DNA Damage and Repair

Question 1

DNA damage vs. mutation

Answer 1

DNA damage is a chemical alteration of the DNA

If DNA damage isn’t repaired before replication, mutation results

Question 2

Effects of mutation on the coding region of the gene

Answer 2

Large effects (altered or nonfunctional protein product)

Question 3

Effects of mutation on an intron

Answer 3

Could cause no effect, splice variants, or alteration of gene expression (introns can contain enhancers)

Question 4

Transition mutation

Answer 4

Purine -> purine or pyrimidine -> pyrimidine

Question 5

Transversion mutation

Answer 5

Purine -> pyrimidine or pyrimidine -> purine

Question 6

Missense mutation

Answer 6

Changes one amino acid to another dissimilar one

Question 7

Nonsense mutation

Answer 7

Changes from an amino acid codon to a stop codon

Question 8

Silent mutation

Answer 8

Changes codon so that the same amino acid is specified

Question 9

Neutral mutation

Answer 9

Changes one amino acid to another similar one

Question 10

Frameshift mutation

Answer 10

Addition or deletion of one or a few base pairs, leading to a change in reading frame

Question 11

2 sources of spontaneous mutation

Answer 11

Mistakes during replication (incorrect nucleotide, extra nucleotides, etc.)
Tautomeric shift (shift from normal form of bases to rare forms)

Question 12

Slippage

Answer 12

Polymerase adds extra nucleotides during replication, often in the form of repeat expansions

Question 13

How tautomeric shift affects base pairing

Answer 13

Rare forms of bases pair to the opposite purine or pyrimidine than normal

Question 14

Mismatch repair in E. coli

Answer 14

1. MutL recognizes the template (contains methyl group, which non-template strand doesn’t) and binds to it (preventing MutH from cutting the template) and MutS recognizes the damage and recruits MutH to correct spot
2. MutH makes a nick upstream of the mutation
3. An exonuclease cuts out the wrong nucleotide
4. DNA pol III fills in the excised area with the proper nucleotides

Question 15

2 methods of hydrolytic DNA damage

Answer 15

Deamination (loss of -NH2)

Depurination (loss of purine leaves apurinic site)

Question 16

Base analogs

Answer 16

Look like nucleotides, causing them to be incorporated into DNA

Question 17

5 BU

Answer 17

Base analog that looks like T

If it is in high abundance, it can be incorporated into DNA base paired with A

Question 18

Intercalating agents

Answer 18

Inserted between bases, distorting DNA helix

Question 19

Base modifying agents

Answer 19

Change covalent bonds, changing structure of base

Question 20

Nucleotide that is the most vulnerable to damage

Answer 20

Guanine

Can be subject to oxidation, alkylation, and deamination

Question 21

Mustard gas

Answer 21

Alkylating agent

Question 22

Nitric oxide

Answer 22

Base modifying agent

Question 23

Base oxidation

Answer 23

Triggered by gamma rays, x-rays, and UV radiation

Ionization of water, forming free radicals, which attack DNA (oxidative damage)

Question 24

Reactive oxygen species

Answer 24

O2- (superoxide)
H2O2 (non-radical oxidant)
OH (hydroxy radical)

Question 25

Oxidative stress

Answer 25

Cells have a way to manage reactive oxygen species, but if the reactive oxygen species become too great in the cell, damage occurs

Question 26

8-oxoG

Answer 26

7,8-dihydro-8-oxoguanine (caused by oxidative DNA damage) | Large source of mutation

Question 27

Pyrimidine dimers from UV light

Answer 27

UV light changes covalent bonding properties of pyrimidines (usually thymine) Bulky legions cause stalling of transcriptional machinery, causing apoptosis

Question 28

5 methods of measuring DNA damage

Answer 28

1. Fluorescent Ab to 8oxoG (measure by flow cytometry) 2. Sequence for mutation 3. Measure amounts of DNA repair machinery/proteins (Western blotting) 4. Histone 2B phosphorylation (H2B is phosphorylated in response to nearby DNA damage) 5. Comet: detects double strand breaks

Question 29

Comet steps

Answer 29

1. Treat cells with DNA damaging agent 2. Permeabilize membrane 3. Run gel electrophoresis (damaged DNA comes out of the cell faster than normal DNA because it is chopped up into small pieces) Damaged DNA streaks on gel, looking like a comet

Question 30

4 basic themes in repair

Answer 30

1. Directly undo the damage (photoreactivation, O6-methylguanine methyltransferase) 2. Excision repair- cut out the lesion and replace (base excision repair, nucleotide excision repair) 3. Double strand break repair (homologous recombination, nonhomologous end joining) 4. Translesion synthesis- replicate through damage (error prone bypass or SOS response)

Question 31

Photoreactivation in E. coli

Answer 31

Light (visible) activates DNA photolyase to repair damage caused by UV light

Question 32

Methyl group removal

Answer 32

Methyltransferase recognizes methyl group and takes it from DNA Methyltransferase is then degraded (suicide enzyme)

Question 33

Base excision repair

Answer 33

Most prevalent type of repair Glycosylase cuts out base, leaving AP (apyrimidinic/apurinic) site AP site is filled in by DNA pol

Question 34

8-oxoguanine repair

Answer 34

Type of base excision repair Guanine is oxidized and replication occurs: mismatch between G and A is recognized by fail-safe glycosylase, which cuts out A A is replaced by C

Question 35

Nucleotide excision repair

Answer 35

Used for bulkier damage (thymine dimers, etc.) Excinuclease cuts on both sides of bulky lesion Lesion is cut out and degraded and non-damaged nucleotides are put in its place

Question 36

2 types of nucleotide excision repair

Answer 36

1. Global genome: scanning all parts of genome for damage | 2. Transcription coupled: detecting when polymerase stalls

Question 37

Transcription coupled repair

Answer 37

1. RNA pol stalls because lesion doesn't fit into active site 2. Pol is released 3. Nucleotide excision repair proteins excise lesion 4. DNA pol makes new DNA in lesion's place

Question 38

Xeroderma pigmentosa

Answer 38

Disease caused by mutations in nucleotide excision repair proteins, rendering them non-functional (can't repair thymine dimers) Children with this disease are 1,000 x more likely than the average child to get skin cancer (UV radiation causes dimers)

Question 39

Only pathway in humans for reparation of thymine dimers

Answer 39

Nucleotide excision repair

Question 40

Nonhomologous end joining steps

Answer 40

1. Double strand break 2. Ku proteins recognize free ends of DNA 3. Ku proteins recruit kinase (DNA Pk): dimerization of DNA Pks and Pks phosphorylate each other 4. Localized unwinding of DNA- microhomology aligns DNA ends 5. Ligase patches ends together

Question 41

Translesion DNA synthesis: 2 methods

Answer 41

Method 1: 1) DNA pol stalls at lesion 2) Clamp changes shape and DNA pol lets go of template 3) Translesion DNA pol adds any nucleotide to growing strand (complementary or not) so that replication can proceed) Method 2: 1) DNA pol skips over lesion 2) Translesion DNA pol fills in gap left by DNA pol