DNA Repair: Damage Flashcards
Genetic Stability
For survival, organisms not only need accurate mechanisms for replicating DNA but also mechanisms for
repairing spontaneously occurring damage in DNA
• DNA damage can be caused by heat, metabolic accidents, radiation, exposure to the environment,
different substances
• Fewer than one in 1000 accidental base changes results in permanent mutation thanks to DNA repair
DNA repair is critical
- Range of coding genes involved in repair
- Inactivation of DNA repair genes causes an increased rate of mutation
- Many of these were originally identified in bacteria
- Many serious human diseases are linked to decreased DNA repair
Human syndromes linked to defective DNA repair
Eg. Cancers, UV sensitivity, leukaemia, growth and development
If errors not fixed during replication:
mutations arise
Errors such as:
• Tautomeric bases
• Mismatch
Mechanisms to prevent replication errors
- Proofreading polymerase (fixing majority of errors) 3’->5’
- Errors occur usually in 1:100,000 to 1:1,000,000 bases
- With proofreading: 1:100,000,000 bases
- Mismatch repair system
- Identifies errors in the secondary structure
- Mismatch repair enzymes recognize this and remove/replace the nucleotide
proofreading polymerase
Proofreading polymerase (fixes majority of errors: up to 99%)
• Incorrect base paired, elongation pauses
• 3’-5’ exonuclease activity of the polymerase removes several bases, including the incorrect one
• Replication resumes 5’-3’
• Occurs during replication (S phase)
Mismatch repair system
- Identifies errors in the secondary structure e.g. tautomeric bases
- Mismatch repair enzymes recognize this and bind to the base (MutS)
- MutL scan DNA to find a nick
- Region between mismatched base and nick excised by exonucleases
- DNA polymerase fills the gap and DNA backbone sealed via DNA ligase
- Occurs mostly in S phase of the cell cycle, follows behind replication
Spontaneous DNA damage
DNA susceptible to mutation if left unrepaired: spontaneous alterations that require repair
• Sites on each nucleotide known to be modified by spontaneous:
• Oxidative damage (red arrows): METABOLIC guanine is more susceptible
• Hydrolytic attack (blue): CLEAVES chemical bonds in DNA- results in removal of a base
• Uncontrolled methylation: (green): ALKYLATION of bases - change the base paring
Spontaneous DNA damage by hydrolysis
Depurination = spontaneous loss of purine bases (adenine and guanine)
by hydrolysis
Deamination = spontaneous conversion of cytosine to uracil by hydrolysis
Hydrolytic Damage: Depurination
loss of a purine from the sugar and phosphate backbone
Depurination leads to loss of a nucleotide pair. When replication machinery encounters missing purine on template, it skips to next nucleotide resulting in a deletion: frameshift
Hydrolytic Damage: Deamination
Deaminated cytosine becomes uracil and mutation propagated as uracil pairs with adenine: base substitution
Alkylation Damage: Methylated Guanine
Results in an altered base that doesn’t follow base pairing rules
• Alteration to the base
– Methyl group attached to O
• Methyl Guanine pairs with thymine, not cytosine
– Base substitution
Induced DNA damage: UV Irradiation
Covalent linkage between two adjacent pyrimidine bases
• Caused by UVB radiation from the sun
• Thymine dimers: covalent linkages on the C-C bonds form lesions
• Can occur between any two neighbouring pyrimidine bases
– (T or C)
Covalent linkage between two adjacent pyrimidine bases
- UV irradiation leads to:
- Sunburn
- ↑melanin production
- If left unrepaired:
- can lead to melanoma (cancer)
DNA double helix can be repaired
yes
We have two separate copies of all genetic information (double helical structure of DNA)
• When one strand is damaged, the complementary strand (copy of same information) remains intact
• This is used to restore correct nucleotides to damaged strand
DNA damage repair pathways
Base lesions: single or double
Base excision repair (BER) • Nucleotide excision repair (NER) • For BER & NER • Damage is excised • Original sequence restored by using undamaged strand as template • Remaining break sealed with ligase • Direct reversal repair (DR) • Direct removal of lesion • No cleavage or ligation
Base Excision Repair
Repairs damage to a single base - depurination
• A set of enzymes acting sequentially
• Specific DNA glycosylases
• recognise specific type of altered base by ‘flipping out’ from helix
• excise/remove base via hydrolysis (breaking the bonds)
• AP endonucleases (AP for apurinic or apyrimidinic)
• recognise ‘missing tooth’ in helix
• cut the phosphodiester backbone
• DNA polymerase adds new nucleotides
• DNA ligase seals the nick
Nucleotide Excision Repair
Repairs larger changes to DNA helix: 2 or more bases
• Multienzyme complex scans DNA for distortion
• Cleaves phosphodiester backbone of abnormal )strand on both sides of distortion (excision nuclease)
• DNA helicase (DNA unwinding enzyme) peels away single-stranded oligonucleotide containing lesion
• Gap filled by DNA polymerase
• Sealed with DNA ligase
Nucleotide Excision Repair- disease example
Xeroderma pigmentosum
• Autosomal recessive genetic defect: nucleotide excision
repair enzymes are mutated
• Prevalence 1 in 250,000
• Symptoms: severe sunburn after minutes of sun exposure, freckle
• UV light can cause mutations via unrepaired DNA
• High risk of developing skin cancers: tumor suppressor genes are
affected
• Life expectancy shorter by ~30 years
Transcription-coupled DNA repair
Ensures cell’s most important DNA is efficiently repaired
• Links excision repair systems with RNA polymerase (enzyme
that transcribes DNA into RNA)
• RNA polymerase stalls at DNA lesions and directs the repair
machinery to these sites
• Targets repair to genes that are actively being transcribed into
mRNA
Transcription-coupled repair and human disease
Cockayne syndrome:
• Autosomal recessive congenital disorder
• Prevalence: 1 in 200,000
• Symptoms: growth retardation, skeletal abnormalities, progressive
neural degeneration and retardation, severe sensitivity to sunlight
• Defect in transcription-coupled repair
• RNA polymerase molecules become permanently stalled at sites of
DNA damage in important genes
• Causes cell apoptosis (programmed cell death)
• Life expectancy 10-20 years
Direct Reversal Repair
Most efficient form of DNA repair
• Rapid removal of certain highly mutagenic or cytotoxic
lesions
• e.g. Alkylation lesion 6-O-methylguanine
• Methyltransferase (MTase) protein accepts methyl
group (CH3) on cysteine residue from alkylated
guanine nucleotide
• Restores normal guanine
• MTase inactivated
• No DNA cleavage or ligation required
Emergency repair of heavily damaged DNA
Highly accurate replicative DNA polymerase (Pol III) stalls when it
encounters damaged DNA
• In emergencies, they employ less accurate back-up polymerases to
replicate through the DNA damage - translesion polymerases (Pol V)
• The back-up polymerases lack exonucleolytic proofreading activity
• These polymerases only add one or a few nucleotides before it falls off
and the replicative polymerase continues from there
• Risky for the cell: responsible for many mutations
DNA damage can delay progression of cell cycle
When does repair occur?
• In most cells, DNA damage causes a delay in
cell cycle
• Ensure that all damaged is repaired before a
cell divides
G1/S check point prevent damage cell to enter into replication phase
Cell Cycle involves critical check points
Cell cycle will not progress pass these check points until damage is repaired
• Orderly progression of cell cycle maintained through use of checkpoints to ensure completion of one step
before next step begins
• Cell cycle stops if damaged DNA is detected
• In mammalian cells, the presence of DNA damage can:
• block entry from G1 to S phase (checkpoint)
• slow S phase (replication) once it has begun
• block transition from G2 phase to M phase (checkpoint)
• Delays facilitate DNA repair by providing time needed for repair to reach completion
DNA damage results in increased synthesis of some DNA repair enzymes
• Special signalling mechanisms that arrest the cell cycle and respond to DNA damage
• ATM protein: large kinase that signals intracellularly to delay the cell cycle in response to DNA damage
• Individuals with ataxia telangiectasia (AT) (defects in ATM protein) suffer from effects of unrepaired DNA lesions
(neurodegeneration, genome instability etc)
• p53: ‘Guardian of the genome’
• Arrests the cell cycle at G1/S checkpoints until damage repaired
• Activates DNA repair enzymes
• Can initiate apoptosis if damage too great
• Huge implication in cancer: tumour suppressor
• Chk1: kinase
• cycle arrest at S and G2/M checkpoints
• DNA repair or cell death
Pyrimidine dimers are repaired by:
Select one:
a. Base Excision Repair
b. Nucleotide Excision Repair
c. Direct Reversal Repair
d. Homologous Recombination
e. Non-Homologous End Joining
Nucleotide Excision Repair
Which DNA repair protein(s) is/are primarily responsible for correcting apurinic or apyrimidinic sites?
Select one:
a. Mismatch repair enzymes
b. A multienzyme/nuclease complex
c. DNA ligase only
d. DNA Glycosylase
e. AP Endonuclease
AP Endonuclease
Which of the following enzymes are utilised in the Direct Reversal Repair (DR) pathway?
Select one:
a. AP Endonuclease
b. RecA/Rad51
c. DNA Glycosylase
d. Ku
e. Methyltransferase
f. Polymerase V
g. DNA Helicase
Methyltransferases are responsible for cleaving methyl groups from alkylated bases in the direct repair pathway
Deaminated bases are repaired by:
Select one:
a. Homologous Recombination
b. Base Excision Repair
c. Nucleotide Excision Repair
d. Non-Homologous End Joining
e. Direct Reversal Repair
Base Excision Repair
Which of the following enzymes are utilised in the Nucleotide Excision Repair (NER) pathway?
Select one:
a. DNA Glycosylase
b. Ku
c. Methyltransferase
d. DNA Helicase
e. RecA/Rad51
f. Polymerase V
g. AP Endonuclease
DNA Helicase
