I.

Mutations

Definition

A mutation is a permanent, heritable change in the DNA sequence of an organism's genome.

Mutations can occur naturally or be induced by external factors, affecting genetic information and,
consequently, the organism's phenotype.

Characteristics of Mutations

1. Heritability: Mutations are passed to the offspring if they occur in germ-line cells or during
DNA replication in unicellular organisms.

2. Randomness: Mutations generally occur at random, without being directed by the

organism's needs.

3. Permanence: Once established, a mutation becomes a fixed part of the genome unless
repaired.

4. Impact: Can be:

o Neutral: No observable effect on the organism.

o Beneficial: Provides an advantage (e.g., antibiotic resistance in bacteria).

o Deleterious: Causes harmful effects or disease.

Classification of Mutations

1. Based on Molecular Changes:

o Point Mutation:

 Affects a single nucleotide.

 Types:

 Substitution:

 Transition: Purine ↔ Purine (e.g., A ↔ G) or Pyrimidine ↔

Pyrimidine (e.g., C ↔ T).

 Transversion: Purine ↔ Pyrimidine (e.g., A ↔ T).

 Insertion/Deletion: Addition or loss of nucleotides, leading to

frameshift mutations if not in multiples of three.

o Chromosomal Mutation:

 Involves large-scale changes in chromosome structure or number.

 Types: Deletions, duplications, inversions, translocations.

 2. Based on Effect on Function:

o Silent Mutation: Alters DNA but does not affect the protein (due to redundancy in
the genetic code).

o Missense Mutation: Leads to the substitution of one amino acid for another in the
protein.

o Nonsense Mutation: Converts a codon into a stop codon, terminating protein

synthesis prematurely.

o Frameshift Mutation: Disrupts the reading frame due to insertion/deletion.

3. Based on Phenotypic Effects:

o Loss-of-function Mutation: Reduces or abolishes protein activity (e.g., tumor

suppressor gene mutations).

o Gain-of-function Mutation: Produces a new or enhanced protein function.

o Lethal Mutation: Causes death of the organism.

4. Based on Origin:

o Spontaneous Mutation: Occurs naturally during DNA replication, repair, or

recombination.

o Induced Mutation: Caused by external agents (mutagens).

Causes of Mutation

1. Errors in DNA Replication:

o Misincorporation of nucleotides by DNA polymerase.

o Slippage during replication leading to expansions or contractions (e.g., trinucleotide

repeat disorders).

2. Spontaneous Chemical Changes:

o Depurination: Loss of purine bases (A or G).

o Deamination: Conversion of cytosine to uracil.

3. Exposure to Mutagens:

o Physical Mutagens:

 UV radiation (causes thymine dimers).

 Ionizing radiation (induces double-strand breaks).

o Chemical Mutagens:

 Alkylating agents (e.g., ethyl methanesulfonate).

 Base analogs (e.g., 5-bromouracil).

 o Biological Agents:

 Viruses and transposons.

Types of Mutations

1. Point Mutations:

o Alter a single nucleotide pair.

o Example: Sickle-cell anemia caused by a missense mutation in the β-globin gene.

2. Structural Mutations:

o Changes affecting chromosome structure:

 Deletion: Loss of a chromosome segment.

 Duplication: Repetition of a segment.

 Inversion: Reversal of a segment's orientation.

 Translocation: Transfer of a segment to another chromosome.

3. Numerical Mutations (Aneuploidy):

o Changes in chromosome number.

o Examples: Down syndrome (trisomy 21), Turner syndrome (monosomy X).

Detection and Isolation of Mutants

1. Selection Methods:

o Mutants are selected based on observable phenotypes, such as antibiotic resistance.

2. Screening Methods:

o Mutants are identified without direct selection.

o Example: Replica plating for auxotrophs.

3. Molecular Techniques:

o Polymerase Chain Reaction (PCR) and DNA sequencing can pinpoint mutations at the
nucleotide level.

Significance of Mutations

1. Source of Genetic Variation:

o Drives evolution and adaptation in populations.

2. Role in Diseases:
 o Mutations underlie many genetic disorders (e.g., cystic fibrosis, cancer).

3. Biotechnological Applications:

o Induced mutations are used in crop improvement, drug resistance studies, and
synthetic biology.

II. Mutagens

Definition

A mutagen is any physical, chemical, or biological agent that can cause a mutation by altering the
DNA sequence. Mutagens can lead to changes that affect the structure of chromosomes, genes, or
regulatory regions, potentially resulting in disease or driving evolution.

Types of Mutagens

1. Physical Mutagens

o Ionizing Radiation:

 Includes X-rays, gamma rays, and cosmic rays.

 Mechanism:

 Ionizing radiation causes double-strand breaks in DNA, which are

difficult for the cell to repair. This leads to chromosomal
aberrations, deletions, or translocations.

 Effects:

 Mutations can occur directly from DNA strand breaks or indirectly

from reactive oxygen species (ROS) formed by radiation interacting
with water molecules in the cell.

 Examples:

 Radiation therapy for cancer can lead to secondary mutations or

cancers.

 Natural background radiation (e.g., radon gas).

o Non-Ionizing Radiation:

 Includes ultraviolet (UV) radiation from sunlight.

 Mechanism:
  UV radiation primarily causes the formation of thymine dimers
(covalent bonds between adjacent thymine bases), which distort the
DNA helix and block replication.

 Repair Mechanism:

 Cells repair thymine dimers using nucleotide excision repair (NER),

but if repair fails, mutations can accumulate, potentially leading to
skin cancer (e.g., melanoma).

2. Chemical Mutagens

o Base Analog Mutagens:

 Chemicals that resemble nitrogenous bases and get incorporated into the
DNA during replication.

 Example: 5-bromouracil (resembles thymine but pairs with guanine), which

can lead to transition mutations.

o Alkylating Agents:

 Chemical mutagens that add alkyl groups (e.g., methyl or ethyl groups) to
the bases of DNA.

 Example: Ethyl methanesulfonate (EMS), which can alter bases to cause

mismatches during replication (often leading to point mutations).

o Intercalating Agents:

 These mutagens insert between the stacked base pairs in the DNA helix,
leading to frameshift mutations.

 Example: Acridine orange and ethidium bromide (used in gel

electrophoresis).

o Deaminating Agents:

 Chemicals that remove an amino group from the DNA bases, causing base
substitutions.

 Example: Nitrous acid can deaminate cytosine to uracil, causing a C to T

mutation.

o Hydroxylating Agents:

 Add hydroxyl groups (-OH) to bases, leading to mutations.

 Example: Hydroxylamine specifically adds a hydroxyl group to cytosine,

promoting C to T mutations.

3. Biological Mutagens

o Viruses:

 Some viruses can integrate their DNA into the host genome, causing
mutations by disrupting host genes or by carrying foreign genetic material.
  Example: Retroviruses like HIV can cause insertional mutagenesis.

o Transposons (Jumping Genes):

 Transposable elements are segments of DNA that can move around within
the genome, causing mutations when they insert into or near genes.

 Effect: Disrupts gene function or regulatory regions, potentially causing

inactivation of genes or creating new genetic combinations.

o Bacteriophages:

 These viruses can induce mutations in bacteria by transferring DNA between

bacterial strains through transduction (a type of horizontal gene transfer).

o Mobile Genetic Elements:

 In bacteria, plasmids and integrons can carry antibiotic resistance genes and
can spread rapidly across populations, often through horizontal gene
transfer.

Mechanisms of Mutagenesis

1. Direct DNA Damage:

o Mutagens interact directly with DNA, causing chemical alterations in nucleotides or

disrupting the structure of the DNA helix (e.g., UV radiation causing thymine
dimers).

2. Indirect DNA Damage:

o Mutagens, such as ionizing radiation, can produce reactive oxygen species (ROS)
that chemically modify DNA, causing oxidative damage. ROS can oxidize bases,
leading to base mispairing or strand breaks.

3. DNA Replication Errors:

o Some chemical mutagens cause replication errors, leading to base mispairing or

frameshift mutations. For example, base analogs can incorporate incorrectly during
DNA replication, causing mismatches.

Types of Mutations Induced by Mutagens

1. Point Mutations:

o Base substitutions (e.g., transitions or transversions) or small insertions/deletions

caused by chemical mutagens like base analogs or alkylating agents.

2. Frameshift Mutations:

o Caused by intercalating agents or insertion/deletion of nucleotides, leading to a shift

in the reading frame and altered protein synthesis.

3. Chromosomal Mutations:
 o Physical mutagens, such as ionizing radiation, can cause deletions, inversions, or
translocations in chromosomes.

4. Gene Amplification:

o Some mutagens can induce gene duplications, leading to overproduction of proteins

(e.g., oncogene amplification in cancer).

Repair Mechanisms for Mutagen-Induced Damage

Cells have evolved a variety of repair systems to counteract DNA damage induced by mutagens:

1. Direct Repair:

o Photoreactivation (repair of UV-induced thymine dimers using light) or O6-

methylguanine methyltransferase (repair of methylated guanine).

2. Excision Repair:

o Base Excision Repair (BER): Fixes small base lesions or modified bases.

o Nucleotide Excision Repair (NER): Removes bulky DNA adducts like thymine dimers.

3. Mismatch Repair:

o Fixes errors that escape proofreading during DNA replication, such as base
mismatches.

4. Double-Strand Break Repair:

o Homologous recombination and non-homologous end joining (NHEJ) repair breaks

in the DNA backbone, a common result of ionizing radiation.

III. Frameshift mutations

What Are Frameshift Mutations?

 A frameshift mutation occurs when nucleotides are added to or deleted from the DNA
sequence, and this insertion or deletion shifts the reading frame of the codons during
translation.

 The reading frame refers to how the sequence of three nucleotides (a codon) is grouped and
translated into an amino acid sequence in proteins. Each codon corresponds to a specific
amino acid or a stop signal.

 In a frameshift mutation, the insertion or deletion of nucleotides other than multiples of

three alters the groupings of codons from the mutation point onward, which can
dramatically change the resulting polypeptide.

Types of Frameshift Mutations:

 1. Insertion Frameshift: Involves the insertion of one or more nucleotides into the DNA
sequence.

2. Deletion Frameshift: Involves the deletion of one or more nucleotides from the DNA
sequence.

Both of these types result in a shift in the reading frame and lead to incorrect protein translation.

How Frameshift Mutations Occur:

 Insertion: If a base is inserted into the sequence, it shifts the downstream codons, resulting
in a completely altered sequence of amino acids.

 Deletion: If a base is deleted from the sequence, the remaining codons downstream are also
shifted, altering the encoded protein.

Consequences of Frameshift Mutations:

1. Altered Protein Sequence: Because the codon groupings are shifted, the entire amino acid
sequence downstream of the mutation site is altered. This often results in a nonfunctional or
malfunctioning protein.

2. Early Termination: Frameshift mutations often introduce a nonsense codon (stop codon)
prematurely. This causes translation to terminate early, resulting in a truncated protein,
which is usually nonfunctional.

3. Loss of Protein Function: A frameshift mutation can result in a protein with a completely
different structure or function, which may be harmful to the organism. In some cases, it can
lead to genetic diseases (e.g., cystic fibrosis, Tay-Sachs disease).

4. Potential for Major Phenotypic Changes: Since proteins carry out nearly all cellular
functions, a frameshift mutation can have a large-scale impact on an organism's phenotype,
causing diseases or developmental defects.

Example of Frameshift Mutation:

Let’s say you have the following original DNA sequence (for simplicity, we’ll focus on the coding
strand):

Original Sequence (coding strand):

 5' ATG ACC GGT TAG 3'

o This codes for the amino acid sequence: Met-Thr-Gly-STOP (start codon, three
amino acids, then stop).

Now, let’s consider an insertion of one nucleotide:

After Insertion (e.g., adding a G):

 5' ATG ACC GGT GAA G 3'

o This sequence now codes for: Met-Thr-Gly-Glu (a different set of amino acids).

o The addition of the nucleotide shifts all subsequent codons and alters the protein
drastically.
Similarly, if a nucleotide is deleted, the effect would be similar, but the downstream codons would
also be shifted.

IV. DNA damage

Introduction to DNA Damage and Repair

DNA is constantly exposed to both internal and external factors that cause damage, including natural
metabolic processes, replication errors, and environmental mutagens (e.g., radiation, chemicals). To
maintain the integrity of the genome, cells have evolved a variety of DNA repair mechanisms to
correct these damages before they result in mutations or cell death.

Types of DNA Damage

1. Base Modifications:

o Deamination: Removal of an amino group from a base, leading to mispairing (e.g.,

cytosine to uracil).

o Oxidation: Reactive oxygen species (ROS) oxidize bases, causing mispairing or strand
breaks.

o Alkylation: Addition of alkyl groups to bases, often leading to mispairing or distorted

DNA.

2. Single-Strand Breaks (SSBs):

o Causes: Replication errors, oxidative stress, or exposure to chemicals.

o Consequence: Can lead to double-strand breaks (DSBs) during replication if not

repaired promptly.

3. Double-Strand Breaks (DSBs):

o Causes: Ionizing radiation, oxidative stress, replication errors.

o Consequence: DSBs are the most lethal form of DNA damage and require precise
repair to prevent genomic instability.

4. Thymine Dimers:

o Caused by UV radiation forming covalent bonds between adjacent thymine bases,

distorting the DNA helix and blocking replication.

5. Crosslinks:

o Chemical crosslinks (e.g., from alkylating agents) can link strands of DNA together,
preventing the separation of strands during replication.

DNA Repair Mechanisms

Cells have developed specialized repair pathways to correct different types of DNA damage. Below
are the main repair mechanisms:

1. Direct Repair Mechanisms

1. Photoreactivation (for UV-induced damage):

o Function: Repair of thymine dimers caused by UV light.

o Mechanism: Photolyase enzyme binds to the thymine dimers and uses visible light
to break the covalent bond between the adjacent thymine bases.

o Limitation: This process is only active in prokaryotes and some eukaryotic organisms
(e.g., plants, fungi).

2. O6-methylguanine Methyltransferase (MGMT):

o Function: Repair of alkylation damage.

o Mechanism: The enzyme directly removes methyl or ethyl groups from the O6
position of guanine, restoring its normal base pairing.

2. Excision Repair Mechanisms

These repair pathways work by recognizing and removing the damaged DNA region and then
replacing it with the correct sequence.

1. Base Excision Repair (BER):

o Function: Corrects small, non-helix-distorting base lesions, such as oxidative damage

or deamination.

o Mechanism:

 DNA glycosylases recognize the damaged base and remove it.

 An AP endonuclease cuts the backbone, and DNA polymerase adds the

correct nucleotide, followed by DNA ligase sealing the strand.

o Example: Repair of 8-oxo-guanine caused by oxidative damage.

2. Nucleotide Excision Repair (NER):

o Function: Corrects bulky, helix-distorting lesions, such as thymine dimers or

crosslinks.

o Mechanism:

 The damaged region is recognized, and a segment of the strand containing

the lesion is excised by a complex of nucleases.

 The gap is filled by DNA polymerase and sealed by ligase.

o Example: Repair of UV-induced thymine dimers.

3. Mismatch Repair (MMR)

 Function: Corrects errors that escape proofreading during DNA replication, such as
mispaired bases.

 Mechanism:

o The MutS complex recognizes the mismatch and binds to the DNA.

o The MutL complex coordinates the repair process by recruiting exonucleases that
remove the incorrect strand.

o DNA polymerase then resynthesizes the correct strand, and ligase seals the DNA.

 Example: MMR is especially important in the correction of base pair mismatches after DNA
replication and is involved in correcting errors caused by DNA polymerase slippage.

4. Homologous Recombination (HR)

 Function: Repairs double-strand breaks (DSBs) by using the undamaged homologous

chromosome or sister chromatid as a template for accurate repair.

 Mechanism:

o The DSB is resected by exonucleases to produce single-stranded DNA, which invades

the homologous chromosome.

o DNA polymerase synthesizes the missing sequence, and the repair is completed by
ligation.

 Example: The BRCA1/BRCA2 genes are involved in homologous recombination repair of

DSBs. Mutations in these genes are associated with an increased risk of breast cancer.

5. Non-Homologous End Joining (NHEJ)

 Function: An error-prone repair mechanism for double-strand breaks when a homologous

template is unavailable, common in non-dividing cells.

 Mechanism:

o The broken ends of DNA are recognized by KU proteins that protect the ends and
bring them together.

o DNA-PKcs (protein kinase) helps in rejoining the ends of the DNA, but this process
can lead to small insertions or deletions (indels) at the break site.

 Example: NHEJ is predominant in cells in G1 phase when a homologous template is

unavailable, and it is crucial for immune system function (e.g., V(D)J recombination).

6. SOS Repair (In Bacteria)

  Function: A last-resort repair mechanism in bacteria that activates when DNA damage is too
severe for normal repair mechanisms.

 Mechanism:

o Involves error-prone DNA polymerases (e.g., DNA polymerase IV and V) that

replicate over damaged sites, potentially introducing mutations.

o This system is induced by the LexA repressor being cleaved in response to DNA
damage, allowing the expression of repair genes.

V. Tandem duplication

What Is Tandem Duplication?

 Tandem duplication is a type of structural variation in which a segment of DNA is

duplicated and inserted adjacent (next to the original segment) in the genome.

 This duplication results in two (or more) copies of a region of the DNA located next to each
other in the genome. The repeated region may include genes, exons, or regulatory regions,
depending on the size and location of the duplication.

Mechanism of Tandem Duplication:

 Tandem duplications can arise due to errors during DNA replication or recombination
events. A common mechanism involves unequal crossing over during meiosis when
homologous chromosomes misalign, causing one chromosome to receive an extra copy of a
segment.

 Replication slippage is another mechanism that can cause duplication of a specific region
within the DNA, particularly in repetitive sequences.

Types of Tandem Duplications:

1. Simple Tandem Duplication: Involves a direct repeat of a segment of DNA. The repeated
region is adjacent to the original, forming a head-to-tail pattern.

2. Complex Tandem Duplication: May involve multiple repeated regions and can be more
irregular in pattern. These duplications can sometimes involve intermediate sequences or a
mix of different parts of the genome.

Consequences of Tandem Duplication:

1. Gene Dosage Effect:

o One of the key consequences of tandem duplication is the increased gene dosage.
This means that there are more copies of the genes in the duplicated region.
 o If the duplicated genes are functional, the increased gene dosage can lead to
overexpression of the encoded protein, which could disturb the balance of cellular
processes.

o Gene amplification (an extreme form of duplication) can be observed in certain

cancers, where the overexpression of oncogenes drives uncontrolled cell division.

2. Altered Protein Function:

o If the duplication includes coding sequences, it could result in the production of an

altered or excess protein. Depending on the gene's function, this could lead to
disease or developmental disorders.

o The extra copies of a gene may cause the resulting proteins to aggregate, interfere
with normal protein function, or disrupt cellular pathways.

3. Disruption of Regulatory Elements:

o Tandem duplications may include not only genes but also regulatory regions (like
promoters or enhancers). This can result in abnormal regulation of gene expression,
potentially causing diseases related to misregulation of critical genes.

4. Increased Genetic Instability:

o Duplicated regions of the genome are often prone to further rearrangements, such
as additional duplications or deletions. This contributes to genetic instability, which
can be a factor in the development of diseases like cancer.

5. Phenotypic Effects:

o Depending on the genes affected, tandem duplications can lead to developmental

abnormalities, neurological disorders, or metabolic defects. The extra copies of
certain genes can contribute to disease phenotypes if they are involved in critical
biological pathways.

Examples of Tandem Duplication in Disease:

1. Charcot-Marie-Tooth Disease Type 1A:

o This is a peripheral neuropathy caused by a duplication of the PMP22 gene. The

gene encodes a protein that is essential for the formation of myelin in peripheral
nerves. The duplication leads to the overproduction of the PMP22 protein, which
disrupts the normal function of the nerves and causes symptoms like muscle
weakness, sensory loss, and neuropathy.

2. Huntington’s Disease:

o Although Huntington’s disease is typically associated with trinucleotide repeat

expansions, there are cases where tandem duplications of the repeat region can
exacerbate the symptoms, leading to more severe forms of the disease.

3. Cancer:
 o In various types of cancers, such as breast cancer or glioblastoma, tandem
duplications of oncogenes or tumor suppressor genes can occur. For example, EGFR
(Epidermal Growth Factor Receptor) is sometimes amplified in cancer, leading to
the production of excess growth factor receptors, which contributes to uncontrolled
cell proliferation.

VI. DNA proofreading

DNA proofreading is a crucial process for maintaining the integrity of the genetic material during
DNA replication. It involves the correction of errors that occur during the DNA synthesis process,
ensuring that the newly synthesized DNA strand is accurate and free from mutations. DNA
proofreading is a part of DNA repair mechanisms, which are critical for preserving genome stability.

DNA Proofreading: The Mechanism

1. DNA Polymerase and Its Role in Proofreading:

o DNA polymerases are the enzymes responsible for synthesizing new DNA strands by
adding nucleotides to the growing strand during replication. They also have an
intrinsic proofreading ability to correct mistakes made during replication.

o During DNA replication, DNA polymerase works by adding nucleotides

complementary to the template strand (A with T, C with G). However, errors can
occasionally occur due to mismatch pairing, such as the incorporation of an
incorrect base (e.g., G pairing with A instead of C).

2. Proofreading Mechanism:

o Exonuclease Activity: Many DNA polymerases have a 3' to 5' exonuclease activity,
meaning they can move backward along the newly synthesized strand (in the 3' to 5'
direction) to remove the incorrectly paired nucleotide.

o When a mismatch is detected, the polymerase pauses, and its exonuclease domain
removes the incorrect base. Once the error is corrected, the polymerase resumes its
forward movement to continue DNA synthesis.

o This proofreading ability significantly reduces the error rate during DNA replication
and contributes to maintaining the accuracy of genetic information.

3. Error Detection:

o Shape Recognition: The DNA polymerase detects errors in base pairing by

recognizing distortions in the helical structure of the DNA caused by mispaired
bases. The mismatch causes a kink or abnormal structure in the DNA helix, which
the polymerase senses.

o Additionally, the base pairing rules (A-T, G-C) are strictly enforced by the
polymerase. If an incorrect base is added, the polymerase detects the distortion
caused by the incorrect base pairing and activates the proofreading mechanism.
DNA Repair After Proofreading:

While proofreading by DNA polymerase prevents many errors, some mistakes are not caught during
replication. These can be repaired by other DNA repair mechanisms that act after replication:

1. Mismatch Repair (MMR):

o If proofreading does not correct a mistake, the mismatch repair system detects and
fixes base-pair mismatches and small insertions/deletions that may occur after
replication.

o MMR proteins (such as MutS, MutL, and MutH in prokaryotes, and MSH, MLH, and
PMS in eukaryotes) recognize mismatches in the DNA and remove the incorrect
segment. The gap is then filled in with the correct bases by DNA polymerase.

2. Base-Excision Repair (BER):

o For single-base errors (e.g., incorrect bases or damaged bases like oxidized guanine),
base-excision repair mechanisms remove the damaged base, and DNA polymerase
fills the gap with the correct nucleotide.

3. Nucleotide Excision Repair (NER):

o This repair pathway corrects larger DNA lesions, such as thymine dimers (caused by
UV radiation), by excising a short segment of the damaged DNA strand and replacing
it with the correct sequence using the undamaged strand as a template.

4. Double-Strand Break Repair:

o If the DNA has double-strand breaks (often caused by radiation or chemical

damage), the cell uses non-homologous end joining (NHEJ) or homologous
recombination (HR) to repair the breaks. These are more complex processes but are
essential for genome stability.

VII. DNA damage and repair

Introduction to DNA Damage and Repair

DNA is constantly exposed to both internal and external factors that cause damage, including natural
metabolic processes, replication errors, and environmental mutagens (e.g., radiation, chemicals). To
maintain the integrity of the genome, cells have evolved a variety of DNA repair mechanisms to
correct these damages before they result in mutations or cell death.

Types of DNA Damage

1. Base Modifications:
 o Deamination: Removal of an amino group from a base, leading to mispairing (e.g.,
cytosine to uracil).

o Oxidation: Reactive oxygen species (ROS) oxidize bases, causing mispairing or strand
breaks.

o Alkylation: Addition of alkyl groups to bases, often leading to mispairing or distorted

DNA.

2. Single-Strand Breaks (SSBs):

o Causes: Replication errors, oxidative stress, or exposure to chemicals.

o Consequence: Can lead to double-strand breaks (DSBs) during replication if not

repaired promptly.

3. Double-Strand Breaks (DSBs):

o Causes: Ionizing radiation, oxidative stress, replication errors.

o Consequence: DSBs are the most lethal form of DNA damage and require precise
repair to prevent genomic instability.

4. Thymine Dimers:

o Caused by UV radiation forming covalent bonds between adjacent thymine bases,

distorting the DNA helix and blocking replication.

5. Crosslinks:

o Chemical crosslinks (e.g., from alkylating agents) can link strands of DNA together,
preventing the separation of strands during replication.

DNA Repair Mechanisms

Cells have developed specialized repair pathways to correct different types of DNA damage. Below
are the main repair mechanisms:

1. Direct Repair Mechanisms

1. Photoreactivation (for UV-induced damage):

o Function: Repair of thymine dimers caused by UV light.

o Mechanism: Photolyase enzyme binds to the thymine dimers and uses visible light
to break the covalent bond between the adjacent thymine bases.

o Limitation: This process is only active in prokaryotes and some eukaryotic organisms
(e.g., plants, fungi).

2. O6-methylguanine Methyltransferase (MGMT):

o Function: Repair of alkylation damage.

 o Mechanism: The enzyme directly removes methyl or ethyl groups from the O6
position of guanine, restoring its normal base pairing.

2. Excision Repair Mechanisms

These repair pathways work by recognizing and removing the damaged DNA region and then
replacing it with the correct sequence.

1. Base Excision Repair (BER):

o Function: Corrects small, non-helix-distorting base lesions, such as oxidative damage

or deamination.

o Mechanism:

 DNA glycosylases recognize the damaged base and remove it.

 An AP endonuclease cuts the backbone, and DNA polymerase adds the

correct nucleotide, followed by DNA ligase sealing the strand.

o Example: Repair of 8-oxo-guanine caused by oxidative damage.

2. Nucleotide Excision Repair (NER):

o Function: Corrects bulky, helix-distorting lesions, such as thymine dimers or

crosslinks.

o Mechanism:

 The damaged region is recognized, and a segment of the strand containing

the lesion is excised by a complex of nucleases.

 The gap is filled by DNA polymerase and sealed by ligase.

o Example: Repair of UV-induced thymine dimers.

3. Mismatch Repair (MMR)

 Function: Corrects errors that escape proofreading during DNA replication, such as
mispaired bases.

 Mechanism:

o The MutS complex recognizes the mismatch and binds to the DNA.

o The MutL complex coordinates the repair process by recruiting exonucleases that
remove the incorrect strand.

o DNA polymerase then resynthesizes the correct strand, and ligase seals the DNA.

 Example: MMR is especially important in the correction of base pair mismatches after DNA
replication and is involved in correcting errors caused by DNA polymerase slippage.
4. Homologous Recombination (HR)

 Function: Repairs double-strand breaks (DSBs) by using the undamaged homologous

chromosome or sister chromatid as a template for accurate repair.

 Mechanism:

o The DSB is resected by exonucleases to produce single-stranded DNA, which invades

the homologous chromosome.

o DNA polymerase synthesizes the missing sequence, and the repair is completed by
ligation.

 Example: The BRCA1/BRCA2 genes are involved in homologous recombination repair of

DSBs. Mutations in these genes are associated with an increased risk of breast cancer.

5. Non-Homologous End Joining (NHEJ)

 Function: An error-prone repair mechanism for double-strand breaks when a homologous

template is unavailable, common in non-dividing cells.

 Mechanism:

o The broken ends of DNA are recognized by KU proteins that protect the ends and
bring them together.

o DNA-PKcs (protein kinase) helps in rejoining the ends of the DNA, but this process
can lead to small insertions or deletions (indels) at the break site.

 Example: NHEJ is predominant in cells in G1 phase when a homologous template is

unavailable, and it is crucial for immune system function (e.g., V(D)J recombination).

6. SOS Repair (In Bacteria)

 Function: A last-resort repair mechanism in bacteria that activates when DNA damage is too
severe for normal repair mechanisms.

 Mechanism:

o Involves error-prone DNA polymerases (e.g., DNA polymerase IV and V) that

replicate over damaged sites, potentially introducing mutations.

o This system is induced by the LexA repressor being cleaved in response to DNA
damage, allowing the expression of repair genes.

Significance of DNA Repair Mechanisms

1. Prevention of Mutations:

o Repair pathways are crucial in maintaining genome stability by preventing mutations

that could otherwise accumulate and lead to diseases like cancer.
 2. Genetic Diversity:

o While some repair mechanisms are error-prone (e.g., NHEJ), they contribute to
genetic variation, which can be advantageous for evolution in certain contexts.

3. Cellular Survival:

o Efficient DNA repair is necessary for the survival of cells under stress (e.g., radiation
or oxidative stress). Defects in repair mechanisms can lead to cell death, aging, or
disease.

Diseases Associated with DNA Repair Defects

1. Cancer:

o Mutations in genes involved in DNA repair, such as BRCA1/2 or MLH1, increase the
risk of developing cancers due to the accumulation of DNA damage.

2. Neurodegenerative Disorders:

o Defective DNA repair can lead to conditions like ataxia-telangiectasia (ATM gene
defect), which results in neurodegeneration and immune deficiencies.

3. Xeroderma Pigmentosum (XP):

o Caused by mutations in NER genes, individuals with XP are extremely sensitive to UV

light and have a high incidence of skin cancer.

4. Progeria (Hutchinson-Gilford Syndrome):

o Caused by defects in DNA repair and nuclear lamina proteins, leading to premature
aging and cardiovascular diseases.

VIII. Gene transfer mechanisms

Gene Transfer Mechanisms in Bacteria

Gene transfer in bacteria plays a crucial role in their adaptability, survival, and evolution. Through
different mechanisms, bacteria can acquire new genetic material, sometimes conferring
advantageous traits such as antibiotic resistance, virulence factors, or metabolic capabilities. There
are three primary mechanisms of gene transfer in bacteria:

1. Transformation

Definition

Transformation is the process by which a bacterium takes up foreign DNA from its environment and
incorporates it into its own genome.

Mechanism:
 1. DNA Uptake:

o Free DNA (often from a lysed bacterial cell) is released into the environment. A
bacterium that is competent (able to take up foreign DNA) binds this DNA to its
surface.

2. DNA Entry:

o The DNA passes through the bacterial cell wall and membrane into the cytoplasm,
typically through a specialized system of proteins that form pores or channels in the
cell envelope.

3. Integration:

o The foreign DNA is integrated into the recipient's genome by recombination. This
integration could either replace a segment of the recipient's genome (homologous
recombination) or insert as a plasmid.

Competence:

 Some bacteria are naturally competent (e.g., Streptococcus pneumoniae, Bacillus subtilis),
while others can be made competent through chemical treatment (e.g., with CaCl2) or
electroporation, where a brief electric shock makes the bacterial membrane permeable to
DNA.

Significance:

 Transformation is an important mechanism for bacteria to acquire new traits such as

antibiotic resistance or virulence factors. It plays a crucial role in horizontal gene transfer
(HGT) in the environment, where bacteria share genetic information.

2. Transduction

Definition

Transduction is the process by which bacterial DNA is transferred from one bacterium to another by
a bacteriophage (a virus that infects bacteria).

Mechanism:

1. Bacteriophage Infection:

o A bacteriophage (phage) infects a donor bacterial cell and injects its DNA into the
host cell.

2. Phage Replication and Assembly:

o The phage DNA takes over the host machinery to replicate its own genome and
assemble new phage particles. During this process, bacterial DNA can become
accidentally packaged inside the phage capsid.

3. Phage Release and Infection of New Host:

 o The phage particles are released from the donor cell and infect a new recipient
bacterium. The bacterial DNA packaged into the phage can integrate into the new
host’s genome via recombination.

Types of Transduction:

1. Generalized Transduction:

o Any gene from the donor bacterium can be transferred to the recipient. This occurs
when the phage mistakenly packages bacterial DNA during replication.

o Example: P1 phage in E. coli.

2. Specialized Transduction:

o Only specific genes near the phage integration site are transferred. This occurs when
the phage DNA excises incorrectly from the host chromosome, carrying adjacent
bacterial genes with it.

o Example: Lambda phage in E. coli.

Significance:

 Transduction allows the transfer of specific genes between bacteria, which can contribute to
genetic diversity, virulence, and the spread of antibiotic resistance.

3. Conjugation

Definition

Conjugation is the direct transfer of genetic material between two bacterial cells through a physical
connection called a pilus.

Mechanism:

1. Contact and Pilus Formation:

o Conjugation requires direct contact between the donor and recipient cells. The
donor cell typically contains a fertility plasmid (F plasmid), which encodes the genes
necessary to form the pilus.

2. Plasmid Transfer:

o The F plasmid in the donor cell is transferred to the recipient cell via the pilus. The
transfer is initiated by a single-stranded copy of the plasmid being transferred.

3. Conjugation Bridge:

o Once the pilus establishes contact, it retracts, forming a conjugation bridge, which
facilitates the transfer of DNA from the donor to the recipient.

4. Plasmid Replication:

o Both the donor and recipient cells now contain a copy of the plasmid, and
replication of the plasmid ensures that both cells retain a copy.
High-Frequency Recombination (Hfr):

 In some cases, the F plasmid integrates into the bacterial chromosome, forming an Hfr (high-
frequency recombination) strain. This allows the transfer of chromosomal DNA to the
recipient, resulting in genetic recombination.

 This process can lead to the exchange of large sections of bacterial chromosomal DNA,
contributing to genetic diversity.

Significance:

 Conjugation is one of the most important mechanisms for the transfer of antibiotic
resistance genes and virulence factors in bacterial populations.

 It is common in Gram-negative bacteria (e.g., E. coli, Klebsiella), but can also occur in Gram-
positive bacteria (e.g., Streptococcus species) with a slightly different mechanism.

Significance of Gene Transfer in Bacteria

1. Antibiotic Resistance Spread:

o Gene transfer mechanisms, especially conjugation, are a major route for the spread
of antibiotic resistance genes among bacterial populations.

o Resistance plasmids can carry multiple resistance genes, leading to the development
of multi-drug resistant (MDR) bacteria.

2. Evolution and Adaptation:

o Gene transfer contributes to genetic diversity in bacteria, allowing rapid adaptation

to new environments (e.g., new nutrients, host immune systems, or environmental
stresses).

3. Horizontal Gene Transfer (HGT):

o These mechanisms enable bacteria to exchange genes across different species,

facilitating the transfer of virulence factors, metabolic traits, and beneficial genes
like those for antibiotic resistance.

4. Bioengineering and Biotechnology:

o Scientists utilize bacterial conjugation and transformation in genetic engineering

(e.g., cloning, gene therapy), and transduction is used in gene mapping and
functional genomics studies.

Key Differences Between the Three Gene Transfer Mechanisms:

Feature Transformation Transduction Conjugation

Direct transfer of plasmids

Free DNA from the Bacteriophage (virus)
DNA Source or chromosomal DNA
environment transfers bacterial DNA
between bacteria

Contact No physical contact No physical contact Physical contact between

Required required required donor and recipient bacteria

Bacteriophage DNA
DNA fragments, plasmids, Plasmids, sometimes
Type of DNA (sometimes bacterial
chromosomal DNA chromosomal DNA
DNA)

Virus-mediated transfer
Mechanism Uptake of extracellular DNA Pilus-mediated DNA transfer
of bacterial DNA

Naturally competent
Bacteria with F-plasmids or
Common in bacteria, artificial induction Phage-infected bacteria
other conjugative elements
in the lab