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DNA damage and repair
•Types of damage
•Direct reversal of damage
•Excision repair in prokaryotes and eukaryotes
base excision
nucleotide excision
•Nonhomologous end-joining in eukaryotes
•Mismatch repair
•Recombination repair, error-prone bypass and error-free
bypass
DNA damage vs. mutation
•DNA damage refers to a chemical alteration of the DNA
(e.g. G-C bp to methyl-G-C is DNA damage)
•Mutation refers to a change in a base-pair (e.g. G-C bp to
A-T bp is a mutation)
•Problems arise when DNA damage is converted to
mutation
Most inherited syndromes in humans are due
to mutations
•Whether a syndrome occurs or not depends on where the
mutation occurs and how a protein altered by the mutation
is affected
•Mutation may cause a protein:
-to be non-functional
-to have an altered function
-to act less efficiently
-to function as the wild type
Causes of gene mutations
•Spontaneous
-errors by DNA Polymerases during replication
can lead to base changes
-slipped strand mispairing can occur at
homopolymeric runs (mono, di, or trinucleotide
repeats)
-chemical modification of bases followed by
mispairing
•Exposure to mutagens
-ionizing radiation
-UV radiation
Slipped Strand Mispairing
Normal replication
Backwards slippage
causes insertion
Forwards slippage
causes deletion
Levinson and Gutman, Nature 322: 652-656, 1987
Spontaneous deamination of C gives rise to U,
and spontaenous deamination of 5-methylC
gives rise to T.
Sponaneous deamination of A gives rise to
hypoxanthine which can base-pair with C
(but with 2 H-bonds instead of 3).
Cytosine
Hypoxanthine
deamination
Electron rich centers in DNA susceptible to
electrophilic attack
Alkyation highly mutagenic
(forms a “noncoding
base”)
Alkyation “harmless”
Alklyation of guanine by EMS leads to base-pairing
with thymine
Pyrimidine dimers
Model for
Photoreactivation
Mechanism of O6-methylguanine methyl
transferase activity
O6-methylguanine methyl transferase is a “suicide enzyme.”
It is irreversibly inactivated after activity.
Base excision repair in E. coli
The human BER pathway
DNA pol b
APE1
DNA pol b
APE1
APE1=apurinic/apyrimidinic endonuclease
Nucleotide excision repair in E. coli
Human global genome NER
Xeroderma pigementosum
Model for nonhomologous end-joining
Ku heterodimer
(Ku70 and Ku80)
DNA-PKcs
Mismatch Repair in Prokaryotes
•Occurs when DNA Polymerase puts in the wrong nucleotide
during replication and the proofreading activity does not
correct it.
•Repair should occur on the correct strand, the newly
synthesized strand.
•E. coli methylates A of GATC sequence.
•There is a time lapse before newly synthesized strand is
methylated.
•Repair occurs on unmethylated (newly synthesized) strand
during this window of time.
Mismatch repair in E. coli
Mismatch Repair in Eukaryotes
•Eukaryotes are also capable of mismatch repair.
•Less well understood than prokaryotes.
•Homologues of mutS and mutL genes exist so enzymes involved in
eukaryotic mismatch repair likely to be similar to prokaryotic enzymes.
•BUT, no homologue of MutH (protein that recognizes
unmethylated newly synthesized strand) so recognition of newly
synthesized strand does not appear to occur via a methylation signal.
•Failure of mismatch repair in humans can lead to hereditary
nonpolyposis colon cancer (HNPCC)
Recombination repair in E. coli
Error prone SOS bypass in E. coli
Reversion of ochre his- mutation in E. coli
umuC- + muc+
umuC+
umuC-
Error-prone and error-free bypass in humans
•Error-prone repair: DNA Pol z (zeta) inserts bases at random to get by
pyrimidine dimers
•Relatively error-free bypass: DNA Pol h (eta) inserts two dAMPs
across from pyrimidine dimers which are often (but not always) T-T
dimers
•The two A’s cannot base pair though because the two T’s are still
joined together
•DNA Pol h cannot synthesize more DNA after adding the two dAMPs
•Another polymerase continues….
Activities of DNA polymerases alpha and eta
on damaged and undamaged bases