DNA Double-Strand Break Repair

Question 1

what is the importance of DSB repair

Answer 1

just one unpaired DSB can kill a cell

misrepair leads to large chromosomal arrangement and cell death

loss of function primarily affects the immune, nervous, and reproductive systems, can cause premature aging and elevate cancer risk

Question 2

What causes DSB in DNA

Answer 2

Endogenous breaks

• immune system rearrangements
• single-strand breaks during DNA replication
• meiosis

Exogenous

• ionizing radiation (cosmic rays and soils) x2 in CO
• medical imaging and treatments

Question 3

DSB during replication

Answer 3

replication fork reaches a single strand break, becomes a double strand break

Question 4

When are double strand breaks required?

Answer 4

cross-over events during meiosis

Question 5

radiation dosage units

Answer 5

milliSievert (mSv)

1 mSv = 1 Joule per kilogram exposure

Question 6

legal dose if you work in radiation

Answer 6

50 mSv per year

Question 7

What are the two principal pathways of DNA DSBs

Answer 7

Non-Homologous End Joining (NHEJ)
Homologous Recombination (HR)

Question 8

Non-Homologous End Joining characteristics

Answer 8

often imperfect, loss of a few nucleotides

occurs throughout cell cycle (G0, G1, S, G2, M)

Question 9

Homologous Recombination characteristics

Answer 9

perfect repair

requires sister chromatid, limited to S and G2 of cell

Question 10

What are steps of NHEJ

Answer 10

1) NHEJ initiates with recognition of the DSB by Ku
2) Ku recruits DNA-PKcs
3) NHEJ variably uses combinations of:
nuclease to remove the damaged DNA if there is damage
polymerase to fill gaps
4) repair finishes with ligase to restore the continuous phosphodiester backbone to both strands

Question 11

What different types of DSB ends are there

Answer 11

simple ends (ligatable)
dirty ends (damaged)
coding ends (hairpin)

Question 12

What are the PIKKs

Answer 12

phosphitidyl inositol kinase kinase
DNA Pkcs
ATM
ATR

Question 13

What is Artemis nuclease ‘flexibility’ during NHEJ

Answer 13

ability to insert or delete nucleotides at a repair site

Question 14

What is the NHEJ sequence?

Answer 14

not linear, many possible outcomes
leads to sequence heterogeneity after repair
very often have little deletions/insertions, i.e., imperfect healing of break

Question 15

simplest example of DSB repair by homologous recombination: repair of a single-strand nick during DNA replication

Answer 15

replication fork encounters DNA nick
replication fork breaks
exonuclease degrades 5' end
strand invasion occurs, non-broken strand used as template
replication fork restarts

Question 16

Alternative outcomes for repair by HR

Answer 16

In germ dells: bias to use homolog and resolve via DSBR -> assure recombination

In somatic cells: bias to use sister chromatid and resolve via SDSA -> avoid recombination and LOH

Question 17

function of meiotic DSBs

Answer 17

increase genetic diversity

Question 18

Key steps of DSB repair

Answer 18

Sensing of break
Signal transduction
Responses
- DNA is repaired correctly, cell is fine
- DNA is misrepaired, cell dies or becomes cancerous