Unit- IV

DNA damage and Repair

mechanism
 Introduction
• DNA damage, due to environmental factors and normal metabolic
processes inside the cell, occurs at a rate of 1,000 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), if left unrepaired can
cause mutations 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 disease states such as cancer.
 Types of DNA damage
There are several types of DNA damage that can occur due to either normal
cellular processes or due to the environmental exposure of cells to DNA
damaging agents. DNA bases can be damaged by:
(1) Oxidative Damage
(2) Alkylation of bases
(3) Base loss caused by the hydrolysis of bases
(4) Thymine Dimer formation
(5) Bulky adduct formation
(6) DNA strand breaks
 Oxidative damage
• Reactive oxygen species (ROS) can cause significant cellular stress and damage
including the oxidative damage of DNA.
• Hydroxyl radicals (•OH) are one of the most reactive and electrophilic of the ROS
and can be produced by ultraviolet and ionizing radiations or from other radicals
arising from enzymatic reactions.
• The •OH can cause the formation of 8-oxo-7,8-dihydroguanine (8-oxoG) from
guanine residues, among other oxidative products.
• Guanine is the most easily oxidized of the nucleic acid bases, because it has the
lowest ionization potential among the DNA bases.
• The 8-oxo-dG is one of the most abundant DNA lesions, and it considered as a
biomarker of oxidative stress.
• It has been estimated that up to 100,000 8-oxo-dG lesions can occur daily in DNA
per cell. increased levels of 8-oxo-dG are frequently found associated with
carcinogenesis and other disease.
 Alkylation of Bases
• Alkylating agents are widespread in the environment and are also produced
endogenously, as by-products of cellular metabolism. They introduce lesions
into DNA or RNA bases that can be cytotoxic, mutagenic, or neutral to the
cell.

• Cytotoxic lesions block replication, interrupt transcription, or signal the

activation of apoptosis, whereas mutagenic ones are miscoding and cause
mutations in newly synthesized DNA.

• The most common type of alkylation is methylation with the major products
including N7-methylguanine (7meG), N3-methyladenine (3meA), and O6-
methylguanine (O6meG) and methyl phosphotriesters (MPT).

•
 Base Loss / Missing Bases

• An AP site (apurinic/apyrimidinic site), also known as an abasic site, is a

location in DNA (also in RNA but much less likely) that has neither a purine
nor a pyrimidine base, either spontaneously or due to DNA damage.

• AP sites can be formed by spontaneous depurination, but also occur as

intermediates in base excision repair

• If left unrepaired, AP sites can lead to mutation during semiconservative

replication. They can cause replication fork stalling and are often
bypassed by translesion synthesis.

Abasic Sites.
 Formation of Pyrimidine Dimers (Thymine Dimers)

• UV light can cause molecular crosslinks to form between two pyrimidine

residues, commonly two thymine residues, that are positioned
consecutively within a strand of DNA.
• Two common UV products are cyclobutane pyrimidine dimers (CPDs) and
photoproducts. These premutagenic lesions alter the structure and
possibly the base-pairing.
• Up to 50–100 such reactions per second might occur in a skin cell during
exposure to sunlight, but are usually corrected within seconds by
photolyase reactivation or nucleotide excision repair.
• Uncorrected lesions can inhibit polymerases, cause misreading during
transcription or replication, or lead to arrest of replication. Pyrimidine
dimers are the primary cause of melanomas in humans.
 Bulky Adduct formation

• Some chemicals are biologically reactive and will form covalent linkages
with biological molecules such as DNA and proteins creating large bulky
adducts, or appendages, that branch off from the main molecule. An
example is Benzopyrene.
• Benzopyrene is actually a procarcinogen that needs to be biologically
activated by metabolism before it forms a reactive metabolite.
• Benzopyrene forms a highly reactive epoxide that forms a bulky adduct
preferentially with guanine residues in DNA that distorts the DNA
backbone.
• This ubiquitous compound can be found in coal tar, tobacco smoke and
many foods, especially grilled meats.
 DNA 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
 DNA repair mechanisms
• Most kinds of damage create impediments to replication or transcription.
Altered bases cause mis-pairing 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
2. Excision repair
3. Mismatch Base Repair
4. Recombination Repair or Retrieval System
5. SOS Repair Mechanism
 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
cyclobutyl ring of the thymine dimers. In this way thymine dimers are
monomerized.
 Excision repair
• It includes base excision repair and nucleotide excision repair. Base excision
repair system involves an enzyme called N-glycosylase which recognizes the
abnormal base and hydrolyses glycosidic bond between base 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 repair system includes three steps, incision, excision and synthesis.
Incision is done by Endonuclease enzyme precisely on either side of the
damaged patch of the strand, Exonuclease removes the damaged strand, DNA
polymerase synthesizes the new strand by using complementary strand as a
template. DNA ligase forms phosphodiester bonds which seal the ends on
newly synthesized strand.
• In this way damaged portion of the strand is cleaved.
 Mismatch Base Repair

• Sometimes wrong bases are incorporated during 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 labelled or tagged by
methyl groups.

• In this way the newly replication DNA helix is hemimethylated. The

excision of wrong bases occur in the non-methylated or daughter strand.
 Recombination Repair or Retrieval System

• In thymine dimer or other type of damage, DNA replication cannot

proceed properly. A gap opposite to thymine dimer is left in the newly
synthesized daughter strand. The gap is repaired by recombination
mechanism or retrieval mechanism called also sister strand exchange.
• During replication of DNA two identical copies are produced. Replicating
DNA molecule 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 daughter strand gap repair mechanism.
 SOS Repair Mechanism

• Sometimes the replicating 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 emergency repair system.

• This mechanism is catalyzed by a special class of DNA

polymerases called Y-family of DNA polymerases which
synthesized DNA directly across the damaged portion.