Download (CH7) DNA Repair

Fundamental
Molecular Biology
Second Edition
Lisabeth A. Allison
Chapter 7
DNA Repair Pathways
Copyright © 2012 John Wiley & Sons, Inc. All rights reserved.
Cover photo: Julie Newdoll/www.brushwithscience.com “Dawn of the
Double Helix”, oil and mixed media on canvas, © 2003
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
BRCA2
Gene
7.1 Introduction
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 MAY???!! 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 XPFERCC1.
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 serinethreonine 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 end-joining
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 XRCC4-DNA
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