DNA damage and repair

•Types of damage

•Direct reversal of damage

•Excision repair in prokaryotes and eukaryotesbase excisionnucleotide 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

•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

Causes of gene mutations

Slipped Strand Mispairing

Backwards slippagecauses insertion

Forwards slippagecauses deletion

Levinson and Gutman, Nature 322: 652-656, 1987

Normal replication

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

APE1

APE1

APE1=apurinic/apyrimidinic endonuclease

DNA pol

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+

umuC-

umuC- + muc+

Error-prone and error-free bypass in humans

•Error-prone repair: DNA Pol (zeta) inserts bases at random to get by pyrimidine dimers

•Relatively error-free bypass: DNA Pol (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 cannot synthesize more DNA after adding the two dAMPs

•Another polymerase continues….

Activities of DNA polymerases alpha and eta on damaged and undamaged bases