DNA Repair

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DNA Repair
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Learning Objectives
Describe the types of damage that can occur in
DNA. Define exonuclease and endonuclease. Describe
methyl-directed mismatch repair in E.
coli. Describe base-excision repair. Describe
nucleotide-excision repair. Describe
recombinational repair. Describe pol eta and its
role in bypass repair.
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DNA damage
Deamination of bases
Several nucleotide bases undergo spontaneous loss
of their exocyclic amino groups (deamination).
Deamination of cytosine to uracil in DNA occurs
approximately 100 times per day in a mammalian
cell. Deamination of adenine and guanine is about
100 times slower.
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N-glycosidic bond
Under normal physiological conditions, the
hydrolysis of the N-glycosidic bond in DNA
results in the loss of as many as 10,000 purine
bases per mammalian cell in a 24 hour period.
Pyrimidine bases are more stable. The location in
DNA where a purine base has been lost is called
an apurinic site (AP site).
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UV induced thymine dimers and 6-4 photoproducts
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The liver can convert non-carcinogens to
carcinogens via detoxification reactions
(oxidations).
Many plants that are common in the human diet
contain carcinogens. Aflotoxin B is produced by
molds that grow on peanuts and corn. Aflotoxins
are potent mutagenic compounds.
X-rays produce extremely reactive hydroxyl free
radicals.
The burning of plant material (including fossil
fuels) generates toxic and/or mutagenic compounds.
Depletion of the ozone layer results in a higher
level of UV exposure.
Oxidative damage from oxygen and reactive oxygen
species produced by normal metabolic processes.
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Enzymes that cut nucleic acids
Endonuclease an enzyme that hydrolyzes the
interior phosphodiester bond of a nucleic acid
that is, it acts at points other than the
terminal bond. Exonuclease an enzyme that
hydrolyzes only the terminal phosphodiester bonds
of a nucleic acid.
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All cells have multiple DNA repair mechanisms
DNA is the only molecule that is repaired all
others are replaced.
DNA repair is possible largely because the DNA
molecule consists of two complimentary strands.
DNA damage in one strand can be repaired by using
the undamaged strand as a template.
Many DNA repair processes use an extraordinary
amount of energy. When the integrity of the
genetic information is at stake, the amount of
energy invested in a repair process seems to be
almost irrelevant.
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DNA Repair Mechanisms
Mismatch repair in E. coli, requires at least 12
different proteins. Base excision repair DNA
glycosylases (creates an apurinic or apyrimidinic
sitean AP site). This repair requires that the
entire nucleotide be excised, and the correct one
put into place. Nucleotide excision repair
(NER) Recombinational DNA repair Bypass repair
(bypass synthesis)
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Methyl-directed mismatch repair in E. coli
Mismatching of base pairs occurs during
replication. If not corrected by the
proofreading capabilities of DNA polymerase,
these can result in a mutation during the next
round of DNA synthesis.
To repair these lesions, the repair system must
be able to distinguish between the damaged and
the undamaged strand of DNA. Strand
discrimination is based on the action of Dam
methylase which methylates DNA at the N6 position
of all adenines within (5)GATC sequences.
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For a short period following replication, the
template strand is methylated and the new strand
is not.
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Hemimethylated DNA
Dam methylase
After a few minutes, the new strand is
methylated, and the two strands cannot be
distinguished.
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Enzymes/proteins required for methyl-directed
mismatch repair in E. coli
Dam methylase MutH, MutL, MutS proteins DNA
helicase II SSB DNA polymerase III Exonuclease
I Exonuclease VII RecJ nuclease Exonuclease X DNA
ligase
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MutL forms a complex with MutS and binds to the
mismatched base pair region.
MutH binds at methylated GATC sites.
DNA is threaded through the MutL-MutS complex
until it reaches a MutH bound at a methylated
GATC site.
MutH then cleaves the unmethylated strand on the
5 side of the GATC sequence. This marks the
strand for repair.
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Base-excision repair
DNA glycosylases enzymes that recognize certain
DNA lesions and remove the affected base by
cleaving the N-glycosidic bond.
DNA glycosylases remove the products of cytosine
and adenine deamination, leaving an apurinic or
apyrimidinic site (an AP site).
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Base-excision repair
A DNA glycosylase recognizes a damaged base and
cleaves between the base and the deoxyribose in
the backbone.
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An AP endonuclease cleaves the phosphodiester
backbone near the AP site.
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DNA polymerase I initiates repair from the 3 OH
at the nick removing a portion of the damaged
strand with 5 3 exonuclease activity.
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The nick remaining after DNA polymerase has
dissociated from the repair site is sealed by DNA
ligase.
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Nucleotide-excision repair
DNA lesions that cause large distortions in the
DNA helix are generally repaired by the
nucleotide-excision system. This includes
pyrimidine dimers (thymine dimers).
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Nucleotide excision repair (NER)
NER involves the following steps Damage
recognition Binding of a multimeric protein
complex at damaged site Double incision on both
the 3 and 5 side of damage Removal of
damage-containing oligonucleotide Filling in the
gap with a DNA polymerase Ligation
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Direct repair
Direct photoreactivation of cyclobutane
pyrimidine dimers promoted by DNA photolyase.
Photolyases uses the energy from absorbed light
to reverse the damage to DNA.
Chromophores FADH- and folate (in E. coli and
yeast)
This repair system is present in lower
eukaryotes, plants, and bacteria, but not in
mammals.
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Xeroderma Pigmentosum
XP is an multigenic, autosomal recessive
condition that results in a sever sensitivity to
the sun (ultraviolet radiation). This results in
progressive degeneration of sun-exposed regions
of the skin and eyes. Neurological degeneration
occurs in a significant number of patients.
Cancer incidence (especially skin cancer) for
those individuals under 20 years of age is 2000
times that seen in the general population.
Cells from the majority of XP patients cannot
carry out nucleotide excision repair (NER) in
which nucleotides damaged by UV light are
removed. This results in the accumulation of
errors as the damaged DNA tries to replicate.
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XP genes
XPA encodes a zinc finger protein involved in
photoproduct recognition. XPB encodes a 3-5
helicase which may be involved in unwinding DNA
5-ward of a damaged site. XPC encodes a
single-stranded DNA binding protein essential fro
repair of the nontranscribed regions of the
genome. XPD encodes a 5-3 helicase which is a
component of transcription factor TFIIH. XPE
not fully characterized. XPF encodes a
structure-specific endonuclease that cuts DNA on
the 5 side of a damaged site. XPG a nuclease
that cuts on the 3 side of DNA damage also
involved in repair of oxidative damage.
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XP-V (variant) about 25 of clinically diagnosed
XP patients do not have NER defects and their
cells are barely UV sensitive. XP-V patients are
still highly susceptible to UV-induced skin
cancers. XP-V cells cannot accurately replicate
past UV-induced TT dimers on the leading
strand. A bypass DNA polymerase, Pol eta, is
defective in XP-V cells. This polymerase can
correctly insert adenines opposite UV-induced TT
dimers. Another member of this new class of
polymerases, Pol zeta, is an error-prone bypass
polymerase that frequently inserts incorrect
bases opposite pyrimidine dimers. This enzyme is
functional in XP-V cells.
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Model for recombinational repair of DNA
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Blooms Syndrome (BS)
An autosomal recessive genetic disorder with a
range of symptoms small body size, sun-sensitive
facial reddening, sub-fertility,
immunodeficiency, and a predisposition to the
full range of human cancers.
Werners Syndrome (WS)
This is associated at an early age with some, but
not all, of the features of the normal aging
process, including atherosclerosis, osteoporosis,
early graying and loss of hair, and loss of skin
elasticity. These individuals are also cancer
prone, but not to the extent as seen in BS
individuals.
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BLM and WRN, the products of the Blooms and
Werners syndrome genes, are RecQ family of DNA
helicases. (E. coli RecQ helicase plays a vital
role in recombinational repair of DNA).
Both BLM and WRN are atypical helicases that are
highly DNA structure-specific and have similar
substrate specificities.
WRN also exhibits 3-exonuclease activity in
contrast to BLM.