L36 DNA Damage and Repair Flashcards
DNA Damage Categories
- spontaneous (endogenous)
- environment (exogenous)
spontaneous (endogenous)
- arise during DNA rep, division and repair
- result from alteration in the chemistry of DNA bases (turomeric shifts, deamination of bases; depuination and depyrimidination)
environment (exogenous)
- exposure to chemical mutagens (e.g. alkylating agens, polycyclic aromatic hydrocarbons, aflatoxis)
- exposure to physical agent mutagens (e.g. UV or ionising radiation)
Polymerase (spontaneous (endogenous) DNA damage)
- normally polymerase can move backward and correct itself if it copies incorrectly
- ‘proofreading’ ability
- D400A mutation (in proofreading domain of DNA polymerase) in mice showed poor tumor progression
Mismatch repair (spontaneous (endogenous) DNA damage)
- usually goes back and repairs mistakes from polymerase
DNA replication error frequency
DNA polymerases have an incorporation error frequency of 1 in 1000000 copied nucleotides
* 3’ to 5’ proofreading by polymerases reduces this to 1 in 100000000 copied nucleotides
* The mismatch repair system reduces this to
1 in 10000000000 copied nucleotides
DNA strand breaks (spontaneous (endogenous) DNA damage)
- during replication the DNA is vulterable to breakage when the replication fork is made
estimated 10 strand breaks are formed per cell during S-phase - failure to repair can lead to TRANSLOCATION AND CHROMOSOMAL BREAKS
Tautomeric shift (spontaneous (endogenous) DNA damage)
When there is alteration to the base pairing
- the hydrogen bonds change
- e.g. Keto (common) and Enol (rare) form OR amine (common) and imine (rare) form
- the other form can pair with something else
Deamination of DNA bases (spontaneous (endogenous) DNA damage)
- losing amine entities
- exocyclic amine groups that aren’t part of the carbon ring are lost
- when this happens, it can result in a different base forming e.g. (cytosine to uracil or adenine to hypoxanthine or guanine to xanthine)
- it can lead to DNA RECOGNIZING IT AS SOMETHING DIFFERENT (Transition mutation)
DNA Alkylatiohn (Environmental DNA Damage)
- the addition of a methyl or ethyl group to the DNA
- The guanine N7 and adenine N3 are the major sites
Preferred sites of methylation and why?
guanine N7 and adenine N3
- because they are the most electro negative sites in the molecule
monofunctional alkylation
- chemical adds 1 methyl/ethyl group
- only interacts with 1 site
- 7 alkylguanine is the major product
(almost HARMLESS) - ## BUT…. 3-alkyadendine (formed less frequently) is very toxic
bifunctional alkylating agents
- can add two methyl/ethyl groups
- interacts with two nucleophilic sites
- could produce CROSSLINKS
- can be interstrand, intrastrand, or DNA protein
- the crosslinks = a locked strand which BLOCKS THE PATH OF THE POLYMERASE so DNA can’t replicate
- highly toxic becuase nothing can happen
3-alkyadendine
- monofunctional alkylation
-DNA minor groove, blocks progression of DNA polyer - more rare
- major toxic alkylation
7 alkylguanine
the major product in monofunctional alkylation
- b/c in the major groove, not much change in structure or pairing
- less chance of mutation happening (almost HARMLESS)
O-6 alkylguanine
- formed even less frequently than N7-alkylguanine and N3-alkyladedine
- base is locked in the enol tautomeric form
- can base pair with either C or T
- can result in a G to A transition muation which is critical in carcinogensis
Why is O-6 alkylguanine carcinogenic?
- it can result in a G to A transition muation which is critical in carcinogensis
metabolism of carcinogens
- cytochrome P-450 that is in liver, enzyme that mainly digests
- compoound is converted to a less toxic form and more easily removable
- SOMETIMES a harmLESS may be converted to a harmFULL molecule
- enzymes create a very reactive compound
enzyme in liver that digests carcinogens
cytochrome P-450
alfatoxin B1
- normally not carcinogenic
- but P-450 in the liver can convert it to a carcinogenic form that reacts with DNA and alkylates them
nitrosamines
- found in tobacco
- linked to adenocarcinoma of ht elung
- liver changes them to be highly reactive
- NNN ad NNK are extensively metabolized and when reactive, interact wiht DNA
China study showing ___ is a mutagen
- area of china had fungal toxin alfatoxiin B1 made by moulds that grow on peanuts and grains stored improperly
UV radiation
- creates covalent crosslinks between adjacent pyrimidine bases in DNA
- cyclobutane pyrimidine dimers are the major photoproductws
- more than 60% are TT (thymine dimes), 30% are CT dimers and the rest are CC
- structures are relatively stable and persist unless they are recognized and repaired (they aren’t easily removed)
ionising radiation
- can be direct or indirect (by creation of free radicats)
- also interact with DNA and cause alkylazing agents
DNA damage from ionising radiaiton
- DNA damage (direct or indirect)
- DNA single and double strand breaks
- double strand breaks are cytotoxic and difficult to repair
oxidation of bases in DNA
- ROS produced by ionising radiaiton = oxidation of DNA bases
- frequent oxidation reaction
involves deoxyguaninosine which is
oxidized to 8-oxo-deoxyguanosine (8
-oxo-dG). - 8-oxo-dG can mispair with
deoxyadenosine, which can lead to a
G to T transversion.
Methods of DNA damage repair
- Direct reverasal of damage
- exision of damage
- double strand break repair
- damage tolerance (acts like nothing is wrong)
- cell cycle arrest or cell death
PHYTOLYASES - Direct reversal of DNA damage (Methods of DNA damage repair)
- e.g. 1 removal of pyridine dimers by PHOTOREACTIVATION
- PHYTOLYASES are enxymes that repaire UV damage
- requrie light, more present in plants/animals and not present in humans
O6-methylguanine methyltransferase- Direct reversal of DNA damage (Methods of DNA damage repair)
- repaires O6-methylguanine
- methyle group transferred from guanine to a cysteine group int he active site of the enzyme
- BUT A SUICIDE ENZYME- SO ONLY OWORKS ONCE
MISMATCH REPAIR (excision of DNA damage)
Can repair:
- basebase mismatches e.g. G:T
- one base insert/delete
- 1 base insertion/deletion loops
- recombination intermediates
MUTS and MUTL collaborate to initiate repair of mismatched DNA
MUT-S and MUT-L
- heterodimers
- repair mismatched DNA
- collaborate together (for each situation a set of 1 S and 1L work together)
MUT-S and MUT-L
- heterodimers
- repair mismatched DNA
- collaborate together (for each situation a set of 1 S and 1L work together)
Base excision repair (excision of DNA damage repair mechanisms)
DNA glycosylases initate it by recognizing an abnormal base
- they cleave its bond to deooxyribose
- each DNA glycosylase is specialized to recognize a unique abnormal base
Uracil DNA-glycosylase
- base excision repair mechanism
- recognized uracil and removed
- base-free site is excised by an apurinic/apyriminic endonuclease (APE)
- gab is filled by a DNA polymerase and sealed by a DNA ligase
Poly-(ADP ribose)polymerase-1 (PARP1)
- DNA repair enzymes involved in base excision
repair are recruited to single strand breaks by
the action of PARP1. - It binds to the breaks and attaches multiple
ADP-ribose units to itself and to other proteins. - The ADP-ribose chains act as docking sites for
the repair enzymes. - TARGETABLE ENZYME IN TREATMENT
nucleotide excision repair
- In contrast to base excision repair, nucleotide excision
repair largely repairs lesions created by exogenous agents. - It repairs bulky, helix-distorting alterations.
- Rather than removing a single base, it removes damage-
containing oligonucleotides from DNA. - It is highly conserved and has a broad specificity. * It involves the product of over thirty genes.
- Transcription factor TFIIH is an essential component.
TFIIH
- transcription factor
- important in nucelotide excision repair
subtypes of nucleotide excision repair
- global genomic repair (GGR)
- Transcription - coupled repair (TCR)
Global Genomic Repair (GGR)
- type of nucleotide excision repair
- repairs ALL regions of the genome
- requires XPC
- defective in p53 mutant cells
Transcription - coupled repair (TCR)
- type of nucleotide exiciion repair
- only repairs template strands during transcription
- requres CSA and CSP and all other nucleotide exision repair proteins except XPC
Homology-directed repair (HR)
(Repair of DNA double strand break)
- Occurs during late S and G2
phases of cell cycle
– Requires undamaged sister
chromatid
– Important components are
proteins RAD51, BRCA1 and
BRCA2
– The process is error free
Nonhomologous end joining (NHEJ)
(Repair of DNA double strand break)
- Used when a sister chromatid is
not available e.g. in G1
– Has a normal function in V,D,J
gene rearrangement
– Important components are
proteins KU70, KU80 and DNA-
PK
– The process is error-prone
Tolerance of DNA damage
- As a last resort cells have distinct
DNA polymerases that can bypass
some types of DNA damage in a
process called translesion
synthesis (TLS). - This process is highly error-prone
due to the high incidence of
misincorporated bases
The role of p53
DNA damage can cause a rapid
increase in p53 levels.
* P53 protein undergoes post-
translational modifications and
induces a number of responses. * This can include cell cycle arrest
which can allow time for DNA
repair, and mobilisation of DNA
repair proteins.
* In certain circumstances, it can
also trigger apoptosis.
