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MOLECULAR BIOLOGY
LESSON 3: DNA REPAIR AND RECOMBINATION
DNA POLYMERASE PROOFREADING
• DNA polymerase has 2 types:
o The polymerizing site
▪ Adds new nucleotide
o The Editing site
▪ Detects bases which are mispaired. If it
detects the mispaired, the DNA polymerase
has an exonuclease activity to remove the
incorrect nucleotide.
• Basically, the DNA polymerase has an own
proofreading mechanism and it allows us to have
low mutation rate.
o Eukaryotic DNA polymerases make errors every
10,000-100,000 nucleotides
o 1 mutation per 10 billion nucleotide base pairs per
cell generation
DNA DAMAGE AND REPAIR
• This image shows how DNA repair happened.
• Researchers from Harvard school of Medicine
labeled the end of the DNA with Fluorescent marker
and they observed that once that the DNA ends
reconnect, they changed in color.
• Without DNA repair, spontaneous DNA damage
would rapidly change the DNA sequences even on
their normal cell condition the DNA is susceptible to
damage.
• These damages may lead to mutation and if they are
not repaired, they will cause certain diseases.
2 nd M
A. SPONTANEOUS DNA DAMAGE
• Oxidative Damage
• Hydrolytic Attack
• Methylation
• This figure shows the representation of the site where
the DNA damage occur.
• Red arrow indicates Oxidative damage, Blue arrow
indicates Hydrolytic attack and the Green arrow
indicates the Methylation.
METHYLATION
• In methylation, due to harmful chemicals, methyl
groups are added to the nucleotide bases.
B. DEPURINATION
• The human cell loses about 18,000 purine bases
every day because of Depurination.
• Depurination is where your glycosyl linkage of
purine to deoxyribose hydrolyzed
o In depurination, it removes the purine. The
purines are Guanine and Adenine.
o Depurination happens only in Purines, Guanine
and Adenine.
• The Glycosyl linkage (links to sugar and nucleotide
bases) is hydrolyzed. The water acts upon glycosyl
linkage therefore displacing the nucleotide base.
o When the glycosyl linkage hydrolyze, the purine
will be removed.
• The purines are substituted with hydroxyl group.
• In depurination, once it is happened, there is mutation
specifically the lesion
• When the nucleotide Is depurinated, the purine
(Adenine) is removed.
• During the DNA replication, when it uses the template
with mutation, it will have a mutation that will occur.
The new DNA will no longer have A-T nucleotide
because of depurinate nucleotide.
C. DEAMINATION
• Deamination is the conversion of nucleotide to
unnatural products by hydrolysis of the amino group.
o In deamination, the amino group is hydrolyzed,
one it is hydrolyzed, it is removed. So, it will
change Cytosine to Uracil.
• The most common example is Cytosine
• The Adenine has amino group and the amino group
is hydrolyzed therefore displacing the amino group
and you will have unnatural products called
“Hypoxanthine”
o If you deaminate the Adenine, you will have
“Hypoxanthine”
• In Guanine, the amino group is hydrolyzed therefore
displacing the amino group and will change to
“Xanthine”
o If you deaminate the Adenine, you will have
“Xanthine”
• In Pyrimidines, there is Cytosine. The amino group
will hydrolyze and will be removed. It will form Uracil
• In Thymine, there is no deamination because there
is no amino group.
• Deamination creates mutation
o Deamination = Substitution Mutation
o Depurination = Deletion Mutation
o In deamination, once the Cytosine is deaminated
to Uracil, there is a change in the template
strand. When it uses the template strand for DNA
synthesis, there will have a new nucleotide base.
D. PYRIMIDINE DIMERS
• In Pyrimidine dimers, the UV light affects the
nucleotide bases. They formed a covalent bond and
this covalent bond, once it is formed, it becomes a
Dimer.
• The most common dimer is the Thymine dimer. This
thymine dimer forms a kink in the DNA strand.

E. BREAKS IN THE DNA

• Breaks in the DNA is caused by X-rays or Gamma-

rays or other environmental factor.
• Single strand break and Double strand break are
the damages in the DNA.

IF THE DNA DAMAGES ARE NOT REPAIRED

AND ACCUMULATE, DISEASE WILL OCCUR
• If the Thymine dimer is still carried on in the
transcription, it can cause diseases. That’s why we
need to repair the damages. Luckily, our cells have
the ability to repair DNA damages.
• Ex. Xeroderma pigmentosum

DNA DOUBLE HELIX IS READILY

REPAIRED
• DNA damages are easily repair because the DNA
double helix is very ideal.
o The DNA double helix is a complementary
strand, so you can use either of the strand to
correct the mutated strand.
TRANSLESION POLYMERASE
• The replicative polymerase is switched out for
specialized DNA polymerases and replicates past the
damage.
• DNA polymerase n (eta)
o DNA polymerase used in eukaryotes
• In this figure, it detects that there is a damage,
o DNA polymerase will be released and will
changed to translesion DNA polymerase
o This translesion DNA polymerase is in the form of
DNA polymerase eta in eukaryotes
o What it does, it continues to replicate the DNA
o The translesion DNA polymerase has the ability
to put nucleotide sequences in the growing
strand even though it has thymine dimer in the
template strand.
▪ Thymine dimers is the usually DNA damage
▪ Add correct base pair in the form of Adenine
REPAIR OF SINGLE STRAND BREAKS
• Mismatch Repair
o The back-up repair during DNA replication
o genetic defects (Lynch Syndrome)
• Base Excision Repair
o carcinogenic exposure (such as tobacco smoking
and/or diesel exhaust)
• Nucleotide Excision Repair
o Most common used repair for DNA damages
which are bulky
▪ For Thymine dimers
o UV light (Xeroderma pigmentosum)
A. MISMATCH REPAIR B. BASE EXCISION REPAIR

• In this figure, A mismatched is detected in the newly

synthesized DNA
o Thymine is the complementary base pair but the
added nucleotide strand is guanine
• The new DNA strand is cut and the mispaired
nucleotide and its neighbor are removed.
• The missing patch is replaced with correct
nucleotides by a DNA polymerase.
o Once the section is removed, the DNA
polymerase puts the right nucleotide bases
• A DNA ligase seals the gap in the DNA backbone.
• Deamination converts a cytosine base into Uracil
• The Uracil is detected and removed, leaving a base-
less nucleotide
• The Sugar phosphate backbone is also removed.
When the base-less nucleotide is removed, leaving a
small gap in the DNA
• The hole is filled with the right base by a DNA
polymerase, and the gap is sealed by a ligase.

• In mismatch repair, it has a protein called MutS and

MutL complex. It detects the mismatch in the DNA
• The MutH detects certain sequence in the DNA called
GATC. If the GATC is detected, the certain sequence
will bind to the complex and it bends the DNA.
o When It is bended, the MutH makes a cut unto
the mismatch base pair • DNA glycosylases
o Once it makes a cut the DNA polymerase can o “flipping out”
now add the nucleotide base pair which are ▪ It flips out the nucleotide base to go outside
correct. o recognizes and removes the unnatural nucleotide
o An enzyme that detects the wrong in the template
• AP endonuclease
o “missing tooth”
▪ Removes phosphodiester and sugar
phosphate that makes it to look like a missing
tooth.
 o Removes the sugar-phosphate backbone
▪ It produces a gap in the DNA
▪ The gap is filled with new nucleotides and the
nick is sealed by DNA ligase
C. NUCELOTIDE EXCISION REPAIR
• “Bulky lesions”
o covalent reactions of DNA bases with large
hydrocarbons (e.g. carcinogen benzopyrene
found in tobacco smoke, coal tar, and diesel
exhaust)
o pyrimidine dimers caused by sunlight
• In the figure, UV radiation produces a thymine dimer
• Once the dimer has been detected, the surrounding
DNA is opened to form a bubble
• Enzymes cut the damaged region out of the bubble
• DNA polymerase replaces the excised (cut-out) DNA
with new correct nucleotide sequence and a ligase
seals the backbone
• Enzymes responsible for Nucleotide excision repair:
• Excision nuclease
o Detects the error
o It marks the section of the DNA to be remove
• DNA Helicase
o Removes the strand of the DNA which has the
pyrimidine dimers
• DNA Polymerase
o Adds the correct DNA bases
• DNA Ligase
o Corrects the gap between the strands
REPAIR OF DOUBLE STRAND BREAK
• Non-homologous End Joining
• Homologous Recombination
A. NON-HOMOLOGOUS END JOIN
• Scientists in Harvard school of Medicine uses the
technique Fluorescence Resonance Energy
Transfer (FRET) microscopy, that once the DNA
connects it changes the color to Red.
o In this way, we can observe the in between
phases of the non-homologous joining.
• Non-homologous end joining is very simple
because it just glues the DNA strand back together,
usually with a small mutation at the break site.
• It is also called “Quick end dirty” because it just
connects the end of the DNA
B. HOMOLOGOUS RECOMBINATION
• In homologous recombination, the damaged region is
replaced via recombination, using sequences copied
from the homologue.
• Based in the figure, it has a double strand break and
when it makes a daughter DNA, it will produce 2
daughter DNA double helix.
o The 1st daughter DNA has a double strand break
o The nuclease will digest the region of the
daughter DNA that has double strand break and
it will have a strand exchange from the other
daughter DNA will go inside to copy and replaced
the damaged DNA.
• Homologous recombination
o General term for crossing over of DNA section to
another DNA section (alleles with same
hereditary information) in Myosis.
o Genetic information from parents will combine
o In this case, Homologous recombination is used
to repair DNA with double strand break
• Once the strand exchange occurs, the repair
polymerase, synthesizes the DNA (represented in
green) using the undamaged DNA as template
• Invading strand release and broken helix re-formed.
• DNA synthesis continues using strands from
damaged DNA as template
• And lastly, it will have DNA Ligation
• Repair of broken replication fork
o The nuclease will degrade the part which has a
nick or damage and once the nuclease degrade,
It will have an overhang which goes to the strand
which is damage. And it is termed as “Grand
Exchange”
▪ In Grand exchange, the damage DNA uses
the undamaged DNA as a template
o Once the strand breakage and additional
synthesis happen, It can already have a DNA
replication.
o You can see at the last part of the picture that the
Replication fork resumes/continues to work.
 CASE STUDY • In Normal tissue (blue line) microsatellite markers
• Hereditary Non-polyposis Colon Cancer do not match to the Tumor tissue (red); therefore, it
has Instability.
• Xeroderma Pigmentosum
• The MSS (Stable) matches the microsatellite
• BRCA1 and BRCA2

HEREDITARY NON-POLYPOSIS COLON

CANCER (HNPCC)
• Lynch Syndrome
o Genetic disorder that causes an increased risk of
developing cancer
o Autosomal dominant
o Mutation in the genes involved in DNA repair
▪ Mismatch Repair Genes (MMR genes)
• Can be diagnosed using Microsatellite Instability
Testing (MSI)
o The presence of MSI is an evidence of mismatch
repair gene is not functioning properly
• Microsatellite
o A condition in which it has hyper mutability in
impaired DNA. XERODERMA PIGMENTOSUM
o A structure which repeated nucleotide mostly • Children of the moon
often seen as GTCA. • It is a rare hereditary disease which children who
o Are repeats in DNA and normal cells. inherited the disease is very sensitive to the sun
o When the mismatch repair is working properly, ultra-violet light / radiation.
the microsatellite will all properly replicates. o Exposure even a few minutes in the sunlight is
o Researchers found out that when we look at the enough to cause severe burning in the skin or it
microsatellite, we can actually determine if the caan cause skin cancer.
person has an HNPCC. • Their suit is designed by NASA to prevent them from
the UV radiation.

• In the Normal satellite replication, you will see the

right replication of the DNA
• In the Abnormal mismatch repair defect, the
microsatellite has a variability in sizes
o Variability in microsatellite is called
Microsatellite Instability.

MICROSATELLITE INSTABILITY TESTING

BY PCR
• In Microsatellite Instability Testing, it amplifies the
Microsatellite (repeats).
• In MSI-H (Height), it is the samples with microsatellite
instability, therefore the variability of the microsatellite
is high.
• The Fam, Hex and Texas red are Chromophores
that use in PCR.
o These chromophores are the one who gives
signal if it detects the section of DNA
• The Bat 25, NR24, NR21, NR27, and Bat25 are only
a microsatellite marker. These are the ones amplified.
• UV radiation can cause DNA damage by triggering
pyrimidine dimer formation
• Excision repair to remove dimer formation is
dysfunctional
• Mutations in seven genes, XPA to XPG, responsible
for the excision repair pathway
o Their body has no capability to exhibit the
excision repair, that’s why the dimers accumulate
to cause a cancer.
• Another type of Xeroderma Pigmentosum is the BREAST CANCER
Mutations in the eighth gene called XPV produce
an alternate form of xeroderma pigmentosum
• Affects DNA polymerase ɳ (eta) responsible for
translesion synthesis
• 2 DNA repair mechanisms affected in XP:
1. Excision Repair
2. Translesion Synthesis

• For diagnosis of breast cancer, the ff can be used:

o Breast Exam
o Needle Biopsy
o MRI

SUMMARY
• Discussed about the different types of DNA damage.
• Learned the various ways in which DNA repair is
conducted for all kinds of damage, such as dimers,
• Xeroderma Pigmentosum can be diagnosed by single and double strand DNA breaks.
o Skin Biopsy • Discussed the mechanism behind DNA
o Genetic Testing recombination.
▪ By giving a sample of the DNA to see if it has • Studied diseases relating to the dysfunction of DNA
a mutation in the genes involve in the repair.
xeroderma pigmentosum
• Very rare, diagnosis not yet fully developed. Usually,
this disease is discovered in grown-ups only.

BRCA1 AND BRCA2

• Brca1 and Brca2 gene compromise the homologous
recombination repair and causes Hereditary Breast
and Ovarian Cancer
o Mutations in Brca1 and Brca2 results to ovarian
and breast cancer because of damage
accumulation

• Brca1 and Brca2 aids in the homologous

recombination.
• Brca1 is the protein that’s initiate or calls the other
enzyme to repair the DNA damage
• Brca2 holds a protein mRad51 which facilitates to
enter the DNA strand to the homologous repair