Transcript (2) Excision Repair

DNA Repair
Dr Derakhshandeh-Peykar, PhD
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For DNA
• information must be
transmitted intact to daughter
cells
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Accuracy is maintained by:
1- High fidelity in replication
• 3’- exonuclease activity of DNA pol I
• Uracil-DNA N-glycosylase pathway
(corrects mutations from deamination of
cytosine)
cytosine deamination
Uracil methylase Thymine
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Accuracy is maintained by:
2-Mechanisms for correcting genetic
info. in damaged DNA
• e.g due to chemical modifications
• Irradiation changes
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2-Mechanisms for correcting
genetic info. in damaged DNA
1. Direct Repair - Damaged
base undergoes a chemical/UV
reaction Restores original
structure (pro)
• e.g. DNA photolyase - E.coli
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2-Mechanisms for correcting
genetic info. in damaged DNA
2. Mismatch Repair (Synthesis + Repairing)
• MM created by replication errors
• DNA Pol III proof reading
• non-homologous recombination are
recognized and corrected
DNA Pol III
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2-Mechanisms for correcting
genetic info. in damaged DNA
3. Base Excision Repair (Euk/Pro)
• Starts at cleavage of glycosidic bond
(connects base to sugar-phosphate
backbone)
glycosidic bond
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2-Mechanisms for correcting
genetic info. in damaged DNA
4. Nucleotide Excision Repair:
(Prok: 12 bp/Euk: 28bp)
- damaged DNA:
• excised
• replaced with normal DNA
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2-Mechanisms for correcting
genetic info. in damaged DNA
5. Recombinational Repair
- Fills gaps in DNA :
- Newly replicated DNA duplexes
undergo genetic recombination
• Removal of damaged segment
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DNA REPAIR
(1) Photoreactivation
(aka Light Repair)
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DIRECT DNA DAMAGE AND
REPAIR
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DNA damage of a variety of sorts:
A variety of irradiation (ionizing, ultraviolet, etc)
U.V. induced formation of Thymine Dimmer
Blocked replication and gene expression until
repaired
• Prohotoreactivation enzyme
• Photolyase
• Prokaryote
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UV induced formation of
Thymine Dimer
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T
C
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Photoreactivation (Light Repair)
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PHR/PRE gene
codes for photolyase
with cofactor folic acid
binds in dark to T dimer
When light shines on cell
folic acid absorbs the light (photon)
uses the energy to break bond of T dimer
photolyase then falls off DNA
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DNA REPAIR
(2) Excision Repair
(aka Dark Repair)
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Excision Repair (Dark Repair)
• 3 different types of repair mechanisms
• use different enzymes
• (a) AP Repair (Base Excision Repair, BER)
• (b) UV Damage Repair (also called NER nucleotide excision repair)
• (c) Mismatch Repair (MMR)
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(a) AP Repair
(Base Excision Repair, BER)
• Repair of apurinic and apyrimidinic sites on
DNA
• in which base: has been removed
• Base removed by:
– DNA glycosylases
– which remove damaged bases
• ung gene codes for uracil-DNA glycosylase
– recognizes and removes U in DNA
– by cleaving the sugar-nitrogen bond to remove the
base
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AP endonucleases:
• class I nick at 3' side of AP site
• class II nick at 5' side of AP site
• Exonuclease removes short region of
DNA
• DNA Pol I and ligase fill in gap
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(b) UV Damage Repair (also called
NER - nucleotide excision repair)
• It uses different enzymes
• NER removes a large "patch" around the
damage
• Even though there may be only a single
"bad" base to correct, its nucleotide is
removed along with many other adjacent
nucleotides
• NER: UV
• BER: Chemicals/Agents
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NER (UV Damage Repair)
• Nuclease:
• can detect T dimer
• nicks DNA strand on 5' end of dimer
(composed of subunits coded by uvrA,
uvrB and uvrC genes)
• UvrA protein and ATP bind to DNA at the
distortion
• UvrB binds to the UvrA-DNA complex and
increases specificity of UvrA-ATP complex
for irradiated DNA
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• UvrC nicks DNA 8 bases upstream and 4
or 5 bases downstream of dimer
• UvrD (DNA helicase II; same as DnaB)
separates strands to release 12-bp
segment
• DNA polymerase I now fills in gap in 5'>3'
direction
• ligase seals
• polA - encodes DNA pol I
– mutant was viable retained normal 5'>3'
exo activity
– only 2% of polymerase activity
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Excision Repair of Thymine dimers
by UvrABC exinuclease of E.coli
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(c) Mismatch Repair (MMR)
• Accounts for 99% of all repairs
• Mismatch from replication
• behind replication fork
• Two ways to correct mistakes made during
replication:
1) 3'>5' exonuclease - proofreading
2) Mismatch repair
• mutL
• mutS
• mutH
• and mutU (same UvrD) gene products involved (mut
for mutator because if gene is mutated, cell has
increased levels of spontaneous mutations)
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How does system recognize progeny
strand rather than parent strand as one
with mismatch?
• Because of methylation
• DNA methylase (coded for by dam [DNA adenine
methylase] locus)
• methylates 5'-GATC-3' sequence in DNA at A residue
• Mismatch from replication recognized by mutL and mutS
gene products
• mutH gene product nicks DNA strand (progeny strand)
on either side of mismatch
• DNA helicase II from mutU gene (also called uvrD gene)
• unwinds DNA duplex and releases nicked region
• Gap filled in by DNA Pol I and ligase
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DNA REPAIR
• (1) Photoreactivation (aka Light Repair)
• (2) Excision Repair (aka Dark Repair)
• (3) Postreplicative (Recombinational)
Translesion Bypass Repair
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DNA REPAIR
(3) Postreplicative
(Recombinational)
Translesion Bypass
Repair
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Translesion bypass
• Recent research in bacteria, yeast and
mammalian cells
• most of the mutations arise by transletion
bypass
• when highly processive semiconservative
DNA replication is arrested at DNA lesions
• translesion synthesis (TLS) polymerases
allows them to insert nucleotides opposite
DNA lesions, but at the expense of frequent
misincorporations
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Postreplicative
(Recombinational) Repair
Translesion Bypass
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SOS response
• If T dimer is not repaired
• DNA Pol III can't make complementary strand
during replication
• leaves large gap (800 bases)
• Gap may be repaired by enzymes in recombination
system
• RecA - coats ssDNA
• it also acts as autocatalysis of LexA repressor
• recA mutants - very UV-sensitive
• Now have sister-strand exchange - a type of
recombination:
Translesion bypass
• Postreplicative repair is part of SOS response
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SOS Response
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• LexA normally represses about 18 genes
sulA and sulB, activated by SOS system
• inhibit cell division in order to increase
amount of time cell has to repair damage
before replication
• Each gene has SOS box in promoter
• LexA binds SOS box to repress expression
• RecA : LexA catalyses its own breakdown
when RecA is stimulated by ssDNA
• due to RecA binding ssDNA in lesions
• could then bind to DNA Pol III complex
passing through this area of the DNA
• RecA no longer catalyzes cleavage of LexA
(which is still being made)
• so uncleaved LexA accumulates and turns
the SOS system off
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Why are DNA Repair Systems
Necessary?
• E.coli
• Xeroderma Pigmentosum (XP)
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E.coli
• repairing thymine dimers
• important to bacteria
• an E. coli strain that is:
– phr (no photoreactivation)
– recA (no translesion by pass or SOS)
– uvrA (no excision repair) is killed by a single
thymine dimer
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Xeroderma Pigmentosum (XP)
• XP is a rare inherited disease of humans
• predisposes the patient to:
– pigmented lesions on areas of the skin
exposed to the sun
– an elevated incidence of skin cancer
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Xeroderma Pigmentosum
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• It turns out that XP can be caused by
mutations in any one of several genes
• all of which have roles to play in NER
• Some of them:
• XPA, which encodes a protein that binds the
damaged site
• assemble the other proteins needed for
NER
• XPB and XPD, which are part of TFIIH
(Helicase)
• XPF, which cuts the backbone on the 5' side
of the damage
• XPG, which cuts the backbone on the 3'
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side
Some mutations in XPB and XPD also
produce signs of premature aging
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Transcription-Coupled repair
• Protein: ERCC6
recognizes RNApol
Mutation in gene:
Cokayne Syndrom:
MR
Nerve disease
Sensibility to sun
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