L7: DNA damage and repair

Question 1

UVB radiation effect on DNA

Answer 1

• Promotes formation of intra-strand crosslinked pyrimidine dimers
• linked pyrimidines aren’t accommodated in AS of replicative DNA pols (DNA polIII, delta, epsilon)
• Replicated by low fidelity TLS polymerases - introduces mismatches
  -> leads to mutation
• 75% of UV damage is cyclobutane dimer (5’-5’ and 6’-6’), the rest is 6’-4’ photoproducts which are less more distorting (and therefore more easily noticed and repaired)

Question 2

IR (X-rays, gamma rays): sources, proportion of the 2 types, effect on DNA

Answer 2

• Natural sources, therapeutic, diagnostic or occupational sources
• Damage bases, break poly-nt strand phosphodiester bb (ss and ds breaks)
• Lethal effects de to strand breaks, particularly DSBs
• 35% direct damage, 65% as a result of ROS (ionisation of cellular water)

Question 3

Ames test (and modified ames test)

Answer 3

• Assaying potential mutagenicity of chemicals
• In a modified ames test, treated w/ a mixture of liver enzymes prior to addition to medium (e.g. benzopyrene which is not mutagenic in the first instance but is converted into a mutagen in the body)
• Utilises a histidine auxotroph, monitoring for frequency of revertants induced by mutagen

Question 4

How do cell repair DNA damage? - Types of repair

Answer 4

Excision of damaged DNA…
1. Mismatch repair (MMR)
2. Base excision repair (BER)
3. Nucleotide excision repair (NER)
Direct reversal of DNA damage..
1. Repair of O6-alkylguanine
2. Enzymatic photoreactivation

Question 5

Goals of a mismatch repair pathway, example in bacteria

Answer 5

• Recognise mismatched base pairs
• Discriminate between the correct (parental strand) base and the incorrect (daughter strand) base in a mismatched pair
• Excise the incorrect base and carry our repair synthesis

MutSLH mismatch repair system

Question 6

MutSLH complex (MMR)

Answer 6

• MutS protein recruited to mismatch bp in newly replicated DNA
• MutL recruited, the two work together along strand to find MutH, which labels the part of the new DNA which is hemi-methylated
• MutSLH complex assembles, introduces a nick, then the strand is coordinately unwound (by UvrD hel), and digested by and exonuclease
• Different exonucleases required depending on which side of complex MutH is on (5’-3’ or 3’-5’)
• DNA is resynthesised by DNA polIII

Question 7

Insertions and deletions in MMR

Answer 7

• Hairpins in region w/ lots of AT repeats often form - result in deletion when not replicated correctly
• Mismatch repair can detect and repair hairpins - the newly synth. strand is degraded, hairpin unfolds and new strand can be remade
• However, defects in this process lead to increased rates of spontaneous mutation and cancer

Question 8

BER

Answer 8

• Damaged base is recognised, flipped out of helix and then removed by cleavage of glycosidic bond between base and sugar
• Cellular glycosylases are specific to type of damage
• Abasic site repaired by apurinic/apyrimidinic endonuclease, which cleave the bb
• Gap is filled by DNA pol and ligated

Question 9

NER

Answer 9

• Specificity for helix distortion. Removes oligo-nts (~13 mer), ie. bulky lesions that distort the helix
• UvrA scans for distorted regions, recruits UvrB when it finds them - unwinds the damaged region and UvrB dimer recruits 2 UvrC proteins which each introduce a nick on their side of the damage
• Damaged section removed by UvrD, resynth. from undamaged strand

Question 10

NER vs BER (conserved proteins). GIev the types of NER

Answer 10

• Whilst the MutS and MutL proteins are highly conserved across organisms, NER proteins aren’t; the mechanism is conserved however
• GGR: Global genomic repair
  -> anywhere in genome
• TCR: Transcription coupled repair, removes stalled RNA pols
  -> preferential repair for damage in genes actively being transcribed

Question 11

Defects in NER, example disorder w/ details

Answer 11

• Result in mutator phenotype
• e.g. Xeroderma pigmentosum…
  Rare, fatal, AR disorder, 1 in 250000. Sun sensitive, predisposition to skin cancer

Question 12

Direct reversal; alkylation damage

Answer 12

• Alkylation on guanine or the DNA bb is repaired by alkyltransferases
• In E.coli, Ada takes the alkyl grp onto itself, which inactivates it (suicide enzyme)
• Methyl-Ada stimulates production of more Ada, and the AlkA glycosylate

Question 13

Direct reversal; enzymatic photoreactivation (about the enzyme, mechanism, which organisms?)

Answer 13

• Enzymatic photoreactivation of pyrimidine dimers by photolyase enzyme (specific for cyclopyrimidine dimers/6-4 photoproducts)
• They contain 2 noncovalently bound chromophores…
  -> An antenna pigment that absorbs sunlight, and a catalytic cofactor (fully reduced Flavin-adenine dinucleotide)
• Carries out electron transfer from FADH- to UV-induced lesion, dimer splitting, transfer of e- back to FADH to generate FADH-
• Enzyme found in all organisms but placental mammals