Lec 4 - DNA damage & repair Notes

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DNA damage & cancer

i) Mutations can change gene function

RAS = GTP-ase proto-oncogenes that act as molecular switches controlling cell proliferation & survival

Frequently mutated in cancer

~20% human tumours relate to RAS malfunction

What happens when RAS is mutated?

RAS becomes unable to hydrolyse GTP

RAS becomes constitutively activated

Leading to cell hyper-proliferation

Mutations in codons 12, 13 & 61 account for almost all RAS hyperactivation

i) Mutations can change gene function RAS = GTP-ase proto-oncogenes that act as molecular switches controlling cell…

ii) DNA damage - both the cause & solution

Causative DNA damage:

UV damage & skin cancer

Cigarettes & lung cancer

Therapeutic DNA damage:

Anti-cancer drugs

Chemotherapy

See pg. 4

iii) Sources of DNA damage

Endogenous sources:

Free radicals

Spontaneous base deamination/depurination

DNA replication errors

Exogenous sources:

Radiation

Chemicals

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Mechanisms of DNA repair

i) Base excision repair

Inappropriate base pairing or damage to base

A pairs with G

U pairs with T

Caused by depurination, deamination & oxidative damage

How does base excision repair occur?

DNA glycosylase recognises damaged base & removes it (including a few nucleotides either side of it)

AP endonuclease cleaves DNA at empty site (sugar phosphate backbone)

DNA polymerase carries out repair synthesis & DNA ligase rejoins sugar-phosphate backbone

i) Base excision repair Inappropriate base pairing or damage to base A pairs with G U pairs with T Caused by depurination…

ii) Nucleotide excision repair

Removes DNA lesions that dramatically alter DNA structure

Thymine dimers

How does NER occur?

Complex containing XPA & XPC (xeroderma pigmentosum) detects DNA distortion

DNA endonuclease cuts out offensive dimer & removes it

DNA polymerase replaces correct nucleotide sequence & DNA ligase seals sugar P bond

ii) Nucleotide excision repair Removes DNA lesions that dramatically alter DNA structure Thymine dimers How does NER occur?…

Xeroderma pigmentosum

Rare autosomal recessive disease

NER system does not work properly

Leads to base-pair substitutions in skin cells

Skin very easily damaged by UV light

Causing cancerous growths to occur

Risk of skin cancer increases approx 1000-fold

Xeroderma pigmentosum Rare autosomal recessive disease NER system does not work properly Leads to base-pair substitutions…

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iii) Mismatch repair

Mistakes made by DNA polymerase during S phase of cell cycle

DNA replication!

How does MMR occur?

Mismatch recognition proteins detect error

Sliding clamp searches for single-strand nick

DNA exonuclease degrades DNA back to mismatch

DNA polymerase & DNA ligase do their thing!

iii) Mismatch repair Mistakes made by DNA polymerase during S phase of cell cycle DNA replication! How does MMR occur?…

MMR role in cancer

Defective MMR = microsatellite instability (**See Genetics lecture Consanginous mating pg.3 iii)**)

HNPCC has defective MMR - (**See mol med lec 2 - Types of cancer & intro to DDR pg.5 b) HNPCC**)

iv) DDSB repair

Breaks in both DNA strands (Lagging & leading)

Very toxic & dangerous due to risk of translocations & loss genetic material

DDSBs are induced in cancer cells using radiotherapy in order to kill them

Telomeres @ chromosome tips give them uniformity

i.e. DDSB repair enzymes do not assume they're broken ends

What causes DDSBs?

Ionising radiation

Oxidative free radicals

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DNA DSB repair mechanisms

i) Non-homologous end joining (NHEJ)

Useful because can function throughout cell cycle

Does not require homologous chromosome

Involves rejoining of remains of DNA strands

Tolerates nucleotide loss or addition @ rejoining site is tolerated

IMPRECISE MECHANISM!

What does this mean?

Can lead to accumulation of randomly located mutations over time in genome

How does NHEJ work?

Ku heterodimer binds

DNA nuclease, polymerase & ligase + MRN complex bind to Ku

DNA is repaired

i) Non-homologous end joining (NHEJ) Useful because can function throughout cell cycle Does not require homologous…

ii) Homologous recombination

Cannot function throughout cell cycle

Requires presence of homologous chromosome to act as template

More accurate than NHEJ

Occurs in meiosis @ chiasma formation

Sequence variation in gametes

BRCA2 plays key role in HR

How does HR work?

Rad50 & Mre11 resect DNA to expose tract SSDNA

Rad51 recombinase carries out homology search

Strand invasion occurs & template DNA synthesis occurs

Holliday junction cleaves to yield repaired sequence

ii) Homologous recombination Cannot function throughout cell cycle Requires presence of homologous chromosome to act as…

iii) Holliday junctions

Cross-shaped structures that form during HR

2 double stranded DNA molecules become separated into 4 strands

Allows segments DNA info to be exchanged

Unresolvement of Holliday junction may lead to mutations

iii) Holliday junctions Cross-shaped structures that form during HR 2 double stranded DNA molecules become separated into 4…

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Using DNA damage to kill cancer

Spontaneous breaks occur during normal DNA replication repaired by HR & PARP-I dependent SSB repair

BRCA-2 defective cells = defective HR

Rely on PARP-I to repair DSBs/SSBs

Inhibition PARP-I in BRCA-2 mutated cells = specific & potent killing mechanism

Spontaneous breaks occur during normal DNA replication repaired by HR & PARP-I dependent SSB repair BRCA-2 defective cells…

Lec 4 - DNA damage & repair

Content of the notes (5)