7 DNA Mutation & Repair

aim

investigates how UV-C irradiation affects yeast DNA

strains used

D02 wild-type - has functional RAD9

D02-Δrad9/Δrad9 - RAD9 deleted from both copies

both strands are diploid

what does this practical measure

Does deleting RAD9 affect yeast survival and mutation rate after UV-C damage?

after UV-c exposure:

1. survival - how many cells survive UV irradiation and form colonies

2. mutation - how many cells survive UV irradiation and form colonies

why is yeast used

- easy to grow

- cheap to culture

- eukaryotic, like human cells

- able to exist as haploid or diploid cells

- useful for studying cell cycle control and DNA repair

- cell cycle only takes 50 mins, fast enough to study cell division experimentally

what wavelength is UV-C

290-180nm

why does UV-C damage DNA

The main damage caused by pyrimidine dimers

- happens when two adjacent pyrimidines on the same DNA strand become covalently bonded. I.e TT, CC and TC

- These dimers distort the DNA double helix and block normal DNA replication.

how does DNA repair itself in yeast

1. photoreactivation

2. excision repair

3. error prone repair

4. recombinational repair

1. photoreactivation

repairs pyrimidine dimers using light.

photolyase uses light energy to break apart the pyrimidine dimer.

2. Excision repair

This removes the damaged section of DNA and replaces it.

1. Damage is recognised.

2. DNA is cut around the damaged region.

3. The damaged piece is removed.

4. DNA polymerase fills the gap using the other strand as a template.

5. DNA ligase seals the nick.

3. Error-prone repair

This is a less accurate repair process.

Sometimes the cell has to repair DNA even when the correct template is unclear.

This can save the cell, but can introduce mistakes . RAD18 involved in this

recombinational repair

used when DNA replication is blocked by damage and leaves a gap.

repairs the missing information by using another similar DNA molecule or chromosome as a template.

RAD51 important as helps DNA molecules find similar partner sequences and begin recombination.

checkpoint

A checkpoint is a surveillance system in the cell cycle.

It checks whether the cell is ready to move to the next stage.

A checkpoint can stop the cell cycle if:

- DNA is damaged

- DNA replication is incomplete

- mitosis has not completed properly

- the spindle is faulty

RAD9

a DNA damage checkpoint gene.

Rad9p is involved in:

- recognising DNA damage

- helping DNA repair

- activating downstream checkpoint signalling

- stopping the cell cycle at G2 if DNA is damaged

- can activate RAD53 which is also needed for G2 arrest after DNA damage

What happens if RAD9 is deleted

So after DNA damage:

- cells do not arrest properly at G2

- they continue into M phase

- DNA may not be repaired before division

- this can reduce survival

- this can increase mutation frequency

therefore more sensitive to UV-C than wild type

ADE2 and red colonies

ADE2/ade2

one working ADE2 allele and one mutant ade2 allele = white colonies

ade2/ade2

A red colony indicates mutation affecting the remaining functional ADE2 copy.

why does the red colony appear

The red colour happens because if the adenine biosynthesis pathway is blocked around the ADE2 step, a compound called AIR accumulates and is oxidised to form a red pigment.

main experiment idea

You expose yeast cells to different UV-C doses:

0 J m⁻²

30 J m⁻²

60 J m⁻²

90 J m⁻²

Then you count:

total colonies

- tells you survival

red or red-sector colonies

- tells you mutation at the ADE2 locus

Then you compare wild-type and rad9 mutant yeast.

step 1

1. place a yeast suspension onto a haemocytometer and count cells under a microscope.

counting rules

count cells touching the top or left boundary lines

do not count cells touching the bottom or right boundary lines

what if the culture is too concentrated

dilute the culture first.

needs to be 20-200 cells per large square

dilute 1:10 and record dilution factor

equation for haemocytometer calculation

cells/ml = average cells per large square × 10 × 1000 × dilution factor

how to calculate cell density

original cell density = calculated cell density × dilution factor

1. count total cells in 4 large squares (in 0.4 mm³)

2. divide by 4 = average cells in 0.1 mm³

3. x10 to give cells per 1.0 mm³

4. x1000 to give cells per ml

5. if you diluted before counting x dilution factor

2. Dilute yeast culture to the target density

goal is 500 cells per plate

plating volume is 200 µl

so final yeast suspension needs to contain 500 cells per 200 µl = 2.5 × 10³ cells/ml

How to calculate how much culture to add

volume of culture needed = desired density / actual density × 1000 µl

3. make a 10-fold dilution seires

100 µl sample + 900 µl sterile water

10⁻¹ dilution

4. plate yeast cells on YED plates

add 200 µl of diluted yeast suspension onto each YED plate.

spread

4. irradiation with different UV-C doses

control = 0J m⁻²

low UV = 30J m⁻²

medium UV = 60J m⁻²

high UV = 90J m⁻²

in session 2 what are the steps

1. Count total colonies on each plate.

2. Count red colonies or red sectors.

3. Record results for each UV dose.

4. Calculate percentage survival.

5. Calculate percentage mutants.

6. Plot survival and mutation graphs.

7. Compare wild-type and rad9 mutant yeast.

What a fully red colony means

A fully red colony may come from a cell where mutation happened before colony growth began.

So all cells in the colony inherited the ade2/ade2 mutant genotype.

What a sectored colony means

both white and red areas.

This suggests the mutation happened after some cell divisions, not at the very beginning. for example

- a white cell starts forming a colony

- during colony growth, one descendant cell gets an ADE2 mutation

- descendants of that mutant cell form a red sector

- the rest of the colony remains white

why are red colonies often smaller

because ade2 mutants cannot make adenine properly.

They require adenine in the growth medium and may grow less efficiently than ADE2 cells.

why do we look fo unusual colony shapes and not just colour

Mutations in many genes can affect colony morphology, not just ADE2.

uv-c van cause other visible phenotypes

percentage survival equation

% survival = colonies after UV dose / colonies on 0 J m⁻² plate × 100

percentage mutants equation

% mutants = colour mutant colonies / total colonies on that plate × 100

Graph 1: survival curve

Plot % survival on y-axis

against:

UV-C dose on x-axis

expected shape:

survival decreases as UV-C dose increases

rad9 mutant likely decreases more steeply than wild-type

Graph 2: mutation frequency

Plot: % mutants on y-axis

against:

UV-C dose on x-axis

Expected trend:

mutation frequency may increase with UV-C dose

rad9 mutants may show more red mutants because damaged DNA is not properly checkpointed/repaired before division

why diploidity matters

Both strains are diploid and heterozygous:

ADE2/ade2

Because they have one functional ADE2 copy, they are white.

To become red, they need loss or mutation of the functional ADE2 copy so the genotype behaves like:

ade2/ade2

This makes red mutants rarer than if you were working with haploid ade2 cells

Expected comparison: wild-type

- better survival after UV-C

- lower mutation frequency

- functional RAD9

- checkpoint allows G2

- arrest and repair

Expected comparison: D02-Δrad9/Δrad9

- lower survival after UV-C

- higher mutation frequency among survivors

- no functional Rad9p

- cells continue into mitosis - despite damaged DNA

Lethal effects of radiation

can cause reproductive death

- the cell may frow but can't divide to form viable progeny

- if a cell can't form a colony, it's treated as not surviving