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BMS1062- > DNA Repair: Damage > Flashcards

DNA Repair: Damage Flashcards

1
Q

Genetic Stability

A

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

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2
Q

DNA repair is critical

A
  • 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
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3
Q

Human syndromes linked to defective DNA repair

A

Eg. Cancers, UV sensitivity, leukaemia, growth and development

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4
Q

If errors not fixed during replication:

A

mutations arise
Errors such as:
• Tautomeric bases
• Mismatch

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5
Q

Mechanisms to prevent replication errors

A
  • 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
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6
Q

proofreading polymerase

A

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)

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7
Q

Mismatch repair system

A
  • 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
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8
Q

Spontaneous DNA damage

A

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

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9
Q

Spontaneous DNA damage by hydrolysis

A

Depurination = spontaneous loss of purine bases (adenine and guanine)
by hydrolysis
Deamination = spontaneous conversion of cytosine to uracil by hydrolysis

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10
Q

Hydrolytic Damage: Depurination

A

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

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11
Q

Hydrolytic Damage: Deamination

A

Deaminated cytosine becomes uracil and mutation propagated as uracil pairs with adenine: base substitution

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12
Q

Alkylation Damage: Methylated Guanine

A

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

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13
Q

Induced DNA damage: UV Irradiation

A

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)

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14
Q

Covalent linkage between two adjacent pyrimidine bases

A
  • UV irradiation leads to:
  • Sunburn
  • ↑melanin production
  • If left unrepaired:
  • can lead to melanoma (cancer)
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15
Q

DNA double helix can be repaired

A

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

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16
Q

DNA damage repair pathways

Base lesions: single or double

A 
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
17
Q

Base Excision Repair

A

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

18
Q

Nucleotide Excision Repair

A

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

19
Q

Nucleotide Excision Repair- disease example

A

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

20
Q

Transcription-coupled DNA repair

A

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

21
Q

Transcription-coupled repair and human disease

A

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

22
Q

Direct Reversal Repair

A

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

23
Q

Emergency repair of heavily damaged DNA

A

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

24
Q

DNA damage can delay progression of cell cycle

A

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

25
Q

Cell Cycle involves critical check points

A

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

26
Q

DNA damage results in increased synthesis of some DNA repair enzymes

A

• 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

27
Q

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

A

Nucleotide Excision Repair

28
Q

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

A

AP Endonuclease

29
Q

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

A

Methyltransferases are responsible for cleaving methyl groups from alkylated bases in the direct repair pathway

30
Q

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

A

Base Excision Repair

31
Q

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

A

DNA Helicase

BMS1062- (28 decks)

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