Mutation

Introduction
• A mutation is a permanent change of the nucleotide sequence of the genome of
an organism, virus, or extra-chromosomal DNA or other genetic elements.
• Mutations result from damage to DNA which is not repaired or to RNA genomes
(typically caused by radiation or chemical mutagens), errors in the process of replication,
or from the insertion or deletion of segments of DNA by mobile genetic elements.
• May or may not produce discernible changes in the observable characteristics
(phenotype) of an organism.
• Mutations play a part in both normal and abnormal biological processes
including: evolution, cancer, and the development of the immune system, including
junctional diversity
• Mutations in genes can either have no effect, alter the product of a gene, or prevent the
gene from functioning properly or completely.
• Mutations can also occur in nongenic regions
• Due to the damaging effects that mutations can have on genes, organisms have
mechanisms such as DNA repair to prevent or correct (revert the mutated sequence
back to its original state) mutations
 Mutagenesis
• Mutagenesis is a process by which the genetic information of
an organism is changed in a stable manner, resulting in
a mutation.
• Occur spontaneously in nature, or as a result of exposure
to mutagens.
• It can also be achieved experimentally using laboratory
procedures.
• In nature mutagenesis can lead to cancer and various heritable
diseases, but it is also a driving force of evolution.
• Mutagenesis as a science developed based on work done
by Hermann Muller, Charlotte Auerbach and J. M. Robson in the
first half of the 20th century.
Biochemical Basis of Mutations
Central Dogma of Molecular Biology

Deoxyribo Nucleic Acids (DNA)

Replication
DNA
Transcription
Ribose Nucleic Acid (RNA)
Translation
Amino Acids

Proteins
Deoxyribo Nucleic Acids (DNA)
Chemical Structure
and Interactions
 Grooves in DNA

Major Grooves Minor Grooves

HYDROGEN BONDING
DNA to Chromosomes
 Molecular Basis
• Protein-coding DNA can be divided into codons —
sets of three bases that specify an amino acid or
signal the end of the protein.
• Codons are identified by the bases that make them
up — in the example at right, GCA, for guanine,
cytosine, and adenine.
• The cellular machinery uses these instructions to
assemble a string of corresponding amino acids (one
amino acid for each three bases) that form a protein.
• The amino acid that corresponds to "GCA" is called
alanine; there are twenty different amino acids
synthesized this way in humans.
• "Stop" codons signify the end of the newly built
protein.
 Causes of Mutation
Four classes of mutations are
(1) spontaneous mutations (molecular decay),
(2) Mutations due to error prone replication bypass of naturally occurring DNA
damage(also called error prone translesion synthesis)
(3) Errors introduced during DNA repair, and
(4) induced mutations caused by mutagens. Scientists may also deliberately introduce
mutant sequences through DNA manipulation for the sake of scientific
experimentation.

Spontaneous Mutation
Spontaneous mutations on the molecular level can be caused by:
A) Tautomerism
B) Depurination
C) Deamination
D) Slipped Strand Pairing
 Tautomerism
• Tautomers are constitutional isomers of organic compounds that readily
interconvert by a chemical reaction called tautomerization
• Tautomerism: A base is changed by the repositioning of a hydrogen atom, altering
the hydrogen bonding pattern of that base, resulting in incorrect base pairing
during replication
• This reaction commonly results in the formal migration of a hydrogen atom or
proton, accompanied by a switch of a single bond and adjacent double bond.
• Because of the rapid interconversion, tautomers are generally considered to be the
same chemical compound.
• Tautomerism is a special case of structural isomerism and can play an important
role in non-canonical base pairing in DNA and especially RNA molecules.

Aromaticity provides some stability to the

triple lactim form of these triple lactam-
lactim tautomers.
 Depurination
• Loss of a purine base (A or G) to form an apurinic site (AP site).
• Depurination is a chemical reaction of purine deoxyribonucleosides, deoxyadenosine and
deoxyguanosine, and ribonucleosides, adenosine or guanosine, in which the β-N-glycosidic
bond is hydrolytically cleaved releasing a nucleic base, adenine or guanine respectively.
• The second product of depurination of deoxyribonucleosides and ribonucleosides is sugar, 2’-
deoxyribose and ribose, respectively.
• More complex compounds containing nucleoside residues, nucleotidesand nucleic acids, also
suffer from depurination.
• Deoxyribonucleosides and their derivatives are substantially more prone to depurination
than their corresponding ribonucleoside counterparts.
• Loss of pyrimidine bases (Cytosine and Thymine) occurs by a similar mechanism, but at a
substantially lower rate.
• When depurination occurs with DNA, it leads to the formation of apurinic site and results in
an alteration of the structure.
• Apurinic sites in double-stranded DNA are efficiently repaired by portions of the base
excision repair (BER) pathway.
• Depurinated bases in single-stranded DNA undergoing replication can lead to mutations,
because in the absence of information from the complementary strand, BER can add an
incorrect base at the apurinic site, resulting in either a transition or transversion mutation.
 Depurination

Chemical structure of apurinic site present in a fragment of single-stranded DNA

 Deamination
Cytosine
• Spontaneous deamination is the hydrolysis reaction of cytosine into uracil,
releasing ammonia in the process.
• This can occur in vitro through the use of bisulfite, which converts
cytosine, but not 5-methylcytosine.
• This property has allowed researchers to sequence methylated DNA to
distinguish non-methylated cytosine (shown up as uracil) and methylated
cytosine (unaltered).
 In DNA, spontaneous deamination is corrected

Removal of uracil (product of cytosine deamination and not part of DNA)

Uracil-DNA glycosylase
Generating an abasic (AP) site.

Recognized by enzymes (AP endonucleases)

Break a phosphodiester bond in the DNA

Permitting the repair of the resulting lesion

Replacement with another cytosine

Cytosine Uracil
A DNA Polymerase may perform this replacement via nick translation

Terminal excision reaction by its 5'-->3' exonuclease activity

Fill-in reaction by its polymerase activity

DNA ligase then forms a phosphodiester bond

Seal the resulting nicked duplex product

A new, correct cytosine.

5-methylcytosine
• Spontaneous deamination of 5-methylcytosine results in thymine and ammonia.
• This is the most common single nucleotide mutation.
• In DNA, this reaction can be corrected by the enzyme thymine-DNA glycosylase prior to
passage of the replication fork, which would fix the cytosine to thymine point mutation
in one of the two daughter strands
Guanine
• Deamination of guanine results in the formation of xanthine.
• Xanthine, in a manner analogous to the enol tautomer of guanine, selectively base
pairs with thymine instead of cytosine.
• This results in a post-replicative transition mutation, where the original G-C base pair
transforms into an A-T base pair.
• Correction of this mutation involves the use of alkyladenine glycosylase (Aag)
during base excision repair
Adenine
• Deamination of adenine results in the formation of hypoxanthine.
• Hypoxanthine, in a manner analogous to the imine tautomer of adenine, selectively
base pairs with cytosine instead of thymine.
• This results in a post-replicative transition mutation, where the original A-T base pair
transforms into a G-C base pair.
 Slipped strand mispairing

• Slipped strand mispairing (SSM) is a mutation process which occurs

during DNA replication.
• Denaturation of the new strand from the template during replication,
followed by renaturation in a different spot ("slipping"). This can lead to
insertions or deletions.

It involves denaturation and displacement of the DNA strands

Mispairing of the complementary bases

One explanation for the origin and evolution of repetitive DNA sequences

Shown to function as a phase variation mechanism in certain bacteria

 Conditional Mutants

• Types involved in this are:

– Error prone replication by-pass
– Errors introduced during DNA repair
– Induced mutation
 Error prone replication by-pass
• DNA damage is an alteration in the chemical structure of DNA, such as a break in a strand of
DNA, a base missing from the backbone of DNA, or a chemically changed base such as 8-OHdG.
• Damage to DNA that occurs naturally can result from metabolic or hydrolytic processes - DNA
damage (naturally occurring).
• Metabolism releases compounds that damage DNA including reactive oxygen species, reactive
nitrogen species, reactive carbonyl species, lipid peroxidation products and alkylating agents,
among others, while hydrolysis cleaves chemical bonds in DNA

DNA Damage Mutation

Abnormal chemical structure in DNA Change in the sequence of standard base pairs
Undergo DNA repair (~100%) No repair
Recognized by Enzymes Not Recognized by Enzymes
Physical abnormalities such as single- and double- Alterations in protein function and regulation
strand breaks, 8-hydroxydeoxyguanosine residues,
and polycyclic aromatic hydrocarbon adducts
No transfer during cell division Replicates when cell replicates
No function, cell death Increase or decrease, cell survives
May lead to aging if no repair May lead to cancer, then death
 Errors introduced during DNA repair
• Naturally occurring double-strand breaks occur at a relatively low frequency in
DNA and their repair often causes mutation.
• Non-homologous end joining (NHEJ) is a major pathway for repairing double-
strand breaks.
NHEJ

Removal of a few nucleotides

Allow somewhat inaccurate alignment of the two ends for rejoining

Addition of nucleotides to fill in gaps

As a consequence, NHEJ often introduces mutations

 Induced mutation
Chemicals
• Hydroxylamine NH2OH
• Base analogs (e.g., BrdU)
• Alkylating agents (e.g., N-ethyl-N-nitrosourea):
– These agents can mutate both replicating and non-replicating DNA.
– In contrast, a base analog can mutate the DNA only when the analog is incorporated in replicating the DNA.
– Each of these classes of chemical mutagens has certain effects that then lead to transitions, transversions, or
deletions.
• Agents that form DNA adducts (e.g., ochratoxin A metabolites)
• DNA intercalating agents (e.g., ethidium bromide)
• DNA crosslinkers
• Oxidative damage
• Nitrous acid converts amine groups on A and C to diazo groups, altering their hydrogen bonding
patterns, which leads to incorrect base pairing during replication.
Radiation
• Ultraviolet radiation (nonionizing radiation).
• Two nucleotide bases in DNA — cytosine and thymine — are most vulnerable to radiation that can
change their properties.
• UV light can induce adjacent pyrimidine bases in a DNA strand to become covalently joined as
a pyrimidine dimer.
• UV radiation, in particular longer-wave UVA, can also cause oxidative damage to DNA.
 Classification of mutations types

• By the effect on structure

• By the effect on function
• By the effect on fitness
• By the impact on protein sequences
• By inheritance
• Suppressor mutation
 By the Effect on Structure
• Small Scale Mutation: such as those
affecting a small gene in one or a few
nucleotides, including:
• Point Mutation:
– Often caused by chemicals or malfunction of
DNA replication, exchange a single
nucleotide for another
– These changes are classified as transitions or
transversions.
– Transition: Most common is
the transition that exchanges a purine for a
purine (A ↔ G) or a pyrimidine for a
pyrimidine, (C ↔ T).
– Transversion: Less common is a transversion,
which exchanges a purine for a pyrimidine or
a pyrimidine for a purine (C/T ↔ A/G). An
example of a transversion is the conversion
of adenine (A) into acytosine (C).
• A point mutation can be reversed by another point mutation, in which the
nucleotide is changed back to its original state (true reversion) or by second-site
reversion (a complementary mutation elsewhere that results in regained gene
functionality).
• Point mutations that occur within the protein coding region of a gene may be
classified into three kinds, depending upon what the erroneous codon codes for:
– Silent mutations, which code for the same (or a sufficiently similar) amino acid.
– Mis-sense mutations, which code for a different amino acid.
– Nonsense mutations, which code for a stop and can truncate the protein.
• Insertions add one or more extra
nucleotides into the DNA.
– They are usually caused by transposable elements,
or errors during replication of repeating elements
(e.g., AT repeats[citation needed]).
– Insertions in the coding region of a gene may
alter splicing of the mRNA (splice site mutation), or
cause a shift in the reading frame (frameshift),
both of which can significantly alter the gene
product.
– Insertions can be reversed by excision of
the transposable element.
• Deletions remove one or more nucleotides
from the DNA.
– Like insertions, these mutations can alter the
reading frame of the gene.
– In general, they are irreversible:
– Though exactly the same sequence might in theory
be restored by an insertion, transposable elements
able to revert a very short deletion (say 1–2 bases)
in any location either are highly unlikely to exist or
do not exist at all.
Large-scale mutations in chromosomal structure, including:
• Amplifications (or gene duplications) leading to multiple copies of all
chromosomal regions, increasing the dosage of the genes located within them.
• Deletions of large chromosomal regions, leading to loss of the genes within those
regions.
• Mutations whose effect is to put side by side previously separate pieces of DNA,
potentially bringing together separate genes to form functionally distinct fusion
genes (e.g., bcr-abl). These include:
– Chromosomal translocations: Interchange of genetic parts from non-homologous chromosomes.
– Interstitial deletions: An intra-chromosomal deletion that removes a segment of DNA from a single
chromosome, thereby apposing previously distant genes.
– For example, cells isolated from a human astrocytoma, a type of brain tumor, were found to have a
chromosomal deletion removing sequences between the "fused in glioblastoma" (fig) gene and the
receptor tyrosine kinase "ros", producing a fusion protein (FIG-ROS). The abnormal FIG-ROS fusion
protein has constitutively active kinase activity that causes oncogenic transformation (a
transformation from normal cells to cancer cells).
– Chromosomal inversions: reversing the orientation of a chromosomal segment.
• Loss of heterozygosity: loss of one allele, either by a deletion or
a recombination event, in an organism that previously had two different alleles.
 By effect on function
• Loss-of-function mutations result in the gene product having
less or no function.
– When the allele has a complete loss of function (null allele) it is often called
an amorphic mutation.
– Phenotypes associated with such mutations are most often recessive.
– Exceptions are when the organism is haploid, or when the reduced dosage of
a normal gene product is not enough for a normal phenotype (this is
called haplo-insufficiency).
• Gain-of-function mutations change the gene product such
that it gains a new and abnormal function.
– When the new allele is created, a heterozygote containing the newly created
allele as well as the original will express the new allele; genetically this defines
the mutations as dominant phenotypes. Often called a neomorphic mutation.
• Dominant negative mutations (also called antimorphic mutations) have
an altered gene product that acts antagonistically to the wild-type allele.
– These mutations usually result in an altered molecular function (often inactive) and are
characterized by a dominant or semi-dominant phenotype.
– In humans, dominant negative mutations have been implicated in cancer (e.g.,
mutations in genes p53, ATM, CEBPA and PPARgamma).
– It was once thought that Marfan syndrome is an example of a the occurrence of a
dominant negative mutation in an autosomal-dominant disease where the defective
glycoprotein product of the fibrillin gene (FBN1) antagonizes the product of the normal
allele.
– However, it may appear this is not that case and that Marfan's may be a result of haplo-
insufficiency due to the absence of one normal allele that causes the disease not the
presence of an abnormal allele (i.e., Dominant negative).
• Lethal mutations are mutations that lead to the death of the organisms
that carry the mutations.
• A back mutation or reversion is a point mutation that restores the
original sequence and hence the original phenotype.
 By effect on fitness
• In applied genetics, it is usual to speak of mutations as either harmful or
beneficial.
• A harmful, or deleterious, mutation decreases the fitness of the
organism.
• A beneficial, or advantageous mutation increases the fitness of the
organism.
– Mutations that promotes traits that are desirable, are also called beneficial.
– In theoretical population genetics, it is more usual to speak of mutations as deleterious
or advantageous than harmful or beneficial.
• A neutral mutation has no harmful or beneficial effect on the organism.
– Such mutations occur at a steady rate.
– Forms the basis for the molecular clock.
– In the neutral theory of molecular evolution, neutral mutations provide genetic drift as
the basis for most variation at the molecular level.
• A nearly neutral mutation is a mutation that may be slightly deleterious
or advantageous, although most nearly neutral mutations are slightly
deleterious.
 By impact on protein sequence
• A frameshift mutation is a mutation caused by insertion or deletion of a
number of nucleotides that is not evenly divisible by three from a DNA
sequence.
• Any insertion or deletion that is evenly divisible by three is termed an in-
frame mutation.
• A nonsense mutation is a point mutation in a sequence of DNA that
results in a premature stop codon, or a nonsense codon in the transcribed
mRNA, and possibly a truncated, and often nonfunctional protein product.
• Missense mutations or non-synonymous mutations are types of point
mutations where a single nucleotide is changed to cause substitution of a
different amino acid.
– This in turn can render the resulting protein nonfunctional. Such mutations are
responsible for diseases such as Epidermolysis bullosa, sickle-cell disease,
and SOD1 mediated ALS (Boillée 2006, p. 39).
• A neutral mutation is a mutation that occurs in an amino acid codon that
results in the use of a different, but chemically similar, amino acid.
– The similarity between the two is enough that little or no change is often rendered in
the protein.
– For example, a change from AAA to AGA will encode arginine, a chemically similar
molecule to the intended lysine.
• Silent mutations are mutations that do not result in a change to the
amino acid sequence of a protein, unless the changed amino acid is
sufficiently similar to the original.
– They may occur in a region that does not code for a protein, or they may occur within a
codon in a manner that does not alter the final amino acid sequence.
– The phrase silent mutation is often used interchangeably with the phrase synonymous
mutation
– Synonymous mutations are a subcategory of the former, occurring only within exons
(and necessarily exactly preserving the amino acid sequence of the protein).
– Synonymous mutations occur due to the degenerate nature of the genetic code.
 Types of mutations
• There are many different ways that DNA can be changed, resulting in
different types of mutation. Here is a quick summary of a few of these:
Substitution
• A substitution is a mutation that exchanges one base for another (i.e., a
change in a single "chemical letter" such as switching an A to a G).
• Such a substitution could: change a codon to one that encodes a different
amino acid and cause a small change in the protein produced.
– For example, sickle cell anemia is caused by a substitution in the beta-
hemoglobin gene, which alters a single amino acid in the protein
produced.
• Change a codon to one that encodes the same amino acid and causes no
change in the protein produced. These are called silent mutations.
• change an amino-acid-coding codon to a single "stop" codon and cause an
incomplete protein. This can have serious effects since the incomplete
protein probably won't function.
Insertion
Insertions are mutations in which extra base pairs are inserted into a
new place in the DNA.

Deletion
Deletions are mutations in which a section of DNA is lost, or deleted.

Frameshift
• Since protein-coding DNA is divided into codons three bases long,
insertions and deletions can alter a gene so that its message is no longer
correctly phrased. These changes are called frameshifts.
• For example, consider the sentence, "The fat cat sat." Each word
represents a codon. If we delete the first letter and phrase the sentence
in the same way, it doesn't make sense.
• In frameshifts, a similar error occurs at the DNA level, causing the codons
to be phrased incorrectly. This usually generates truncated proteins that
are as useless as "hef atc ats at" is uninformative.
• There are other types of mutations as well, but this short list should give
you an idea of the possibilities.
 By Inheritance
• In multicellular organisms with dedicated reproductive cells
Mutations

Germ line mutations

Descendants

Reproductive cells Somatic mutations (also called acquired

mutations)

Involve cells outside the dedicated reproductive group

(which are not usually transmitted to descendants).

• A germline mutation gives rise to a constitutional mutation in the offspring, that is, a
mutation that is present in every cell.
• A constitutional mutation can also occur very soon after fertilization, or continue
from a previous constitutional mutation in a parent.
• Diploid organisms (e.g., humans) contain two copies of each gene — a
paternal and a maternal allele. Based on the occurrence of mutation on
each chromosome, we may classify mutations into three types.
– A heterozygous mutation is a mutation of only one allele.
– A homozygous mutation is an identical mutation of both the paternal and maternal
alleles.
– Compound heterozygous mutations or a genetic compound comprises two different
mutations in the paternal and maternal alleles.
• A wildtype or homozygous non-mutated organism is one in which neither
allele is mutated.
 Suppressor Mutation
• A suppressor mutation is a second mutation that alleviates or reverts
the phenotypic effects of an already existing mutation.
• Genetic suppression therefore restores the phenotype seen prior to the original
background mutation.
• They are useful for identifying new genetic sites which affect a biological process
of interest.
• They also provide evidence between functionally interacting molecules and
intersecting biological pathways
• Types of Suppressor Mutation:
• Intragenic suppression results from suppressor mutations that occur in the
same gene as the original mutation.
• In a classic study, Francis Crick (et al.) used intragenic suppression to study the
fundamental nature of the genetic code. From this study it was shown that genes
are expressed as non-overlapping triplets (codons).
 – Mutations caused by either a single base insertion (+) or a single base deletion (-) could
be “suppressed” or restored by a second mutation of the opposite sign, as long as the
two mutations occurred in the same vicinity of the gene.
– This led to the conclusion that genes needed to be read in a specific “reading frame” and
a single base insertion or deletion would shift the reading frame (frameshift mutation) in
such a way that the remaining DNA would code for a different polypeptide than the one
intended.
– Therefore, researchers concluded that the second mutation of opposite sign suppresses
the original mutation by restoring the reading frame, as long as the portion between the
two mutations is not critical for protein function.
• Intergenic (also known as extragenic) suppression relieves the effects of a
mutation in one gene by a mutation somewhere else within the genome.
– The second mutation is not on the same gene as the original mutation.
– Intergenic suppression is useful for identifying and studying interactions between
molecules, such as proteins.
– For example, a mutation which disrupts the complementary interaction between protein
molecules may be compensated for by a second mutation elsewhere in the genome that
restores or provides a suitable alternative interaction between those molecules.
– Several proteins of biochemical, signal transduction, and gene expression pathways have
been identified using this approach.
– Examples of such pathways include receptor-ligand interactions as well as the interaction
of components involved in DNA replication, transcription, and translation
 DNA Damage
• DNA damage, due to environmental factors and normal metabolic processes inside
the cell, occurs at a rate of 10,000 to 1,000,000 molecular lesions per cell per day.
• While this constitutes only 0.000165% of the human genome's approximately 6
billion bases (3 billion base pairs), unrepaired lesions in critical genes (such
as tumor suppressor genes) can impede a cell's ability to carry out its function and
appreciably increase the likelihood of tumor formation and contribute to tumor
heterogeneity.
• The vast majority of DNA damage affects the primary structure of the double helix;
that is, the bases themselves are chemically modified.
• These modifications can in turn disrupt the molecules' regular helical structure by
introducing non-native chemical bonds or bulky adducts that do not fit in the
standard double helix.
• Unlike proteins and RNA, DNA usually lacks tertiary structure and therefore
damage or disturbance does not occur at that level.
• DNA is, however, supercoiled and wound around "packaging" proteins
called histones (in eukaryotes), and both superstructures are vulnerable to the
effects of DNA damage.
 Sources of Damage
• DNA damage can be subdivided into two main types:
• Endogenous damage such as attack by reactive oxygen species produced
from normal metabolic byproducts (spontaneous mutation),
– especially the process of oxidative deamination
– also includes replication errors
• Exogenous damage caused by external agents such as
– ultraviolet [UV 200-400 nm] radiation from the sun
– other radiation frequencies, including x-rays and gamma rays
– hydrolysis or thermal disruption
– certain plant toxins
– human-made mutagenic chemicals, especially aromatic compounds that act as
DNA intercalating agents
– viruses
• The replication of damaged DNA before cell division can lead to the
incorporation of wrong bases opposite damaged ones.
• Daughter cells that inherit these wrong bases carry mutations from which
the original DNA sequence is unrecoverable (except in the rare case of a back
mutation, for example, through gene conversion).
 Types of DNA Damage
• There are several types of damage to DNA due to endogenous cellular processes:
– Oxidation of bases [e.g. 8-oxo-7,8-dihydroguanine (8-oxoG)] and
generation of DNA strand interruptions from reactive oxygen species,
– Alkylation of bases (usually methylation), such as formation of 7-
methylguanine, 1-methyladenine, 6-O-Methylguanine
– Hydrolysis of bases, such as deamination, depurination, and
depyrimidination.
– “Bulky adduct formation" (i.e., benzo[a]pyrene diol epoxide-dG
adduct, aristolactam I-dA adduct)
– Mismatch of bases, due to errors in DNA replication, in which the
wrong DNA base is stitched into place in a newly forming DNA strand,
or a DNA base is skipped over or mistakenly inserted.
– Monoadduct damage cause by change in single nitrogenous base of
DNA
– Diadduct damage
• Damage caused by exogenous agents comes in many forms. Some examples are:
– UV-B light causes crosslinking between adjacent cytosine and thymine bases creating pyrimidine
dimers. This is called direct DNA damage.
– UV-A light creates mostly free radicals. The damage caused by free radicals is called indirect DNA
damage.
– Ionizing radiation such as that created by radioactive decay or in cosmic rays causes breaks in DNA
strands.
– Low-level ionizing radiation may induce irreparable DNA damage (leading to replicational and
transcriptional errors needed for neoplasia or may trigger viral interactions) leading to pre-mature
aging and cancer.
– Thermal disruption at elevated temperature increases the rate of depurination (loss of purine bases
from the DNA backbone) and single-strand breaks.
– For example, hydrolytic depurination is seen in the thermophilic bacteria, which grow in hot
springs at 40-80 °C. The rate of depurination (300 purineresidues per genome per generation) is too
high in these species to be repaired by normal repair machinery, hence a possibility of
an adaptive response cannot be ruled out.
– Industrial chemicals such as vinyl chloride and hydrogen peroxide, and environmental chemicals such
as polycyclic aromatic hydrocarbons found in smoke, soot and tar create a huge diversity of DNA
adducts- ethenobases, oxidized bases, alkylated phosphotriesters and crosslinking of DNA, just to
name a few.
– UV damage, alkylation/methylation, X-ray damage and oxidative damage are examples of induced
damage. Spontaneous damage can include the loss of a base, deamination, sugar ring puckering and
tautomeric shift.
 DNA Repair
• Cells cannot function if DNA damage corrupts the integrity and accessibility of
essential information in the genome (but cells remain superficially functional when
non-essential genes are missing or damaged).
• Depending on the type of damage inflicted on the DNA's double helical structure,
a variety of repair strategies have evolved to restore lost information.
• If possible, cells use the unmodified complementary strand of the DNA or the
sister chromatid as a template to recover the original information.
• Without access to a template, cells use an error-prone recovery mechanism
known as translesion synthesis as a last resort.
• Damage to DNA alters the spatial configuration of the helix, and such alterations
can be detected by the cell.
• Once damage is localized, specific DNA repair molecules bind at or near the site of
damage, inducing other molecules to bind and form a complex that enables the
actual repair to take place.
 Direct Reversal
• Cells are known to eliminate three types of damage to their DNA by chemically reversing it.
• These mechanisms do not require a template, since the types of damage they counteract can occur in
only one of the four bases.
• Such direct reversal mechanisms are specific to the type of damage incurred and do not involve
breakage of the phosphodiester backbone.
UV light
Irradiation
Pyrimidine dimers
(an abnormal covalent bond between adjacent pyrimidine bases)

Photoreactivation process

Photolyase

Obligately dependent on energy absorbed from blue/UV light (300–500 nm wavelength)

Catalysis
Direct reversal

• Photolyase, an old enzyme present in bacteria, fungi, and most animals no longer functions in
humans, who instead use nucleotide excision repair to repair damage from UV irradiation.
• Another type of damage, methylation of guanine bases, is directly reversed by the
protein methyl guanine methyl transferase (MGMT), the bacterial equivalent of
which is called ogt.
• This is an expensive process because each MGMT molecule can be used only once;
that is, the reaction is stoichiometric rather than catalytic
• A generalized response to methylating agents in bacteria is known as theadaptive
response and confers a level of resistance to alkylating agents upon sustained
exposure by upregulation of alkylation repair enzymes.
• The third type of DNA damage reversed by cells is certain methylation of the bases
cytosine and adenine.
 Single-strand damage
•
a) Base excision repair (BER)
When only one of the two strands
of a double helix has a defect, the
other strand can be used as a
template to guide the correction of
the damaged strand.
• In order to repair damage to one of
the two paired molecules of DNA,
there exist a number of excision
repair mechanisms that remove the
damaged nucleotide and replace it
with an undamaged nucleotide
complementary to that found in the
undamaged DNA strand.
 b) Nucleotide Excision Repair
Bulky, helix-distorting damage, such as pyrimidine
dimerization caused by UV light
Global Genomic NER
 Transcription coupled NER

Bulky, helix-distorting damage, such as pyrimidine

dimerization caused by UV light
 Mismatch Repair
• DNA mismatch repair is a system for recognizing and repairing erroneous insertion, deletion,
and mis-incorporation of bases that can arise during DNA replication and recombination, as
well as repairing some forms of DNA damage
• Mismatch repair is strand-specific.
• During DNA synthesis the newly synthesised (daughter) strand will commonly include errors.
• In order to begin repair, the mismatch repair machinery distinguishes the newly synthesized
strand from the template (parental).
• In gram-negative bacteria, transient hemimethylation distinguishes the strands (the parental
is methylated and daughter is not).
• However, in other prokaryotes and eukaryotes, the exact mechanism is not clear.
• It is suspected that, in eukaryotes, newly synthesized lagging-strand DNA transiently contains
nicks (before being sealed by DNA ligase) and provides a signal that directs mismatch
proofreading systems to the appropriate strand.
• This implies that these nicks must be present in the leading strand, and evidence for this has
recently been found.
• Recent work has shown that nicks are sites for RFC-dependent loading of the replication
sliding clamp PCNA, in an orientation-specific manner, such that one face of the donut-shape
protein is juxtaposed toward the 3'-OH end at the nick.
• Oriented PCNA then directs the action of the MutLalpha endonuclease to one strand in the
presence of a mismatch and MutSalpha or MutSbeta.
Eukaryotes Prokaryotes E. coli
 Double Strand Breaks
• Double-strand breaks, in which both strands in the double
helix are severed, are particularly hazardous to the cell
because they can lead to genome rearrangements.
• Three mechanisms exist to repair double-strand breaks
(DSBs):
– Non-homologous end joining (NHEJ),
– Microhomology-mediated end joining (MMEJ)
– Homologous recombination.
 Non-homologous end joining (NHEJ)
• In NHEJ, DNA Ligase IV, a specialized DNA ligase that forms a complex with the
cofactor XRCC4, directly joins the two ends.
• To guide accurate repair, NHEJ relies on short homologous sequences called
microhomologies present on the single-stranded tails of the DNA ends to be
joined. If these overhangs are compatible, repair is usually accurate.
• NHEJ can also introduce mutations during repair.
• Loss of damaged nucleotides at the break site can lead to deletions, and joining of
nonmatching termini forms translocations.
• NHEJ is especially important before the cell has replicated its DNA, since there is
no template available for repair by homologous recombination.
• There are "backup" NHEJ pathways in higher eukaryotes.
• Besides its role as a genome caretaker, NHEJ is required for joining hairpin-capped
double-strand breaks induced during V(D)J recombination, the process that
generates diversity in B-cell and T-cell receptors in the vertebrate immune system
Micro-homology-mediated end joining (MMEJ)
DNA Double Strand Repair
MRE11 nuclease
Short-range end resection

Either side of double-strand break

Reveal
Micro-homology regions

PARP1

Pairing of micro-homology regions

Recruitment
Flap structure-specific endonuclease 1 ( FEN1)
Remove
Overhanging flaps

Recruitment of XRCC1–LIG3

Ligating the DNA ends

Intact DNA
 Homologous Recombination
• Homologous recombination requires the presence of an identical or nearly
identical sequence to be used as a template for repair of the break.
• The enzymatic machinery responsible for this repair process is nearly identical to
the machinery responsible for chromosomal crossover during meiosis.
• This pathway allows a damaged chromosome to be repaired using a
sister chromatid (available in G2 after DNA replication) or a homologous
chromosome as a template.
• DSBs caused by the replication machinery attempting to synthesize across a single-
strand break or unrepaired lesion cause collapse of the replication fork and are
typically repaired by recombination
RecBCD pathway of recombination
 RecBCD pathway of recombination
Chi hotspot: 5’ GCTGGTGG 3’

Holliday junction