction

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 site) as compared to no sequence specificity in case of other Nucleases. The REase
are further categorized into four types (Type I-IV) based upon their properties.

 Applications:
 Restriction endonucleases are used in the process of molecular cloning
 ExonucleaseIII is used for generating single stranded templates

2. Deoxy Ribonuclease(DNase)
 A nuclease enzyme that can catalyse the hydrolytic cleavage of phosphodiester bonds in
the DNA backbone are known as deoxyribonuclease (DNase).
 Based on the position of action, these enzymes are broadly classified as
endodeoxyribonuclease (cleave DNA sequence internally) and exodeoxyribonuclease
(cleave the terminal nucleotides).
 Unlike restriction enzymes, DNase does not have any specific recognition/restriction
site and cleave DNA sequence at random locations.
 There is a wide variety of deoxyribonucleases known which have different substrate
specificities, chemical mechanisms, and biological functions. They are:

I. Deoxyribonuclease I (DNaseI): An endonuclease which cleaves double-stranded

DNA or single stranded DNA. The cleavage preferentially occurs adjacent to
pyrimidine (C or T) residues. The major products are 5'-phosphorylated bi-, tri- and
tetranucleotides. It requires divalent ions (Ca2+ and Mn2+/Mg2+) for its activity and
creates blunt ends or 1-2 overhang sequences.
DNase I is the most widely used enzyme in cloning experiments to remove DNA
contamination from mRNA preparation (to be used for cDNA library preparation,
northern hybridization, RT-PCR etc). The mode of action of DNase I varies according
to the divalent cation used. Some of the common applications of DNase I are
Eliminating DNA contamination (e.g. plasmid) from preparations of RNA, Analyzing
the DNA-protein interactions via DNA footprinting, Nicking DNA prior to radio-
labeling by nick translation etc.

II. Deoxyribonuclease II (DNase II): It is a non-specific endonuclease with optimal

activity at acidic pH (4.5-5.5) and conserved from human to C. elegans. It does not
require any divalent cation for its activity. DNase II initially introduces multiple single
stranded nicks in DNA backbone and finally generates 3’ phosphate groups by
hydrolyzing phosphodiester linkages. This enzyme releases 3’phosphate groups by
hydrolyzing phosphodiester linkage and creating nicks in the DNA backbone. DNase
II acts by generating multiple single stranded nicks followed by production of acid
soluble nucleotides and oligonucleotides. The catalytic site of the enzyme contains
three histidine residues which are essential for enzyme activity.
Some of the common applications of DNase II are in DNA fragmentation, as molecular
weight marker, in cell apoptosis assays etc.

3. Ribonuclease(RNase)
 Nuclease that can catalyse hydrolysis of ribonucleotides from either single stranded or
double stranded RNA sequence are called ribonucleotides (RNase).
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  RNase are classified into two types depending on position of cleavage, i.e.
endoribonuclease (cleave internal bond) and exoribonuclease (cleave terminal bond).
 Two common types of Ribonucleases are:

I. Ribonuclease A (RNase A): It is an endo-ribonuclease that cleaves specifically

single-stranded RNA at the 3' end of pyrimidine residues. Optimal temperature for
RNaseA is 60 ̊C (activity range 15-70 ̊C) and optimal pH is 7.6.

II. Ribonuclease H(RNase H): It is a Non-specific endoribonuclease that degrades

RNA by hydrolytic mechanism from DNA/RNA duplex resulting in single stranded
DNA. Enzyme bound divalent metal ion is a cofactor here. The product formed is 5’
phosphorylated ssDNA. During cDNA library preparation from RNA sample, RNaseH
enzyme is used to cleave RNA strand of DNA-RNA duplex.

 Significance:
 RNase is important for RNA maturation and processing.
 RNaseA and RNaseH play important role in initial defence mechanism against RNA
viral infection.
 They can be used to remove RNA contamination from DNA samples.

4. DNA Polymerases
 They are group of enzymes that are used to make copies of DNA templates, essentially
used in DNA replication mechanisms.
 These enzymes make new copies of DNA from existing templates and also function by
repairing the synthesized DNA to prevent mutations.
 DNA polymerase catalyses the formation of the phosphodiester bond which makes up
the backbone of DNA molecules.
 Types of DNA polymerases:

I. DNA polymerase I:
 This is a type A or family A polymerase enzyme.
 Its main function is excision repair of DNA strands in 3’ to 5’ and 5’ to 3’ direction.
 It also helps with maturation of Okazaki fragments, which are short DNA strands
that makeup the lagging strand during DNA replication.
 Its role during DNA replication is the addition of nucleotides at the RNA primer as it
moves along the 5’ to 3’ direction.
 Proteolytic cleavage of DNA Polymerase I resulted in a fragment without 5’ to 3’
exonuclease activity known as Klenow fragment.

II. DNA polymerase II:

 It belongs to type A or family A polymerase enzyme.
 Its major function is the 3’ to 5’exonuclease activity and to also restart replication
after replication stops due to DNA strand damage.
 It is found in the replication fork to help in directing the activities of other
polymerases.

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 III. DNA polymerase III:
 This is the primary enzyme that is used in DNA replication belonging to the family C
or type C.
 It is responsible for the synthesis of new strands by adding nucleotides to the 3’OH
group of the primer.
 It has 3’ to 5’ exonuclease activity hence, it can also proof read the errors that may
arise during DNA strand replication.

IV. Taq DNA Polymerase:

 It is a thermostable DNA polymerase I that was isolated from thermophilic bacteria,
Thermus aquaticus.
 It is commonly used in PCR to amplify short strands of DNA.
 Due to its thermophilic nature it can with stand protein denaturation that is required
during PCR.

5. Ligases
 DNA ligase catalyses the formation of phosphodiester bond between two
deoxynucleotide residues of two DNA strands.
 DNA ligase enzyme requires a free hydroxyl group at the 3 ́ -end of one DNA chain and a
phosphate group at the 5 ́-end of the other and requires energy in the process.
 The role of DNA ligase is to seal nicks in the backbone of double-stranded DNA after
lagging strand formation to join the Okazaki fragments.
 This joining process is essential for the normal synthesis of DNA and for repairing
damaged DNA. It has been exploited by genetic engineers to join DNA chains to form
recombinant DNA molecules.

 The most widely used DNA ligase is isolated from T4 bacteriophage. T4 DNA ligase needs
ATP as a cofactor. The enzyme from E. coli uses cofactor NAD. Except this, the catalysis
mechanism is somewhat similar for both the ligases.

 Significance
 DNA ligase enzyme is used by cells to join the “Okazaki fragments” during DNA
replication process.
 Used in repair of discontinuity- a missing phosphodiester bond in one strand.
 Joining two molecules of dsDNA together
 In molecular cloning, ligase enzyme has been routinely used to construct a recombinant
DNA.

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6. Methyl transferases/Methylases
 Methyltransferase or methylase catalyzes the transfer of methyl group (-CH3) to its
substrate. The process of transfer of methyl group to its substrate is called methylation.
 Methylation is a common phenomenon in DNA and protein structure.
 Methyltransferase uses a reactive methyl group that is bound to sulfur in S- adenosyl
methionine (SAM) which acts as the methyl donor.
 Methylation normally occurs on cytosine (C) residue in DNA sequence. In protein,
methylation occurs on nitrogen atom either on N-terminus or on the side chain of
protein.
 DNA methylation regulates gene or silence gene without changing DNA sequences, as a
part of epigenetic regulation.
 In bacterial system, methylation plays a major role in preventing their genome from
degradation by restriction enzymes. It is a part of restriction – modification system in
bacteria.
 Restriction enzymes cleaves within the recognition sequence if the DNA is unmethylated.
On methylation by methylases, the restriction enzymes are inhibited from cleaving
within the restriction site.
 Significance:
 Inhibit binding of transcriptional
machinery proteins to DNA thereby
blocking gene expression.
 Recruit specific repressors at site that then
switch offs nearby genes (gene silencing)
often by recruiting histone modifying
enzymes.
 Allows discrimination between parent and
daughter strands during DNA repair
(maintenance methylases)
 Protect DNA from action of endonucleases
which are part of Restriction modification
system

7. Phospahtases
 Phosphatase catalyses the cleavage of a phosphate (PO4-2) group from substrate by using
a water molecule (hydrolytic cleavage).
 This reaction is not reversible.
 This shows totally opposite activity from enzyme like kinase and phosphorylase that add
a phosphate group to their substrate.
 On the basis of their activity there are two types of phosphatase i.e acid phosphatase and
alkaline phosphatase.

i. Acid phosphatase: It shows its optimal activity at pH between 3 and 6, e.g. a lysosomal
enzyme that hydrolyse organic phosphates liberating one or more phosphate groups.
They are found in prostatic epithelial cells, erythrocyte, prostatic tissue, spleen, kidney
etc.

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 ii. Alkaline phosphatase: It is a Homodimeric enzyme which catalyses reactions like
hydrolysis and transphosphophorylation of phosphate monoester. They show their
optimal activity at pH of about 10.

During post-translational modification, alkaline phosphatase is modified by N-

glycosylation. It undergoes a modification through which uptake of two Zn+2 ion
and one Mg+2 ion occurs which is important in forming active site of that enzyme.
Alkaline phosphatases are isolated from various sources like microorganisms, tissue of
different organs, connective tissue of invertebrate and vertebrate, and human body.

8. Polynucleotide Kinases
 It is a homotetramer with phosphatase activity at 3’ end and kinase activity at 5’ end with
a tunnel like active site.
 Polynucleotide kinase (PNK) catalyzes the transfer of a phosphate group (PO4-2) from γ
position of ATP to the 5' end of either DNA or RNA and nucleoside monophosphate.
 PNK can convert 3' PO4/5' OH ends into 3' PO4/5' PO4 ends which blocks further ligation
by ligase enzyme.
 PNK is used to label the ends of DNA or RNA with radioactive phosphate group.
 T4 polynucleotide kinase is the most widely used PNK in molecular cloning experiments,
which was isolated from T4 bacteriophage infected E.coli.

9. DNA Glycosylases
 These are the enzymes that generally recognize the
damaged base/nucleotides present in DNA and remove
them
 They hydrolyse the glycosidic bond between sugar and
Nitrogen base (part of DNA repair system) producing a
basic (AP) site in the DNA.
 They are one of the key components of general DNA repair
system.

10. Topoisomerases
 These are enzymes that change supercoiling, i.e., topology
of the DNA molecule.
 They generally remove supercoils relieving the torsional stress present in the
overwound DNA during replication.
 They transiently break the DNA backbone, thereby allowing the DNA supercoils to relax.

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  Topoisomerases are classified according to mechanism they use for changing DNA
Topology

i. Type IA and IB: They break one of the two strands of DNA, rotate the end of the
broken strand around the intact strand, and then seal the ends. The reaction does not
require ATP.

ii. Type II: They cleave both strands during reaction rotate both ends by 360 degrees
and then reconnect the respective ends. The reaction is powered by ATP hydrolysis.
e.g., "DNA gyrase" of E. coli.

2.3. Repair

2.3.1. Major kinds of damage to DNA and causes

 DNA is the source of genetic information and preserving its integrity is essential in order to
sustain life.
 Any modification in the physical or chemical structure of DNA resulting in an altered DNA
molecule which is different from original DNA molecule with regard to its physical,
chemical or structural properties is referred to as DNA damage.
 DNA damage may occur under the influence of factors external to the cell (factors of
exogenous origin, e.g. environmental factors) or potentially aggressive agents produced by
normal cell metabolism (factors of endogenous origin).
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 The consequences for the cell's DNA damage caused by the action of endogenous factors
may be more serious and/or more extensive than the effect of most of the exogenous DNA
damaging factors.
 DNA damaging events caused by endogenous factors generally occur much more frequently
than damage caused by exogenous factors.
 Examples of factors of exogenous origin are ionizing radiations i.e. X-rays, cosmic radiation,
UV light and mutagenic chemicals like polycyclic aromatic hydrocarbons.
 Examples of factors of endogenous origin include reactive oxygen or nitrogen species.
 DNA damaging agents can also be classified based on their nature as physical or chemical.
 The physical agents include short wavelength electromagnetic radiations such as UV and
ionizing radiation.
 Among DNA damaging agents of chemical nature prominent are alkylating agents oxidising
agents, chemicals creating DNA-DNA or DNA-protein cross links.

Types of DNA damages

I. Damages of endogenous origin

i. Conversion of one base to another producing a mismatch

 The four nitrogenous bases in DNA may be subject to direct conversion into one another or

into rare bases which may have pairing affinity to bases different from the original pairing
partner.
 Base conversion often results from hydrolysis of nitrogenous bases in DNA.
 Deamination of nitrogenous bases is a very common type of hydrolytic damage in DNA. For
example, deamination of cytosine produces uracil.
 Uracil pairs more efficiently with A than with G, thereby creating a mismatch.
 In the next round of DNA replication, this will result in a substitution of a C:G pair with an
A:T pair. Adenine may be spontaneously deaminated to hypoxanthine, the later pairing
more readily with C than with T.
 Mismatched bases in DNA are among the critically important alterations of DNA.
 Mismatches may introduce a premature stop signal in mRNA resulting in production of
truncated or unstable protein r no protein at all.

ii. Loss of nitrogenous bases due to hydrolysis

 Typical example of hydrolytic loss of nitrogenous bases is DNA depurination, a very
common type of DNA damage occurring spontaneously.
 In vitro experiments show DNA depurination occurs much faster than depyrimidination.
 Loss of nitrogenous bases in DNA is strongly dependent on the temperature.

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  Loss of nitrogenous bases results in abasic sites in DNA. This, in turn, may promote strand
breakage and/or mispairing.

iii. Alkylation of nitrogenous bases

 Base alkylation usually affects a nitrogen or an oxygen atom in a nitrogenous base in DNA,
producing a variety of modified bases - 5-methylcytosine, 7-methylguanine, 1-
methyladenine, etc. as well as alkyl phosphates.
 Alkylated bases may have different pairing properties from their unmodified counterparts.
 Alkylated bases may be subject to further modification, producing bases different from the
original base (e.g. 5- methylcytosine being deaminated to thymine)

iv. Oxidation of nitrogenous bases

 Oxidation of nitrogenous bases affects the purine bases (A, G) as well as the pyrimidines (C,
T). Purine base oxidation results in 8-oxopurines.
 Oxidation products of pyrimidines are usually thymine glycol, 5-hydroxycytosine, 5-
hydroxyuracil and uracil glycol.
 Base oxidation in DNA is often accompanied by strand breaks, as the same damaging agent
(most often, free radical species) can cause both types of DNA damage.
 Accumulation of oxidative DNA damage (oxidative stress) is believed to be one of the major
mechanisms in ageing and disease.

v. DNA breaks of endogenous origin

 Single-strand breaks (SSBs) result from disruption of the phosphodiester bond between
two adjacent deoxyribose residues in the backbone of DNA.
 SSBs are among the most common instances of DNA damage. Double-strand breaks (DSBs)
are less common in living cells.
 Strand breaks frequently occur as a result of normal manipulation of DNA during
untangling DNA during transcription and replication, relaxation of supercoils, etc.
 Strand breakage may also result from genotoxic stress resulting from the normal cell
metabolism - e.g. from oxidative damage caused by accumulation of free radical species.
 DSB in DNA occur when the phosphodiester backbone of both the strands of the same DNA
helix is broken, the breakpoints being in proximity of each other so that the broken ends
may become physically separated.

II. Damages of exogenous origin

Exogenous agents may cause many different types of damage to DNA, depending on the
nature of agent and the substrate on which it works. Some of this damage is specific to the action
of exogenous agents only (e.g. dimerisation) while others may be caused by endogenous factors
as well (e.g. base alkylation, strand breaks, etc.)

i. Dimerisation
 Dimers of any type do not normally exist in DNA.
 Dimer formation usually results from electromagnetic radiation in the UV range.
 The type of UV induced damage to DNA is dependent on the wavelength of UV.

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 Dimers are most often caused by high energy, short wavelength UV in the range of 100-
300nm (UV-B).
 Under certain conditions, UV-A light (300-400nm) may also cause direct damage to DNA,
including dimers, although the risk is lower than UV-B.
 Dimerisation may occur may occur between bases belonging to the same strand of DNA or
between bases from different DNA strands.
ii. Formation of bulky adducts in DNA
 Bulky adducts in DNA create steric impediment for processes involving DNA replication,
transcription.
 Bulky adducts getting in the way of transcription cause stalling of DNA polymerase II at the
damage site and recruitment of the cell repair machinery.
 The presence of bulky adducts in the DNA of a dividing cell would normally cause
replication arrest.
 Aromatic compounds and, specifically, polycyclic aromatic hydrocarbons (PAH) are among
the commonly encountered and potent adduct-forming environmental agents.
 Another common adduct-forming agent is benzopyrene diol epoxide, which is produced in
vivo from benzopyrene (a compound of tobacco smoke).
 Some compounds commonly used in polishing liquids, cleaning liquids, disinfectants or
industrial solvents are adduct forming agents.

iii. Free radical species of exogenous origin

 Free radical species, including reactive oxygen species (ROS - superoxide radicals, free
hydroxyl radicals, etc.) are generated in large quantities as a result of normal cell
metabolism, but they may be generated by environmental insults as well.
 The majority of ROS of exogenous origin result from UV irradiation, usually with UV-A (300-
400nm). ROS are also generated by radiolysis of water by ionising radiation (alpha, beta or
gamma).

iv. DNA breaks induced by exogenous origin

 SSBs may result from a variety of chemical agents, long-wavelength UV and even infrared
energy.
 Exogenously induced DSBs in DNA are usually product of high-energy electromagnetic
radiation (specifically ionising radiation) and certain chemicals such as benzenes some
dyes such as acrydine yellow etc.
 Double-strand breaks may be generated directly (physical breakage of the phosphodiester
bond in the DNA backbone - for example, by high-energy particles) or indirectly (e.g. by
generation of free radical species).

v. Alkylation of exogenous origin

 Among the more commonly encountered alkylating agents are the haloalkanes
(dichloromethane, chloroform etc.), alkyl sulfonates, nitrosoureas, and others.
 One of the most infamous warfare chemical agents, mustard gas used in World War I and
II is an alkylating agent.
 Some of the most potent and widely used anticancer agents (e.g. cyclophosphamide and
dacarbazine) work by alkylation of DNA.
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III. Mutations in DNA
Mutations are random changes that occur within sequence of bases in DNA. They can
be large scale, altering the structure of the chromosomes called chromosomal mutations or
small scale when they only alter a few or single bases or nucleotides referred to as point
mutations.
i. Point mutations
 Point mutation is a type of mutation in which one single nucleotide base is inserted,
deleted or substituted
 Substitution of a nucleotide base leads to missense, nonsense or silent mutation.
 Insertion or deletion of nucleotide base results in frame shift mutation

a. Missense mutation
 A missense mutation occurs when there is a
mistake in the DNA code and one of the DNA
base pairs is changed, for example, A is
swapped for C.
 This single change means that the DNA now
encodes for a different amino acid, known
as a substitution.
 Sometimes a change in the amino acid has no effect on the resulting protein's function at
all.
 On some occasions, the change in amino acid actually enhances the protein's function,
but in other cases it can ultimately render the protein as "faulty".

b. Nonsense mutation
 Nonsense mutations convert a normal triplet
coding for an amino acid residue to stop codon.
 This may result in premature degradation of the
mRNA containing the illegitimate stop codon or in
premature termination of mRNA translation,
producing a truncated non-functional protein or a
protein with altered properties.
 In most cases this adversely effects the properties of
the synthesized protein resulting in severe phenotype
of genetic diseases.

c. Silent mutation
 Silent mutations are a type of point mutation resulting
in a codon that codes for the same amino acid without
any functional change in the protein product
d. Frame shift mutation
 A frameshift mutation occurs when the "addition" or "deletion" mutations result in a
change to the gene's reading frame, which includes groups of three bases that encode
for an amino acid.

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  The change in the reading frame alters the
grouping of the bases and subsequently
changes the amino acids that are encoded.
 Often, the encoded protein is non-functional.

ii. Chromosomal mutations

 The chromosomal mutation is a process of
change in the chromosomes as a result of rearranged chromosome parts and changes
in the number of individual chromosomes or chromosome set present in the genome.
 They are the result of certain accidents or irregularities in the chromosomes at the
time of cell division, crossing over or fertilisation.
 Chromosomal mutations can be broadly classified into two groups Chromosomal
mutation I and chromosomal mutation II

A. Chromosomal mutation I
 Chromosomal mutations I include structural mutations that arise as a result of
alterations in the structure of the chromosomes.

a. Inversion
 Inversion is a type of structural mutation where
a part of chromosomes or a set of genes rotates
by 180° on its own axis.
 There is no net loss or gain of genes but simply
a rearrangement of the sequence. A part of the
chromosome is broken and then rejoined in a
different direction.
b. Deletion
 Deletion is a type of structural mutation that occurs due to the loss of a part of a
chromosome as a result of the breakage of the
chromosome.
 Chromosomes that have undergone deletion
cannot revert back to normal and, if transmitted
to the next generation, can be hereditary.
 Deletion can either be terminal or intercalary.
 Example cri du chat syndrome occurring due deletion of the short arm of
chromosome 5 in human results in a distinctive cat-like cry in babies.

c. Duplication/Amplification
 Duplication is a type of structural mutation where
a part of a chromosome is present in excess of the
normal composition.
 The genes present in a cell might exist in more
than two doses as a result of duplication.

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 d. Translocation
 Translocation is a type of structural mutation
resulting from the shift or transfer of a part of
a chromosome or a set of genes to a
nonhomologous chromosome.
 There is no net gain or loss of chromosomes
or genes during translocation but a
rearrangement.
 There are three different types of translocations depending on the pattern of
rearrangement; simple translocation, shift translocation, and reciprocal
translocation

B. Chromosomal mutation II
 Chromosomal mutations II include mutations that are caused by the alterations in the
number of chromosomes in a cell.
 The change in the number of whole chromosomes is called heteroploidy.
 Heteroploidy can be further divided into two different categories depending on the
changes in the entire set of chromosomes or in the single whole chromosome.
a. Aneuploidy
 Aneuploidy is a type of mutation that
changes parts of a chromosome set,
resulting in either the loss of one or
more chromosomes or the addition of
chromosomes.
 Aneuploidy resulting from the loss of
chromosomes is called hypoploidy,
whereas that due to the addition of
chromosomes is called hyperploidy.
 Hypoploidy usually occurs due to the
loss of a single chromosome
(monosomy) or due to the loss of a
pair of chromosomes (nullisomy).
 Hyperploidy, in turn, might involve
the addition of a single chromosome
(trisomy) or the addition of a pair of
chromosomes (tetrasomy).
 Aneuploids are caused as a result of
nondisjunction during mitosis or
meiosis.
 Down’s Syndrome is an example of aneuploidy which is a trisomic condition of 21st
chromosome.

b. Polyploidy
 Polyploidy is a type of euploidy (changes in the entire set of chromosomes) where
an organism has more than two sets of genomes (2x).

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  Polyploidy includes different combinations like triploid, tetraploid, pentaploid,
hexaploid, and octoploid.
 Polyploidy can be further divided into three groups; autopolyploids, allopolyploids,
and autoallopolyploids.
 Autopolyploids are polyploids that consist of the same basic set of chromosomes but
multiplied to form multiple sets.
 Allopolyploids are the polyploids that result from the doubling of chromosome
number in a hybrid from two different species.

2.3.2. Repair mechanisms: direct reversal, mismatch repair, excision repair,

recombination, SOS response

DNA repair
 Since most mutations are deleterious, DNA repair systems are vital to the survival of all
organisms.
 If DNA repair systems did not exist, spontaneous and environmentally induced mutations
would be so prevalent that few species, if any, would survive.
 In humans, an individual who is defective in only a single DNA repair system may manifest
various disease symptoms, including a higher risk of skin cancer.
 This increased risk is due to the inability to repair UV-induced mutations.
 Living cells contain several DNA repair systems that can fix different types of DNA
alterations.
 Each repair system is composed of one or more proteins that play specific roles in the repair
mechanism.
 In most cases, DNA repair is a multistep process.
 First, one or more proteins in the DNA repair system detect an irregularity in DNA
structure.
 Next, the abnormality is removed by the action of DNA repair enzymes.
 Finally, normal DNA is synthesized via DNA replication enzymes.

Types of DNA repair mechanisms

1. Direct reversal repair

 Direct reversal repair is when an enzyme recognizes an incorrect alteration in DNA
structure and directly converts it back to a correct structure.
 An example of repair by simple reversal of damage is photoreactivation.
 Photoreactivation directly reverses the formation of pyrimidine dimers that result from
ultraviolet irradiation.
 In photoreactivation, the enzyme DNA photolyase captures energy from light and uses it to
break the covalent bonds linking adjacent pyrimidines.

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  Another example of direct reversal is
the removal of the methyl group from
the methylated base O6-
methylguanine.
 In this case, a methyltransferase
removes the methyl group from the
guanine residue by transferring it to
one of its own cysteine residues.
 This is costly to the cell because the methyltransferase is not catalytic; having once accepted
a methyl group, it cannot be used again.

2. Mismatch repair
 The structure of the DNA double helix obeys the AT/GC rule of base pairing.
 During the normal course of DNA replication, however, an incorrect nucleotide may be
added to the growing strand by mistake.
 This produces a mismatch between a nucleotide in the parental and the newly made strand.
 Various DNA repair mechanisms can recognize and remove this mismatch.
 DNA polymerase has a 3ʹ to 5ʹ proofreading ability that can detect mismatches and remove
them.
 However, if this proofreading ability fails, cells contain additional DNA repair systems that
can detect base mismatches and fix them.
 An interesting DNA repair system that exists in all species is the mismatch repair system.

 The molecular mechanism of mismatch repair has been studied extensively in E. coli.
 Three proteins, designated MutS, MutL, and MutH, detect the mismatch and direct the
removal of the mismatched base from the newly made strand.
 These proteins are named Mut because their absence leads to a much higher mutation rate
than occurs in normal strains of E. coli.
 The role of MutS is to locate mismatches. Once a mismatch is detected, MutS forms a
complex with MutL.
 MutL acts as a linker that binds to MutH by a looping mechanism.
 This stimulates MutH, which is bound to a hemimethylated site, to make a cut in the newly
made, nonmethylated DNA strand.
 After the strand is cut, MutU, which functions as helicase, separates the strands, and an
exonuclease then digests the non-methylated DNA strand in the direction of the mismatch
and proceeds beyond the mismatch site.
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Edited by Dr. Arshad Keethadath, Department of Zoology, PSMO College, Tirurangadi
 This leaves a gap in the daughter strand that is repaired by DNA polymerase and DNA ligase.
 The net result is that the mismatch has been corrected by removing the incorrect region in
the daughter strand and then resynthesizing the correct sequence using the parental DNA
as a template.

3. Excision repair
There are two types of excision repair mechanisms:
i. Base excision repair
 Base excision repair (BER), involves the
function of a category of enzymes known
as DNA N-glycosylases.
 This type of enzyme can recognize an
abnormal base and cleave the bond
between it and the sugar in the DNA
backbone, creating an apurinic or
apyrimidinic site.
 Living organisms produce multiple types
of DNA N-glycosylases, each recognizing
particular types of abnormal base
structures.
 Depending on the DNA N-glycosylase, this
repair system can eliminate abnormal
bases such as uracil, 3-methyladenine, 7-
methylguanine, and pyrimidine dimers.
 Base excision repair is particularly
important for the repair of oxidative DNA
damage.
 Figure above depicts the general steps involved in DNA repair via n N-glycosylases.
 Here the DNA contains uracil in its sequence. This could have happened spontaneously or
by the action of a chemical mutagen.
 N-glycosylase recognizes a uracil within the DNA and cleaves (nicks) the bond between the
sugar and base.
 This releases the uracil base and leaves behind an apyrimidinic site.
 This abnormality is recognized by a second enzyme, AP endonuclease, which makes a cut
on the 5ʹ side.
 Following this cut by AP endonuclease, one of three things can happen.
 In some species such as E. coli, DNA polymerase I, which has a 5ʹ to 3ʹ exonuclease activity,
removes a DNA segment containing the abnormal region and, at the same time, replaces it
with normal nucleotides.
 This process is called nick translation (although DNA replication, not mRNA translation,
actually occurs).
 Alternatively, in eukaryotic species such as humans, the DNA is repaired in two possible
ways.

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  DNA polymerase β has the enzymatic ability to remove a site, which is missing a base, and
then insert a nucleotide with the correct base in its place.
 Another possibility is that DNA polymerase б or ε can synthesize a short segment of DNA,
which generates a flap.
 The flap is then removed by flap endonuclease.
ii. Nucleotide excision repair
 An important general process for DNA
repair is the nucleotide excision repair
(NER) system.
 This type of system can repair many
different types of DNA damage, including
thymine dimers, chemically modified
bases, missing bases, and certain types of
crosslinks.
 In NER, several nucleotides in the damaged
strand are removed from the DNA, and the
intact strand is used as a template for
resynthesis of a normal complementary
strand.
 NER is found in all eukaryotes and
prokaryotes, although its molecular
mechanism is better understood in
prokaryotic species.
 In E. coli, the NER system requires four key
proteins, designated UvrA, UvrB, UvrC, and
UvrD, plus the help of DNA polymerase and
DNA ligase.
 UvrA, B, C, and D recognize and remove a
short segment of a damaged DNA strand.
 They are named Uvr because they are
involved in Ultraviolet light repair of
pyrimidine dimers, although the UvrA–D proteins are also important in repairing
chemically damaged DNA.
 The Figure outlines the steps involved in the E. coli NER system. A protein complex
consisting of two UvrA molecules and one UvrB molecule tracks along the DNA in search of
damaged DNA.
 Such DNA has a distorted double helix, which is sensed by the UvrA/UvrB complex. When
a damaged segment is identified, the two UvrA proteins are released, and UvrC binds to the
site.
 The UvrC protein makes cuts in the damaged strand on both sides of the damaged site
 Typically, the damaged strand is cut eight nucleotides from the 5ʹ end of the damage and
four to five nucleotides away from the 3ʹ end.
 After this process, UvrD, which is a helicase, recognizes the region and separates the two
strands of DNA.

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  This releases a short DNA segment that contains the damaged region, and UvrB and UvrC
are also released.
 Following the excision of the damaged DNA, DNA polymerase fills in the gap, using the
undamaged strand as a template.
 Finally, DNA ligase makes the final covalent connection between the newly made DNA and
the original DNA strand.

4. Recombination
 Of the many types of DNA damage that can occur within living cells, the breakage of
chromosomes—called a DNA double-strand break (DSB)—is perhaps the most dangerous.
 DSBs can be caused by ionizing radiation (X-rays or gamma rays), chemical mutagens, and
certain drugs used for chemotherapy.
 In addition, reactive oxygen species that are the by-products of aerobic metabolism can
cause double-strand breaks.
 The two main types of recombination repair are:

a. Homologous recombination repair(HRR)

 Homologous recombination repair, also called homology-directed repair, occurs when
homologous DNA strands, usually from a sister chromatid, are used to repair a DSB in
the other sister chromatid.
 First, the DSB is processed by the short digestion of DNA strands at the break site.
 This processing event is followed by the exchange of DNA strands between the broken
and unbroken sister chromatids.
 The unbroken strands are then used as templates to synthesize DNA in the region where
the break occurred.
 Finally, the crisscrossed strands are resolved, which means they are broken and then
rejoined in a way that produces separate chromatids.

b. Non-homologous end joining(NHEJ)

 During nonhomologous end joining, the two broken ends of DNA are simply pieced back
together.
 This mechanism requires the participation of several proteins that play key roles in the
process.
 First, the DSB is recognized by end- binding proteins.
 These proteins then recognize additional proteins that form a cross-bridge that prevents
the two ends from drifting apart.
 Next, additional proteins are recruited to the region that may process the ends of the
broken chromosome by digesting particular DNA strands.
 This processing may result in the deletion of a small amount of genetic material from the
region. Finally, any gaps are filled in via DNA polymerase, and the DNA ends are ligated
together

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5. SOS response
 Despite having multiple repair system, sometimes the damage to an organism’s DNA is so
great that the normal repair mechanisms just described cannot repair all the damage.
 As a result, DNA synthesis stops completely.
 In such situations, a global control network called the SOS response is activated.
 The prokaryotic SOS system is regulated by two main protein i.e. Lex A and Rec A.
 The most common cellular signals activating the SOS response are regions of single-stranded
DNA (ssDNA), arising from stalled replication fork or double-strand breaks, which are
processed by DNA helicase to separate the two DNA strands.
 During normal growth the SOS genes are negatively regulated by LexA repressor protein
dimers.
 For initiation of SOS response, RecA protein binds to ssDNA in an ATP hydrolysis driven
reaction creating RecA–ssDNA filaments.

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 RecA binds to single or double stranded DNA breaks and gaps generated by cessation of DNA
synthesis. RecA binding initiates recombination repair.
 RecA–ssDNA filaments activate LexA auto protease activity, which ultimately leads to
cleavage of LexA dimer and subsequent LexA degradation.
 The loss of LexA repressor induces transcription of the SOS genes and allows for further
signal induction, inhibition of cell division and an increase in levels of proteins responsible
for damage processing
 The first SOS repair mechanism to be induced is nucleotide excision repair whose aim is to
fix DNA damage without commitment to a full-fledged SOS response.
 SOS repair system operates only under potentially lethal conditions caused by extensive UV
irradiation.
 Therefore, SOS repair system works on the principle that survival with mutation is better
than no survival at all.
 This repair is also called Error prone repair.

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