DAMAGE AND REPAIR

Introduction to DNA Damage and Repair:

DAMAGE

DNA is a stable and versatile molecule.
But sometimes the damage is caused to it,
it is able to maintain stability by the information contained in it.
DNA has many elaborate mechanisms to repair any damage or distortion.
The most frequent sources of damage to DNA are the inaccuracy in DNA replication and chemical changes in DNA.

Malfunction of the process of replication can lead to incorporation of wrong bases, which are mismatched with the complementary strand.
The damage causing chemicals break the backbone of the strand and chemically alter the bases.
Alkylation, oxidation and methylation cause damage to bases. X-rays and gamma radiations cause single or double stranded breaks in DNA.

A change in the sequence of bases if replicated and passed on to the next generation becomes permanent and leads to mutation.
At the same time mutations are also necessary which provide raw material for evolution.
Without evolution, the new species, even human beings would not have arisen.
Therefore a balance between mutation and repair is necessary.

Types of Damage:

Damage to DNA includes any deviation from the usual double helix structure.

1. Simple Mutations:

Simplest mutations are switching of one base for another base.
In transition one pyrimidine is substituted by another pyrimidine and purine with another purine.
Trans-version involves substitution of a pyrimidine by a purine and purine by a pyrimidine such as T by G or A and A by C or T.
Other simple mutations are detection, insertion of a single nucleotide or a small number of nucleotides.
Mutations which change a single nucleotide are called point mutations.

2. Deamination:

The common alteration of form or damage includes deamination of cytosine (C) to form uracil (u) which base pairs with adenine (A) in next replication instead of guanine (G) with which the original cytosine would have paired.

As uracil is not present in DNA, adenine base pairs with thymine (T).
Therefore the C-G pair is replaced by T-A in the next replication cycle. Similarly, hypoxanthine results from adenine deamination.

3. Missing Bases:

Cleavage of the N-glycosidic bond between purine and sugar causes loss of purine base from DNA.
This is called depurination.
This apurinic site becomes a non-coding lesion.

4. Chemical Modification of Bases:

Chemical modification of any of the four bases of DNA leads to modified bases.
Methyl groups are added to various bases. Guanine forms 7- methylguanine, 3-methylguanine. Adenine forms 3-methyladenine.
Cytosine forms 5- Methylcytosine.
Replacement of amino group by a keto group converts 5-methylcytosine to thymine.

5. Formation of Pyrimidine Dimers (Thymine Dimers):

Formation of thymine dimers is very common in which a covalent bond (cyclobutyl ring) is formed between adjacent thymine bases.
This leads to loss of base pairing with opposite stand.
A bacteria may have thousands of dimers immediately after exposure to ultraviolet radiations.

6. Strand Breaks:

Sometimes phosphodiester bonds break in one strand of DNA helix.
This is caused by various chemicals like peroxides, radiations and by enzymes like DNases.
This leads to breaks in DNA backbone.
Single strand breaks are more common than double strand breaks.

Sometimes X-rays, electronic beams and other radiations may cause phosphodiester bonds breaks in both strands which may not be directly opposite to each other.
This leads to double strand breaks.
Some sites on DNA are more susceptible to damage. These are called hot-stops.

Repair Mechanisms:

Most kinds of damage create impediments to replication or transcription.
Altered bases cause mispairing and can cause permanent alteration to DNA sequence after replication.
In order to maintain the integrity of information contained in it, the DNA has various repair mechanisms.

1. Direct Repair:

The damage is reversed by a repair enzyme which is called photoreactivation.
This mechanism involves a light dependent enzyme called DNA photolyase.
The enzyme is present in almost all cells from bacteria to animals.
It uses energy from the absorbed light to cleave the C-C bond of the cyclobutyl ring of the thymine dimers.
In this way thymine dimers are monomerized.

2. Excision Repair:

It includes

base excision repair (BER)
nucleotide excision repair.(NER)

Base excision repair system (BER)

It involves an enzyme called N-glycosylase which recognizes the abnormal base and hydrolyzes glycosidic bond between it and sugar.
Another enzyme, an endonuclease cleaves the DNA backbone on the 5′-side of the abnormal base.
Then the DNA polymerase by its exonuclease activity removes the abnormal base.
DNA polymerase then replaces it with normal base and DNA ligase seals the region.

Nucleotide excision repair system (NER)

It includes three steps, incision, excision and synthesis.
Incision is done by endonuclease enzymes precisely on either side of the damaged patch of the strand.
In this way the damaged portion of the strand is cleaved.
Endonuclease enzymes involved are UvrA, UvrB which recognize the damaged stretch of the strand.
UvrC makes two cuts (incisions) on either side.
Exonuclease removes the damaged strand. Enzyme involved is UvrD.
Later, DNA polymerase synthesizes the new strand by using complementary strands as a template.
DNA ligase forms phosphodiester bonds which seal the ends on newly synthesized strands.

3. Mismatch Base Repair:

Sometimes wrong bases are incorporated during the replication process, G-T or C-A pairs are formed.
The wrong base is always incorporated in the daughter strand only.
Therefore in order to distinguish the two strands for the purpose of repair, the adenine bases of the template strand are labeled or tagged by methyl groups.
In this way the newly replicated DNA helix is hemimethylated.
The excision of wrong bases occurs in the non-methylated or daughter strand.
In this repair mechanism, three gene-products (proteins) are involved — Mut S, Mut L and Mut H.
The first step of this repair process consists of binding of the Mut S protein to the mismatched base- pair.
The second step involves the recognition of a specific sequence of the template which is -GATC- in E. coli in which A (adenine) is methylated in N-6 position.
The proteins Mut L and Mut H which bind to the unmethylated -GATC- sequence of the new strand form a complex with Mut S which is bound to the mismatch pair.
Thereby the mismatch pair is brought close to the -GATC- sequences.
The Mut H protein then nicks the unmethylated DNA strand at the GATC site and the mismatch is removed by an exonuclease.

4. Recombination Repair or Retrieval System:

In thymine dimer or other types of damage, DNA replication cannot proceed properly.
A gap opposite to the thymine dimer is left in the newly synthesized daughter strand.
The gap is repaired by a recombination mechanism or retrieval mechanism called sister strand exchange.

During replication of DNA two identical copies are produced.
Replicating DNA molecules has four strands A, B, C and D.
Strands A and C have same DNA , sequence.
Strands B and D also have same sequence as they are identical.
A thymine dimer is present in strand A.
The replication fork passes the dimmer as it cannot form hydrogen bonds with incoming adenine bases, thus creating a gap in the newly synthesized strand B.
In recombination repair system a short identical segment of DNA is retrieved from strand D and is inserted into the gap of strand B.
But this creates a gap in strand D which is easily filled up by DNA polymerase using normal strand C as a template.
This event is dependent on the activity of a special protein Rec A.
The Rec A protein plays its role in retrieving a portion of the complementary strand from other side of the replication fork to fill the gap.
Rec A is a strand exchange protein.
After filling both gaps, thymine is monomerised.
So in this repair mechanism a portion of DNA strand is retrieved from the normal homologous DNA segment.
This is also known as the daughter strand gap repair mechanism.

5. SOS Repair Mechanism:

Sometimes the replication machinery is unable to repair the damaged portion and bypasses the damaged site.
This is known as translesion synthesis also called bypass system and is an emergency repair system.
This mechanism is catalyzed by a special class of DNA polymerases called Y-family of DNA polymerases which synthesize DNA directly across the damaged portion.
The SOS response is set in action by the interaction of two proteins, — Rec A protein which is a product of the recA gene and Lex A protein, the product of lexA gene.
The Rec A protein in addition to having a role in genetic recombination and recombinational repair also has a protease function.
The Lex A protein acts as a repressor for a number of genes, known as SOS genes including the recA gene.
Under normal conditions i.e. when the SOS response is not necessary, these genes remain repressed by the Lex A repressor.
The initial event in the SOS response is the activation of RecA protease activity induced by DNA damage.
The activation of Rec A protease activity occurs within a few minutes of DNA damage.
The protease activity catalyzes cleavage of the Lex A repressor making it inactive.
As a result, the SOS genes can now be expressed to produce the enzymes required for DNA repair.
The SOS response, as the name suggests, is an emergency measure to repair mutational damage.
It makes it possible for the cell to survive under conditions which would have been otherwise lethal.
However, the possibility of generating new mutations increases in the repair of DNA molecules.
This is because the SOS repair system allows DNA synthesis bypassing the damaged site.
When the DNA polymerase III reaches a damaged site to which Rec A binds, the protein (Rec A) interacts with the epsilon subunit of the DNA polymerase molecule.
This subunit is responsible for insertion of the correct base into the growing DNA strand. As a result, chain elongation continues bypassing the damaged site, but the chance of incorporation of a wrong base increases.
SOS repair, therefore, enhances the chance of mutation due to mis-pairing of bases.
This is known as error-prone bypass repair.
A more recent model based on SOS repair of UV-irradiated DNA in bacteriophages has been proposed.
UV-irradiation is known to produce dimers of not only thymine, but also of thymine and cytosine and cytosine.
During replication when they are reached, the SOS repair system is halted temporarily and cytosine is deaminated to uracil.
Uracil pairs with adenine, bringing about a transition mutation by changing C-G base pair to T-A.
This is an error-free bypass repair though it still causes a mutation.
It is called error-free because the template DNA strand is faithfully copied in the newly synthesized strand.
The change from C to U occurs in the template strand itself.

MCQ’S

Introduction to DNA Damage and Repair

Which of the following is the most frequent cause of DNA damage?
a) DNA replication errors
b) Protein synthesis errors
c) tRNA malfunction
d) Cytoplasmic degradation
Answer: a) DNA replication errors
What type of mutation occurs when a wrong base is incorporated during replication?
a) Point mutation
b) Frameshift mutation
c) Silent mutation
d) Large-scale chromosomal mutation
Answer: a) Point mutation
Which type of radiation is most likely to cause double-strand breaks in DNA?
a) UV radiation
b) X-rays and gamma rays
c) Infrared radiation
d) Radio waves
Answer: b) X-rays and gamma rays
What is the role of DNA repair mechanisms?
a) Prevent mutations from being inherited
b) Ensure perfect replication
c) Destroy damaged DNA completely
d) Convert all mutations into beneficial ones
Answer: a) Prevent mutations from being inherited
Which of the following DNA damages is necessary for evolution?
a) Mutation
b) Depurination
c) Deamination
d) DNA strand breakage
Answer: a) Mutation

Types of DNA Damage

What is a transition mutation?
a) Purine is replaced by another purine or pyrimidine by another pyrimidine
b) Purine is replaced by a pyrimidine
c) Large segments of DNA are deleted
d) A segment of DNA is inverted
Answer: a) Purine is replaced by another purine or pyrimidine by another pyrimidine
Which type of mutation replaces a pyrimidine with a purine?
a) Transition
b) Transversion
c) Deamination
d) Depurination
Answer: b) Transversion
Deamination of cytosine results in the formation of:
a) Thymine
b) Uracil
c) Adenine
d) Guanine
Answer: b) Uracil
Depurination results in:
a) A missing purine base
b) A missing pyrimidine base
c) The insertion of an extra base
d) A double-strand break
Answer: a) A missing purine base
Which chemical modification leads to the formation of thymine from 5-methylcytosine?
a) Alkylation
b) Deamination
c) Oxidation
d) Phosphorylation
Answer: b) Deamination
Thymine dimers are caused by:
a) X-rays
b) UV radiation
c) Gamma rays
d) Chemical mutagens
Answer: b) UV radiation
Which type of DNA damage involves the breaking of phosphodiester bonds?
a) Point mutation
b) Thymine dimers
c) Strand breaks
d) Base mismatch
Answer: c) Strand breaks

DNA Repair Mechanisms

Which of the following is a direct repair mechanism?
a) Base excision repair
b) Photoreactivation
c) Mismatch repair
d) Nucleotide excision repair
Answer: b) Photoreactivation
The enzyme responsible for repairing thymine dimers through photoreactivation is:
a) DNA ligase
b) DNA polymerase
c) DNA photolyase
d) DNA helicase
Answer: c) DNA photolyase
Which of the following mechanisms corrects alkylated bases?
a) Mismatch repair
b) Direct repair
c) Recombination repair
d) SOS repair
Answer: b) Direct repair
What is the function of DNA glycosylase in base excision repair?
a) Breaks phosphodiester bonds
b) Recognizes and removes abnormal bases
c) Seals nicks in DNA strands
d) Creates an incision in the DNA strand
Answer: b) Recognizes and removes abnormal bases
Which repair system uses UvrA, UvrB, UvrC, and UvrD proteins?
a) Base excision repair
b) Nucleotide excision repair
c) Direct repair
d) SOS repair
Answer: b) Nucleotide excision repair
Mismatch repair distinguishes between old and new DNA strands by:
a) DNA polymerase activity
b) Methylation status
c) Hydrogen bonding strength
d) The presence of thymine dimers
Answer: b) Methylation status
The proteins MutS, MutL, and MutH are involved in:
a) Mismatch repair
b) Direct repair
c) Recombination repair
d) Nucleotide excision repair
Answer: a) Mismatch repair
What is the role of RecA in recombination repair?
a) Removes mismatched bases
b) Creates single-strand DNA nicks
c) Promotes strand exchange
d) Synthesizes new DNA
Answer: c) Promotes strand exchange
Which repair mechanism is also known as the bypass system?
a) Excision repair
b) Recombination repair
c) SOS repair
d) Direct repair
Answer: c) SOS repair
In SOS repair, the LexA repressor regulates:
a) DNA polymerase I activity
b) Expression of repair genes
c) Thymine dimer excision
d) Homologous recombination
Answer: b) Expression of repair genes
Which enzyme is involved in filling gaps left by excision repair mechanisms?
a) DNA ligase
b) DNA polymerase
c) DNA photolyase
d) Endonuclease
Answer: b) DNA polymerase

Additional Questions

Which process is error-prone and may introduce mutations?
a) Base excision repair
b) Mismatch repair
c) SOS repair
d) Photoreactivation
Answer: c) SOS repair
A mutation that changes a codon but does not affect the amino acid sequence is called:
a) Missense mutation
b) Nonsense mutation
c) Silent mutation
d) Frameshift mutation
Answer: c) Silent mutation
Which of the following is not a type of DNA repair?
a) Nucleotide excision repair
b) Base excision repair
c) Mismatch repair
d) RNA excision repair
Answer: d) RNA excision repair
The repair mechanism that restores normal base pairing after replication errors is:
a) Photoreactivation
b) Direct repair
c) Mismatch repair
d) SOS repair
Answer: c) Mismatch repair
In nucleotide excision repair, which enzyme is responsible for cutting the damaged DNA?
a) Ligase
b) Endonuclease
c) Exonuclease
d) DNA polymerase
Answer: b) Endonuclease

DNA Damage Types

Which type of mutation occurs due to the insertion or deletion of nucleotides?
a) Point mutation
b) Frameshift mutation
c) Transition mutation
d) Transversion mutation
Answer: b) Frameshift mutation
Which of the following is not a cause of DNA damage?
a) UV radiation
b) DNA replication errors
c) Protein synthesis errors
d) Chemical mutagens
Answer: c) Protein synthesis errors
What happens when cytosine undergoes deamination?
a) It converts into thymine
b) It converts into uracil
c) It forms guanine
d) It forms adenine
Answer: b) It converts into uracil
Which of the following damages DNA by causing single-strand and double-strand breaks?
a) X-rays and gamma rays
b) UV light
c) Hydrogen peroxide
d) Methylation
Answer: a) X-rays and gamma rays
What is the effect of alkylation on DNA?
a) It removes purines from the strand
b) It causes base modification
c) It induces thymine dimer formation
d) It prevents transcription
Answer: b) It causes base modification
Thymine dimers are primarily caused by:
a) Gamma radiation
b) UV radiation
c) X-rays
d) Chemical exposure
Answer: b) UV radiation
Which of the following types of DNA damage is reversible?
a) Deamination
b) Depurination
c) Thymine dimers
d) Double-strand breaks
Answer: c) Thymine dimers

Direct DNA Repair Mechanisms

Which repair mechanism directly reverses damage without removing bases?
a) Nucleotide excision repair
b) Base excision repair
c) Photoreactivation
d) Mismatch repair
Answer: c) Photoreactivation
The enzyme responsible for photoreactivation is:
a) DNA ligase
b) DNA photolyase
c) DNA polymerase
d) Topoisomerase
Answer: b) DNA photolyase
Photolyase repairs:
a) Depurination
b) Thymine dimers
c) DNA double-strand breaks
d) Frameshift mutations
Answer: b) Thymine dimers

Base Excision and Nucleotide Excision Repair

Base excision repair (BER) removes:
a) Large sections of DNA
b) Individual damaged bases
c) Double-strand breaks
d) Entire nucleotides
Answer: b) Individual damaged bases
Which enzyme is responsible for removing abnormal bases in base excision repair?
a) DNA polymerase
b) N-glycosylase
c) DNA ligase
d) DNA helicase
Answer: b) N-glycosylase
In nucleotide excision repair (NER), what is the first step?
a) DNA synthesis
b) Endonuclease incision
c) Methylation
d) DNA replication
Answer: b) Endonuclease incision
Which enzyme is not directly involved in base excision repair?
a) DNA polymerase
b) DNA ligase
c) DNA glycosylase
d) Helicase
Answer: d) Helicase
Nucleotide excision repair is most effective for:
a) Mismatch mutations
b) Large DNA distortions
c) Point mutations
d) Transversions
Answer: b) Large DNA distortions

Mismatch Repair (MMR)

Mismatch repair corrects errors that escape:
a) RNA proofreading
b) DNA polymerase proofreading
c) DNA photolyase activity
d) Exonuclease digestion
Answer: b) DNA polymerase proofreading
The enzyme responsible for mismatch recognition is:
a) MutS
b) MutL
c) MutH
d) UvrA
Answer: a) MutS
What feature allows mismatch repair to distinguish between old and new DNA strands?
a) Methylation pattern
b) DNA sequence
c) Strand size
d) Number of thymine dimers
Answer: a) Methylation pattern
Which of the following is not a part of mismatch repair?
a) Exonuclease activity
b) Ligase sealing
c) Photoreactivation
d) Polymerase synthesis
Answer: c) Photoreactivation

Recombination and SOS Repair

Which DNA repair pathway uses homologous recombination?
a) Nucleotide excision repair
b) Base excision repair
c) Recombination repair
d) Direct repair
Answer: c) Recombination repair
Which protein plays a key role in recombination repair?
a) UvrA
b) MutS
c) RecA
d) DNA photolyase
Answer: c) RecA
What is the function of RecA in recombination repair?
a) Recognizes mismatches
b) Creates thymine dimers
c) Promotes strand exchange
d) Removes methyl groups
Answer: c) Promotes strand exchange
Which repair mechanism is error-prone?
a) Photoreactivation
b) Mismatch repair
c) SOS repair
d) Base excision repair
Answer: c) SOS repair
The SOS repair system is activated by:
a) DNA damage stress
b) ATP hydrolysis
c) DNA polymerase accuracy
d) Ligase activity
Answer: a) DNA damage stress
Which protein regulates the SOS repair system?
a) RecA
b) LexA
c) DNA ligase
d) DNA helicase
Answer: b) LexA
What does error-prone bypass repair increase?
a) Mutation rates
b) DNA stability
c) DNA polymerase activity
d) DNA repair accuracy
Answer: a) Mutation rates

Final Questions

DNA polymerase has:
a) Only synthesis activity
b) Exonuclease proofreading activity
c) No proofreading
d) Ligase function
Answer: b) Exonuclease proofreading activity
In mismatch repair, which base is preferentially removed?
a) The methylated base
b) The unmethylated base
c) The uracil
d) The thymine
Answer: b) The unmethylated base
Which repair pathway corrects double-strand breaks?
a) Direct repair
b) Nucleotide excision repair
c) Homologous recombination
d) Base excision repair
Answer: c) Homologous recombination
DNA ligase is essential for:
a) Breaking phosphodiester bonds
b) Joining Okazaki fragments
c) Degrading damaged DNA
d) Transcription
Answer: b) Joining Okazaki fragments