DNA Repair Flashcards
different categories of DNA damage?
replication errors
spontaneous damage
environmental damage
what do mutations lead to?
cancer & heritable disease
restored DNA
senescence/apoptosis
how is DNA damage fixed?
direct reversal
base excision repair
nucleotide excision repair
mismatch repair
how many mismatches per cell division?
100 mismatches per cell division
how many lesions per cell per day?
10^5
lesion definition?
modification to DNA
mutation definition?
misincorporation of nucleotides during replication
how does DNA repair affect no of lesions per cell per day?
Reduces 1,000 fold
transition mutations?
A G (purines) C T (pyrimidines)
transversion mutations?
A C or T
G C or T
replication errors?
mismatches
tautomerisation
strand slippage
intercalation
spontaneous and environmental damage?
hydrolysis (depurination/deamination) oxidation alkylation bulkyl adduct formation radiation
error rate of typical DNA polymerase?
1 in 10^5 nucleotides
1 in 10^7 after DNA repair
what are the wobble pairs?
A-C especially in lower pHs, maybe in nucleus
G-T
are the wobble pairs isomorphic?
no, they don’t overlay. Allows some enzymes to recognise that they aren’t the correct base pair
what is tautomerisation?
alternate hydrogen positioning.
usual keto form of T can be in the alternative enol form, can allow it to pair with G
imino A can also pair with C
what can enol T pair with?
G
what can imino A pair with?
C
what is strand slippage?
newly synthesised strand can slip out slightly and cause an extra nucleotide to be incorporated
or for deletion - template strand slips
where is strand slippage prevalent?
in trinucleotide repeats and Huntington’s
what is intercalation?
causes insertions.
intercalation is exhibited by flat/aromatic compounds which can sit between base pairs.
disrupt the replication machinery and can lead to insertion mutations (e.g. frameshift)
example of intercalation substance?
ethidium bromide found in dyes and cigarette smoke
what is an insertion due to transposons?
elements which come from viruses can catalyse their removal and insertion into different regions of genes
what is a transposon?
cut and paste
what is a retrotransposon?
copy and paste
what can chemical modifications lead to?
mismatches structural alterations to double helix single/double stranded breaks blockage/stalling of machinery influence gene expression by altering chromatin architecture
what percentage of genetic mutations are caused by simple replication errors?
66%
what is depurination?
spontaneous hydrolysiss of the glycosidic bond between a purine and the formation of an ABASIC site
20 times more likely to happen with purines
increased susceptibility to strand breaks
what is deamination?
converts C to U by hydrolysis
adenine –> hypoxanthine
guanine –> xanthine
5-methylcytosine –> T
why does DNA contain T, but RNA contains U?
to detect cytosine deamination
what is base oxidation by reactive oxygen species?
guanine can often be attacked at 8 position and add oxygen.
leads to bond formation with A
what is base oxidation by reactive nitrogen species?
adenine –> hypoxanthine which pairs with C
what is base alkylation?
transfer of methyl, ethyl or alkyl groups to nitrogen or oxygen of base
often used in chemotherapy
G pairs with T
forms 3-methyl A
what does 3-methyl A do?
blocks polymerase from processing
what is base adduct formation?
modifies N7 of guanine and causes GC to TA transversions
example of base adduct chemical?
alfatoxin B
what makes aflatoxin B reactive?
metabolism by cytochrome P450 in liver
what is base dimerisation?
caused by UV exposure and causes dimerisation of pyrimidines next to each other
Cs and Ts
really distort the DNA so easily fixed
Ames test?
examines the mutagenic potential of a compound
contains defective histidine gene but requires histidine to live
mix with mutagen
frameshift mutations will reactivate histidine gene and colonies will grow
mechanisms for repairing replication errors and DNA damage?
direct reversal base excision repair nucleotide excision repair mismatch repair double strand break repair translesion synthesis
direct reversal?
acts directly on modification (alkylation/UV)
human example
removes a methyl group using MGMT. Irreversible reaction once methyl is transferred (suicidal enzyme).
Methylated form of MGMTis a transcription activator, upregulates synthesis of other repair proteins
base excision repair?
removes specific small non helix distorting base lesions. (deamination/oxidation/alkylation)
removes base.
DNA glycosylase generates an abasic site. AP endonuclease leaves a hydroxyl group.
ligase/DNA pol I seal the gap
e.g. uracil DNA glycosylase
MutM?
removes 8-oxo-G from the GC lesion
MutY?
removes A opposite 8-oxo-G following DNA replication
MutT?
breaks down 8-oxo-dGMP and ppi before DNA synthesis
nucleotide excision repair?
cleaves backbone either side, removes oligonucleotide (bulkyl, helix distorting lesions)
endonuclease/helicase then DNA poly I/ligase
UvrABCD recognise and remove pyrimidine dimers
UvrA recognises lesion
UvrB opens and binds DNA
UvrC creates incisions either side of lesion
UvrD excises the oligonucleotide
can see a big difference in the DNA surface when there is a TT dimer
difference in nucleotide excision repair in humans?
single stranded segment is longer
can get recruited to an RNA polymerase which is stalled at the site of a lesion
mismatch repair?
error reducing process of 1000 fold
repairs main GT, GG, AC and CC mismatches
removes a long stretch of nucleotides
MutSLH recognises distortion. straight after replication, daughter strand isn’t methylated yet so this means it can find the mismatch and replace the unmethylated DNA
MutH nicks the daughter strand, MutS recognises the distortion. MutL is the communicator between the two
exonuclease, UvrD (helicase) and DNA polymerase
double strand break repair?
before the two sister chromosomes have been generated, NHEJ protects and processes the broken ends and joins them together but usually results in small deletions and is mutagenic
translesion polymerases?
non processive, template dependent polymerases that synthesis acrss a blockage, introduce nucleotides independent of base pairing
some enzymes fix certain lesions - open active site so allows any nucleotide to be added
p53 protein?
transcription factor that regulates the expression of genes involved in growth arrest, DNA repair and apoptosis
50% of cancers have a defective p53
Xeroderma Pigmentosum?
Increased sensitivity to UV light
1000 fold increased risk of skin cancer
