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MB 207 – Molecular Cell Biology
DNA Damage and Repair
DNA recombination
DNA Damage and Repair
• Maintaining genetic stability is very important
- accurate mechanism for replicating DNA.
- mechanism for repairing DNA alterations that arise both
spontaneously and from exposure to DNA-damaging environmental
agents.
• Nearly all DNA damage is harmful but occasionally beneficial
because mutations provide genetic variability.
How important is DNA repair?
– DNA is the only biomolecule that is specifically repaired. All others are
replaced.
– DNA damage is repaired shortly after it occurs and hence it does not
affect future generations
– >100 genes participate in various aspects of DNA repair, even in
organisms with very small genomes.
– Cancer is caused by mutations as well as many other diseases.
DNA damage
Spontaneous
Mutagens
Depurination Deamination Chemicals
Radiation
Spontaneous mutations
• Hydrolysis reactions caused by random interactions between DNA
and the molecules around it.
• Two types of spontaneous mutations:
 Depurination
 Deamination
•
Depurination
 the loss of a purine base by spontaneous hydrolysis of
glycosidic bond that links it to deoxyribose.
 this glycosidic bond is labile under physiological conditions.
Therefore, susceptible to hydrolysis that DNA loss thousands of
purine bases in the human cell everyday.
• Deamination
 Primary amino groups of nucleic acid bases are unstable. They can
be converted to keto groups in the hydrolysis reactions and become
deaminated.
 Involve cytosine, adenine and guanine, changes the base pairing
properties of the affected base.
 Cytosine is more susceptible to deamination, giving rise to uracil.
Others: Adenine to Hypoxanthine, Guanine to Xanthine, and 5-methyl
cytosine to Thymine.
 Usually caused by random collision of a water molecule with the
bond that links the amino group of the base to the pyrimidine or
purine ring.
 Rate is about 100 deaminations per day.
 If not repaired, the error base sequence may be propagated when
the strand serves as a template in the next round of replication.
Depurination & Deamination
Deamination of DNA nucleotides
A. Deamination of cytosine
produces uracil
B. Depurination
Missing
purine
Results in the substitution of one
base for another when the DNA
is replicated
If uncorrected, can lead to either
the substitution or the loss of a
nucleotide pair.
Mutagens (mutation-causing agents)
• Two major categories
 Chemicals
 Radiations
• Chemicals
- alter DNA structure by a variety of mechanisms.
 Base analogs
- resemble nitrogenous bases in structure and are incorporated into
DNA.
 Base modifying agents
- reacts chemically eith DNA bases to alter their structures.
 Intercalating agents
- insert themselves between adjacent bases of the double helix.
• Radiations
 Sunlight (ultraviolet radiation)
- alters DNA by triggering pyrimidine dimer formation (formation of
covalent bonds between adjacent pyrimidine bases).
- blocked replication and transcription.
 X-rays and related form of radiation emitted by radioactive
substances
- ionizing radiation because it removes electrons from biological
molecules.
- generating highly reactive intermediates that cause various types
of DNA damage.
DNA damages
• Distortion of double helix structure
The thymine dimer
– Photodamage
• UV light absorbed by the nucleic
acid bases can induce bond
formation between adjacent
pyrimidines (C or T) within one
strand.
• The two adjacent pyrimidines are
pulled closer to each other than in
normal DNA
• Strand breaks
– Single-strand and double-strand
breaks are produced at low frequency
during normal DNA metabolism by
topoisomerases, nucleases and repair
processes as well as by ionizing
radiation.
This type of damage is introduced
into DNA in cells that are exposed
to ultraviolet irradiation
Types of DNA damages
• Spontaneous oxidative damage (red arrows)
• hydrolytic attack (blue arrows)
• Uncontrolled methylation (green arrows))
DNA Repair
Mechanism
DIRECT
REVERSAL
SINGLE
STRAND
DAMAGE
BASE
EXCISION
REPAIR
NUCLEOTIDE
EXCISION
REPAIR
DOUBLE
STRAND
BREAKS
MISMATCH
REPAIR
TRANSLESION
SYNTHESIS
NONHOMOLOGOUS
HOMOLOGOUS
RECOMBINATION
END JOINING
Repairing Damaged Bases
Damaged or inappropriate bases can be repaired by several mechanisms:
1. Direct chemical reversal of the damage
2. Excision Repair, in which the damaged base or bases are removed
and then replaced with the correct ones in a localized burst of DNA
synthesis. There are three modes of excision repair, each of which
employs specialized sets of enzymes.

Base Excision Repair (BER)

Nucleotide Excision Repair (NER)

Mismatch Repair (MMR)
3. Double strand breaks
 Non-homologous end joining
 Homologous recombination
4. Translesion synthesis- DNA damage tolerance process that allows
the DNA replication machinery to replicate past DNA lesions such as
thymine dimers or AP sites
1. Direct Reversal of Base Damage
The most frequent cause of point mutations in humans is the
spontaneous addition of a methyl group (CH3-) (an example of
alkylation) to Cs followed by deamination to a T. Fortunately, most
of these changes are repaired by enzymes, called glycosylases,
that remove the mismatched T restoring the correct C. This is
done without the need to break the DNA backbone (in contrast to
the mechanisms of excision repair described below).
Some of the drugs used in cancer chemotherapy ("chemo") also
damage DNA by alkylation. Some of the methyl groups can be
removed by a protein encoded by our MGMT gene. However, the
protein can only do it once, so the removal of each methyl group
requires another molecule of protein.
This illustrates a problem with direct reversal mechanisms of DNA
repair: they are quite wasteful.
2. Excision Repair
A.
Base excision repair (BER), which repairs damage to a single
nucleotide caused by oxidation, alkylation, hydrolysis, or
deamination. The base is removed with glycosylase and
ultimately replaced by repair synthesis with DNA ligase.
B. Nucleotide excision repair (NER), which repairs damage
affecting longer strands of 2–30 bases. This process recognizes
bulky, helix-distorting changes such as thymine dimers as well as
single-strand breaks (repaired with enzymes such UvrABC
endonuclease). A specialized form of NER known as
Transcription-Coupled Repair (TCR) deploys high-priority NER
repair enzymes to genes that are being actively transcribed.
C. Mismatch repair (MMR), which corrects errors of DNA replication
and recombination that result in mispaired (but normal, that is
non- damaged) nucleotides following DNA replication
DNA repair mechanisms
1) Base excision repair (BER)
– Removal of the incorrect base by an
appropriate DNA glycosylase to create
a deoxyribose sugar lacking it’s base
(AP site - apurinic / apyrimidinic)
– Nicking of the damaged DNA strand
by AP endonuclease upstream of the
AP site, thus creating a 3'-OH
terminus adjacent to the AP site,
removal of sugar phosphate.
– Extension of the 3'-OH terminus by a
DNA polymerase, DNA ligase seals
nick.
– e.g. removal of uracil from DNA
DNA repair mechanisms
2) Nucleotide excision repair (NER)
– Removes a whole oligonucleotide
that contain the damage.
– Steps:
• Multienzyme complex recognizes
damaged regions based on their
abnormal structure as well as on
their abnormal chemistry (eg.
pyrimidine dimer)
• Double incision of the damaged
strand several nucleotides away from
the damaged site, on both the 5' and
3' sides
• An associated DNA helicase
removes the entire damaged strand,
in-between the nicks.
• Bacteria multienzyme complex
leaves a 12nt gap; doubles the size
in human DNA
• Filling in of the resulting gap by a
DNA polymerase
• Ligation by DNA ligase.
(bulky lesion)
Repairing Strand Breaks
Ionizing radiation and certain chemicals can produce double-strand
breaks (DSBs) in the DNA backbone.
Double-Strand Breaks (DSBs)
There are two mechanisms by which the cell attempts to repair a
complete break in a DNA molecule:
i. Direct joining of the broken ends.
-This requires proteins that recognize and bind to the exposed ends and bring
them together for ligasing. They would prefer to see some complementary
nucleotides but can proceed without them so this type of joining is also called
Nonhomologous End-Joining (NHEJ).
-Errors in direct joining may be a cause of the various translocations that are
associated with cancers.
(Translocation: Type of mutation in which a portion of 1 chromosome
is broken off and attached to another)
ii. Homologous Recombination. Here the broken ends are
repaired using the information on the intact
-sister chromatid (available in G2 after chromosome
duplication), or on the
-homologous chromosome (in G1; that is, before each
chromosome has been duplicated). This requires searching
around in the nucleus for the homolog — a task sufficiently
uncertain that G1 cells usually prefer to mend their DSBs by
NHEJ. or on the
-same chromosome if there are duplicate copies of the gene
on the chromosome oriented in opposite directions (head-tohead or back-to-back).
-Two of the proteins used in homologous recombination are
encoded by the genes BRCA1 and BRCA2. Inherited mutations in
these genes predispose women to breast and ovarian cancers.
DNA repair mechanisms
Two different types of end-joining for repairing double-strand
breaks
1. Nonhomologous end-joining
– permits joining of double-strand breaks even if there is no sequence
similarity between them
– Broken ends are rejoined by DNA ligation with the loss of one or
more nucleotides at the joining site
– Alters the original DNA sequence either by deletions or short
insertions.
2. Homologous end-joining
– More difficult to accomplish but is more precise
– cells are diploid – contain 2 copies of each double helix
– Recombination mechanisms used to transfer nucleotide sequence
information from the homologous intact DNA double helix to the site
of the double-strand break
• Both system involve a lot of different proteins and the processes are
much more complicated
DNA end-joining for repairing ds breaks
Accidental break (ionizing radiation,
oxidizing agents, replication errors)
DNA ligation
Loss of nucleotides due to
degradation from ends
Copying process involving
homologous recombination
Region with altered segment
due to missing nucleotides
Nonhomologous end-joining
- Common in mammalian cells
Complete sequence restored by copying
from second chromosome
(replication process uses the undamaged
chromosome as the template for transferring
genetic information to the broken chromosome,
repairing it with no change in the DNA
sequences)
Homologous end-joining
Summary of DNA repair systems
Type
Damage
Enzyme
Mismatch repair
Replication errors
MutS, MutL, and MutH in E. coli
MSH, MLH and PMS in humans
Photoreaction
Pyrimidine dimers
DNA photolyase
Base excision
repair
Damaged base
DNA glycosylase
Nucleotide
excision repair
Pyrimidine dimer
Bulky adduct on base
UvrA, UvrB, UvrC and UvrD in E. coli
XPC, XPA, XPD, ERCI-XPF and XPG
in humans
Double strand
break repair
Double strand breaks
RecA and RecBCD in E.coli
Translesion DNA
synthesis
Pyrimidine dimer or
apurinic site
Y-family DNA polymerase, such as
UmuC in E. coli
DNA Recombination
• A process that a DNA segment moves from one DNA
molecule to another DNA molecule
– DNA molecules recombine by breaking and rejoining
– Phosphodiester bonds are broken and rejoined.
• Importance of DNA recombination:
– the process of introducing genetic variation: Genetic variation is
crucial to allow organisms to evolve in response to a changing
environment. E.g., genetic recombination results in the exchange of
genes between paired homologous chromosomes during meiosis.
– an important mechanism for repairing damaged DNA.
– involved in rearrangements of specific DNA sequences that alter the
expression and function of some genes during development and
differentiation.
• Two broad classes are commonly recognized general recombination & site-specific recombination.
A heteroduplex joint
General recombination in meiosis
General Recombination
• Allow large section of the DNA double
helix to move from one chromosome to
another
• Responsible for the crossing-over of
chromosomes during meiosis
• Chromosome must synapse (pair) in
order for chiasmata to form where
crossing-over occurs
– DNA synapsis: base pairing between
complementary strands from 2 DNA
molecules
– Chiasmata: regions where paired
homologous chromosomes
exchange genetic material during
meiosis, a cross-shaped structure
• Only occurs between homologous DNA
molecules
General Recombination
• Two homologous DNA molecules line up.
• Nicks (single or double??) are introduced.
• Each nicked strand then invades the other DNA molecule by
complementary base pairing.
• The cut strands cross and join homologous strands, forming the
Holliday structure (or Holliday junction) (R. Holliday (1964).
• Once a Holliday junction is formed, it can be resolved 2 ways by
nicking and rejoining of the crossed strands to yield 2 different
heteroduplexes:
– recombinant heteroduplexes: resulting DNA molecules are a
combination of both parental DNA molecules.
– non-recombinant heteroduplexes: resulting DNA molecules contain
only DNA from one parent molecule with a small portion of
heteroduplex.
Paternal
chromosome A
Maternal
chromosome B
Holliday junction
cleavage
DNA clearage
‘splice’ or crossover
products
reassortment or
flanking genes
Non-recombinant
Recombinant
‘patch’ or noncrossover products
no reassortment
DSB repair model for
homologous
recombination. The figure
shows the step leading to
generation of
recombination intermediate
with 2 Holliday junctions.
General Recombination: example
•
Various enzymes (homologues) are involved in the
recombination process:
–
–
–
–
Rec A: catalyze the exchange of strands between homologous DNAs that
causes heteroduplexes to form
RecB, C, & D: complex of three proteins
1. acts as a helicase and transiently unwinds the double-stranded DNA
2. When it encounters the specific nucleotide sequence GCTGGTGG (the
chi site), the enzyme acts as a nuclease to introduce a single-stranded
nick
3. Continue to unwind the double helix, forming a displaced single strand to
which RecA can bind to initiate strand exchange.
Ruv A, B: catalyze the movement of the crossed-strand site in Holliday
junctions
RuvC: resolves the Holliday junction by cleaving the crossed strands, which
are then joined by ligase
The different resolutions of a general recombination
intermediate in mitotic and meiotic cells.
Site-Specific Recombination
• Occurs between sequences with a limited stretch of similarity; involves
specific sites
• Mediated by proteins that recognize the specific DNA target sequences
rather than by complementary base pairing
• Transposons/transposable elements/ “Jumping genes“: mobile
genetic elements that can move throughout the genome
• Two distinct mechanisms:
1. Transpositional site-specific recombination: insertion of mobile
genetic elements into any DNA sequence, no formation of
heteroduplex
2. Conservative site-specific recombination: site specific
recombination that requires a short DNA sequence that is the same
on both donor and recipient, involve formation of heteroduplex
Transposon (cont’ next)
• Transposase / Integrase: act on the specific sequence at the end of
transposon and disconnecting it from the flanking sequence and then
inserting it to a new target site
Cut and Paste transposition (DNA-only transposons )
Steps of cut and paste transposition:
1. Binding of transposase subunits to the
terminal inverted repeats
2. Transpososome formation (synaptic complex)
3. Excision of the transposon (contrast to
replicative mechanism)
4. DNA strand transfer
5. Gap repair – DNA polymerase
Replicative transposition (DNA-only transposons)
Steps of Replicative
transposition mechanism:
nick
1. Binding of transposase
protein to transposon
sequence.
2. Transposon DNA is
replicated and a copy is
inserted at a new
chromosomal site,
leaving the original
chromosome intact.
The life cycle of a retrovirus
Retrovirus-like transposition
1. LTR on the two ends of the
element
2. Transcription to generate RNA
copiy
3. RNA template to synthesize DNA
using reverse transcriptase
4. cDNA is recognized by integrase
5. Gap repair
Non-retroviral transposition
-poly-A retrotransposons move by a ‘Reverse Splicing’ mechanism
called target site primed reverse transcription
• A significant fraction of vertebrate chromosomes is made
up of repeated DNA sequences
• In human chromosomes, these repeats are mostly
mutated/truncated versions of a retrotransposon called L1
element (LINE= lone interspersed nuclear element)
• L1 element are mostly immobile
• Translocation result in human disease
eg. Hemophilia – L1 insertion into a gene for blood
clotting factor VIII.
• Mechanism: require a complex of endonuclease and a
reverse transcriptase
Non-retroviral transposition
Generates a ssDNA element
directly linked to target DNA
Processing of ssDNA to
produce dsDNA of L1
Conservative site-specific recombination
- Breaking and joining occur at two special sites, one on each participating
DNA molecules
- enzymes involve can break and rejoin two DNA helix, often reversible,
ie. DNA integration, DNA excision or DNA inversion can occur
- eg. Bacteriophage lambda/ bacterial viruses – mobile DNA element,
moving in and out of host chromosomes
eg. Bacteriophage
eg. Salmonella typhimurium
Inversion of DNA segment changes
the type of flagellum produced
Conservative site-specific recombination
Insertion of a circular bacteriophage
lambda DNA chromosome into
bacteria chromosome:
1. Integrase binds to specific
‘attachment site’ on each
chromosome
2. Cuts and switch the partner
strands
3. Re-seals forming heteroduplex
joint (7nt bp long)
4. Phosphodiester bond breakage
release energy used for strand
joining
5. Intergrase dissociates.
How a conservative site-specific recombination enzyme is used to turn on
a specific gene in a group of cells in a transgenic animal:
(used in mice or Drosophila to study the effect of expressing a gene of interest
in the animal, using Cre recombination enzyme & loxP recognition sites)
Major types of transposable elements
Type
Structural features
Mechanism of movement
Bacterial replicative transposons
Terminal inverted repeats that flank
antibiotic resistance and transposase
genes
Copying of element DNA
accompanying each round of insertion
into a new target site
Bacterial cut and paste
transposons
Terminal inverted repeats that flank
antibiotic resistance and transposase
genes
Excision of DNA from old target site
and insertion into new site
Eukaryotic transposons
Inverted repeats that flank coding
region with introns
Excision of DNA from old target site
and insertion into new site
Viral-like retrotransposons
~250 to 600bp direct terminal repeats
(LTRs) flanking genes for reverse,
transcriptase, integrase and
retroviral-like Gag protein
Transcription into RNA from promoter
in left LTR by RNA polymerase II
followed by reverse transcription and
insertion at target site
Poly-A retrotransposons
3’ A-T rich sequence and 5’ UTR
flank genes encoding an RNAbinding protein and reverse
transcriptase
Transcription into RNA from internal
promoter; target primed reverse
transcription initiated by endonuclease
cleavage
DNA-mediated transposition
RNA-mediated transposition