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Chapter 7:
DNA Repair Pathways
We totally missed the possible role of
enzymes in DNA repair…. I later came to
realize that DNA is so precious that
probably many distinct repair mechanisms
would exist. Nowadays one could hardly
discuss mutation without considering repair
at the same time.
Francis Crick, Nature (1974), 248:766
7.1 Introduction
• DNA damage poses a continuous threat to
genomic integrity.
• Cells have evolved a range of DNA repair
enzymes and repair polymerases as complex
as the DNA replication apparatus itself.
• DNA replication, repair, and recombination
share many common features.
7.2 Mutations and DNA damage
Spontaneous mutations
• Occur as a result of natural processes in cells.
e.g. DNA replication errors
Induced mutations
• Occur as a result of interaction of DNA with an
outside agent that causes DNA damage.
Mutations are of fundamental importance
• Mutations are important as the major source of
genetic variation that drives evolutionary
change.
• Mutations may have deleterious or (rarely)
advantageous consequences to an organism
or its descendents.
• Mutant organisms are important tools for
molecular biologists in characterizing the
genes involved in cellular processes.
• The simplest type of mutation is a
nucleotide substitution.
• Mutations that alter a single nucleotide
are called point mutations.
Transitions and transversions can lead
to silent, missense,
or nonsense mutations
• Transition mutations replace one pyrimidine
base with another, or one purine base with
another.
• Transversion mutations replace a pyrimidine
with a purine or vice versa.
• In humans, the ratio of transitions to
transversions is approximately 2:1
• A transition or transversion mutation
can be permanently incorporated by
DNA replication.
Whether or not nucleotide substitutions
have a phenotypic effect depends on:
• Do they alter a critical nucleotide in a gene
regulatory region?
• Do they alter a critical nucleotide in the
template for a functional RNA molecule?
• Are they silent, missense, or nonsense
mutations in a protein-coding gene?
Silent mutations
• Mutations that change the nucleotide
sequence without changing the amino
acid sequence are called synonymous
mutations or silent mutations.
Missense mutations
• Nucleotide substitutions in protein-coding
regions that do result in changed amino acids
are called nonsynonymous mutations or
missense mutations.
• May alter the biological properties of the protein.
• Sickle cell anemia is an AT→TA transversion:
– Glutamic acid codon in the -globin gene replaced by
a valine codon
Nonsense mutations
• A nucleotide substitution that creates a new
stop codon is called a nonsense mutation.
• Causes premature chain termination during
protein synthesis.
• Nearly always a nonfunctional product.
Insertions or deletions can cause
frameshift mutations
• If the length of an insertion or deletion is not an
exact multiple of three nucleotides, this results
in a shift in the reading frame of the resulting
mRNA.
• Usually leads to production of a nonfunctional
protein.
Expansion of trinucleotide repeats leads
to genetic instability
• Trinucleotide repeats can adopt triple helix
conformations and unusual DNA secondary
structures that interfere with transcription and
DNA replication.
• Expansion of trinucleotide repeats leads to
certain genetic neurological disorders.
Repeat expansion can occur by two
different mechanisms:
• Unequal crossing over.
• Slippage during DNA replication.
Unequal crossing over
• A trinucleotide repeat in one chromosome
misaligns for recombination during meiosis with
a different copy of the repeat in the
homologous chromosome.
• Recombination increases the number of
repeats on one chromosome, resulting in a
duplication.
• On the other chromosome, there is a deletion.
Slippage during DNA replication
• During DNA replication the DNA melts and
then reanneals incorrectly in the repeated
region, resulting in re-replication of an
additional repeat.
General classes of DNA damage
• Spontaneous damage to DNA can occur
through the action of water in the aqueous
environment of the cell.
• A mutagen is any chemical agent that causes
an increase in the rate of mutation above the
spontaneous background.
Three general classes of DNA damage
• Single base changes
• Structural distortion
• DNA backbone damage
Single base changes
• A single base change or “conversion” affects
the DNA sequence but has only a minor effect
on overall structure.
• Deamination is the most frequent and
important kind of hydrolytic damage.
• Methylated cytosines are “hotspots” for
spontaneous mutation in vertebrate DNA
because deamination of 5-methylcytosine
generates thymine.
• Alkylating agents such as nitrosamines lead to
the formation of O6-methylguanosine.
• This modified base often mispairs with
thymine.
• Can result in a GC→GT→AT point mutation
after DNA replication.
• Oxidizing agents generated by ionizing
radiation and chemicals that generate free
radicals can lead to formation of 8-oxoguanine
(oxoG)
• OxoG can form a Hoogsteen base pair with
adenine.
• Gives rise to a GC→TA transversion.
• One of the most common mutations found in
human cancers.
Structural distortion
• UV radiation induces that formation of a
cyclobutane ring between adjacent thymines,
forming a T-T dimer.
• The T-T dimer distorts the double helix and can
block transcription and replication.
• UV radiation can also induce dimers between
cytosine and thymine.
• Other bulky adducts can be induced by
chemical mutagenesis.
• Structural distortion can be caused by
intercalating agents and base analogs:
– Ethidium bromide has several flat polycyclic rings
that insert between the DNA bases.
– 5-bromouracil, an analog of thymine, can mispair
with guanine.
DNA backbone damage
Formation of abasic sites
• Loss of the nitrogenous base from a
nucleotide.
• Generated spontaneously by the formation of
unstable base adducts.
Double-stranded DNA breaks
• Induced by ionizing radiation and a wide range
of chemical compounds.
• The most severe type of DNA damage.
Cellular responses to DNA damage
• Damage bypass
• Damage reversal
• Damage removal
7.3 Lesion bypass
Translesion synthesis (TLS)
• Specialized low-fidelity, “error-prone” DNA
polymerases transiently replace the replicative
polymerases and copy past damaged DNA.
• Typical error rates range from 10-1 to 10-3 per
base pair.
Error-prone DNA polymerases
• May insert incorrect nucleotides opposite the
lesion: nucleotide substitution
• May skip past and insert correct nucleotides
opposite bases downstream: frameshift
• A trade-off between death and a risk of high
mutation rate.
DNA polymerase eta ()
• Performs translesion synthesis past TT dimers
by inserting AA.
• Has an extra wide active site that can
accommodate two dNTPs instead of one.
• Van der Waals forces and hydrogen-bonding
interactions hold the TT dimer so that the two
thymines can be paired with two adenines.
7.4 Direct reversal of DNA
damage
Reversal of thymine-thymine dimers by
DNA photolyase
• In most organisms, UV radiation damage
to DNA can be directly repaired.
• DNA photolyase uses energy from near
UV to blue light to break the covalent
bonds holding two adjacent pyrimidines
together.
• DNA photolyase has two cofactors:
– A pigment that absorbs UV/blue light
– Fully reduced flavin dinucleotide (FADH-)
• Splitting of the TT dimer is initiated by an
electron transferred from photoexcited
FADH- to the TT dimer already bound to
the enzyme.
• The TT dimer is flipped out of the DNA helix
and brought very close to FADH-.
• An electron is transferred from FADH- and the
dimer is split.
• The electron is then returned to the transiently
formed flavin radical in less than a
nanosecond.
• Photolyases are an ancient and efficient
means of repairing UV-damaged DNA.
• Placental mammals including humans,
however, do not have a photoreactivation
pathway.
Damage reversal by DNA
methyltransferase
• Methyltransferase catalyzes the transfer
of the methyl group on O6-methylguanine
to the sulfhydryl group of a cysteine
residue on the enzyme.
Damage reversal by DNA
methyltransferase
• DNA methyltransferase binds the minor groove
of the DNA.
• The minor groove widens and the DNA bends
by 15° away from the enzyme.
• The O6-methylguanine flips out from the double
helix into the active site.
• A sulfhydryl group of a cysteine in the active
site accepts the methyl group from guanine.
• Does DNA methyltransferase fit the
classic definition of an enzyme?
7.5 Repair of single base changes
and structural distortions by
removal of DNA damage
• Multiple dynamic protein interactions are
involved in all repair processes.
• Ordered hand-off of damaged DNA from
one protein or protein complex to
another.
• DNA repair proteins are modular.
The repair machinery must gain access to
the DNA
• Upon sensing DNA damage, nucleosomes are
disassembled by histone modification and
chromatin remodeling.
• After repair, PCNA recruits chromatin
assembly factors to restore nucleosomes.
Pathways for repair of single base changes
and structural distortion
• Single base changes
– Base excision repair
– Mismatch repair
• Structural distortion
– Nucleotide excision repair
Base excision repair
• The correction of single base changes that are
due to conversion of one base to another.
• Specific DNA glycosylases recognize and
excise the damaged base.
• How do DNA repair proteins find the rare sites
of damage in a vast expanse of undamaged
DNA?
Model for DNA damage recognition by
8-oxoguanine DNA glycosylase 1
(hOGG1)
• A series of “gates” within the hOGG1
enzyme
• hOGG1 first binds nonspecifically to DNA.
• If the enzyme encounters a normal GC base
pair, then:
The G is transiently extruded into a G-specific
pocket and returned to the double helix.
• If the enzyme encounters a oxoG-C base pair,
then:
The oxoG is extruded into the G-specific
pocket and then inserted into a lesion
recognition pocket where it is excised.
Base excision repair pathway in
mammalian cells
1. A DNA glycosylase recognizes and excises
the damaged base.
2. An endonuclease cleaves the phosphodiester
bond either 3′ or 5′ of the abasic site.
3. An endonuclease removes 1-10 nucleotides.
4. DNA polymerase replaces the missing
nucleotides.
5. DNA ligase seals the gap.
Mismatch repair
• The correction of mismatched base pairs which
result from DNA polymerase errors during
replication.
• A large region of DNA including the mismatch
is excised.
• The method of strand discrimination in
mammalian cells is currently unknown.
Hereditary nonpolyposis colorectal
cancer: a defect in mismatch repair
3 to 5% of all colorectal cancers
•
•
•
•
Inherit one inactive mismatch repair allele.
Somatic loss of wild-type allele.
Defective mismatch repair mechanism.
Accumulation of mistakes during DNA
replication.
• Microsatellite instability.
80% lifetime risk of developing colorectal cancer.
Mismatch repair pathway in mammalian
cells:
1. Damage recognition by the MutS/MutL
complex.
• One model proposes that MutS /MutL then
diffuses for several thousand nucleotides either
5′ or 3′.
2. A 5′ or 3′ single-strand break is generated by
EXO1 in association with PCNA and RFC.
3. 5′→3′ or 3′→5′ progessive exonuclease
activity of EXO1 removes the mismatch.
4. 5′→3′ repair synthesis is mediated by DNA
polymerase and associated factors.
5. Ligation of the remaining gap is catalyzed by
DNA ligase I.
Recurrent theme in DNA repair
• Hand-off of damaged DNA from a
complex with nuclease activity to a
complex with polymerase activity to a
complex with ligase activity.
Reconstitution of mismatch repair in an in
vitro system showed that:
• The recognition protein MutS, the
exonuclease EXO1 and DNA polymerase  are
indispensible for repair.
Nucleotide excision repair
• Repair of structural distortion
– e.g. bulges from thymine-thymine dimers
induced by UV irradiation.
• Global genome repair (GGR) pathway: repair
of lesions in the whole genome.
• Transcription coupled repair (TCR) pathway:
repair of lesions in the transcribed strand of
active genes.
Mammalian nucleotide excision repair
pathway
1. Damage recognition by the cooperative
binding of XPC, RPA, XPA, and TFIIH.
•
XPC binds first, followed by the other proteins.
•
TFIIH is a multiprotein complex that also plays
an important role in gene transcription.
2. Unwinding of the duplex DNA is promoted by
the action of XPB and XPD helicases, which
are subunits of the TFIIH complex.
3. The endonuclease XPG makes a 3′ incision,
and a 5′ incision is made by the endonuclease
XPF-ERCC1.
4. The damaged strand is released (24 to 32 nt).
5. Repair synthesis is mediated by DNA
polymerase  or .
6. Ligation of the remaining gap in the DNA
backbone is mediated by DNA ligase I.
Xeroderma pigmentosum and related
disorders: defects in nucleotide
excision repair
Xeroderma pigmentosum
• Autosomal recessive disorder.
• Photosensitivity.
• Greatly increased risk of sunlight-induced skin
cancer.
• Neurological degeneration.
• Defects in nucleotide excision repair or in T-T
dimer translesion synthesis.
Which of the repair pathway components
are defective in xeroderma pigmentosum?
XPA
RPA
XPG
XPC
TFIIH complex (with XPB, XPD helicases)
XPF/ERCC1
PCNA
RFC
DNA polymerase /
DNA ligase I
DNA polymerase 
Xeroderma pigmentosum complementation
groups
• Seven complementation groups (XPA to XPG)
and xeroderma pigmentosum variant (XPV).
• Complementation group: When fibroblast cells
of two different patients with the same defect
are fused in vitro, the DNA damage is not
repaired.
• Nucleotide excision repair is determined by the
uptake of radiolabeled thymidine into DNA.
• If the two patients have different gene defects,
the cells correct each other and the DNA
damage is repaired.
Treating xeroderma pigmentosum
• Early diagnosis and light-protective lifestyle.
• Topical application of DNA repair enzymes.
Two other nucleotide excision repair
deficiency syndromes
• Trichothiodystrophy
• Cockayne syndrome
7.6 Double-strand break repair by
removal of DNA damage
• Double-strand breaks in DNA are induced by
reactive oxygen species, ionizing radiation, and
chemicals the generate reactive oxygen
species (free radicals).
• Repaired by homologous recombination or
nonhomologous end-joining.
Homologous recombination
•
Repairs double-strand breaks by retrieving
genetic information from an undamaged
homologous chromosome.
Nonhomologous end-joining (NHEJ)
•
Rejoins double-strand breaks via direct
ligation of the DNA ends without any
requirement for sequence homology.
• Homologous recombination plays a major role
in double-strand break repair in prokaryotes
and single-cell eukaryotes.
• In mammalian cells, double-strand breaks are
primarily repaired through NHEJ.
• In mammalian cells, the main function of
homologous recombination is to repair doublestrand breaks at the replication fork.
Homologous recombination
Many essential roles in eukaryotic organisms
• Crossing-over during meiosis.
• Transposition.
• Mating-type switching in yeast.
• Antigen-switching in trypanosomes.
• DNA repair.
Model for mammalian DNA double-strand
break repair by homologous
recombination
1.Double-strand break (DSB)
• A DSB is induced by ionizing radiation.
2. End-processing and recognition:
•
Recruitment of MRN (Mre11-Rad50-Nbs1) to
the DSB.
•
The 3′, 5′ exonuclease activity of Mre11
generates 3′ ssDNA tails that are recognized
by Rad52.
3. Strand invasion and DNA synthesis:
•
The 3′ tails invade homologous intact
sequences.
•
Strand exchange generates a hybrid
molecule.
•
Missing sequence information at the DSB is
restored by DNA synthesis.
4.Branch migration
• Processing of the interlinked molecules.
5.Holliday junction resolution and ligation
ATM activation at double-strand break
(DSB) sites
• ATM (ataxia telangiectasia mutated) is a
serine-threonine kinase
• MRN complexes form a bridge between free
DNA ends via Rad50.
• Inactive ATM is recruited to the DSBs through
interaction with Nsb1.
• ATM is activated by phosphorylation.
• ATM phosphorylates proteins involved in DNA
repair and cell cycle control.
• Patients that lack ATM suffer from a syndrome
called ataxia telangiectasia
–
–
–
–
–
Extreme sensitivity to radiation
Increased susceptibility to developing cancer
Immunodeficiency
Premature aging
Neurodegenerative disorders
Holliday junctions
• Early 1960s: Robin Holliday proposed a model
for general recombination based on genetic
data obtained in fungi.
• The model has survived the test of time…
• Heteroduplex DNA: duplex DNA formed during
recombination is composed of single DNA
strands originally derived from different
homologs.
• Holliday junction: an intermediate in which the
two recombining duplexes are joined covalently
by single crossovers.
• The Holliday junction is resolved into two
duplexes by an enzyme complex called
the resolvasome.
• The resolvasome has “resolvase” activity.
E. coli RuvABC complex
• Unfolds the Holliday junction arms.
• Uses ATPase activity to promote branch
migration.
• RuvC cuts the junction symmetrically in a
sequence-specific manner.
• 2004: The human Holliday junction
resolvasome was purified from 50 liters of
HeLa cells passed through 6 chromatographic
steps.
• Rad51C is required for Holliday junction
processing in mammalian cells.
• Rad51C forms a complex with the XRCC3
protein.
Hereditary breast cancer syndromes:
mutations in BRCA1 and BRCA2
• About 5-10% of all cases of breast cancer.
• Mutations in BRCA1 and BRCA2 “tumor
suppressor genes”.
• Lifetime risk for breast (and ovarian) cancer:
BRCA1: 50 to 87%
BRCA2: 15 to 44%
Nonhomologous end-joining
• This double-strand break repair process
can lead to mutation.
• Two broken ends can be ligated together
regardless of whether they came from
the same chromosome.
Nonhomologous end-joining
• Frequently results in insertions or
deletions at the break site.
• Trade-off between repair and otherwise
lethal breaks in the genome.
Model for mammalian DNA double-strand
break repair by nonhomologous endjoining
1.Double-strand break
• Induced by ionizing radiation.
2.End recognition
• Broken ends are recognized by heterodimers
of Ku70/Ku80.
3. End processing:
•
The endonuclease Artemis is activated by the
DNA-dependent protein kinase catalytic
subunit (DNA-PKCS).
•
DNA polymerase (pol)  or pol  fill-in gaps
and extend 3′ or 5′ overhangs.
4. End bridging
•
The ligase complex XRCC4-DNA ligase IV is
recruited to the damaged site and forms a
bridge.
5. Ligation
•
The broken ends are ligated by the XRCC4DNA ligase IV complex.
In vitro assays suggest that there is
flexibility in the order of the three key
enzymatic steps on each strand:
•
Nucleolytic action
•
Polymerization
•
Ligation