Homologous recombination: DNA resection

Question 1

What is DNA resection? (2)

Answer 1

• The first step of HR which produces a 3’ ssDNA tail used to search for a homologous sequence and initialise DNA synthesis
• Requires nucleolytic degradation of the 5’ terminated strands

Question 2

What direction does resection occur?

Answer 2

5’ to 3’

Question 3

Would 3’ to 5’ resection work?

Answer 3

No because this would leave a 5’ end for strand invasion and DNA replication is 5’ to 3’ so you can’t add nucleotides to a 5’ end

Question 4

Which enzyme performs nucleolytic degradation of the 5’ terminated strands?

Answer 4

A deoxyribonuclease (DNase)

Question 5

What is the action of a DNase?

Answer 5

Catalyses the hydrolytic cleavage of phosphodiester linkages in the DNA backbone

Question 6

What are the 2 types of DNases?

Answer 6

• Endonuclease: cleaves the phosphodiester bond within a DNA chain
• Exonuclease: cleaves one nucleotide at a time from one end of a DNA chain either 5’-3’ or 3’-5’ (polarity of digestion)

Question 7

What characteristics would be expected of a protein involved in initiation of DNA resection? (4)

Answer 7

• Play a role in DNA damage repair so its mutation would likely make cells sensitive to DNA damage
• Bind DNA damage sites at an early stage of recombination
• Posses nuclease activity
• Its mutation prevents the generation of 3’ ssDNA during DSB repair

Question 8

What evidence suggests that MRE11 is involved in DNA resection? (3)

Answer 8

• Deplete MRE11 from cells using siRNA and irradiate cells using increasing doses of ionising radiation which induces DSBs (clonogenic survival assay)
• Stain and count colonies after 10 days
• MRE11 knockdown sensitises cells to ionising radiation as the cells fail to repair DSBs so growth is inhibited

Question 9

What complex contains MRE11?

Answer 9

MRN complex

Question 10

What are the components of the MRN complex? (3)

Answer 10

• MRE11
• RAD50
• NBS1

Question 11

What are the main features of the components of the MRN complex? (4)

Answer 11

• Phosphoesterase domain of MRE11 binds NBS1and has both single strand DNA endonuclease and double strand DNA exonuclease activity
• Antiparallel coiled-coil domain of RAD50 extends the protein across the break site
• Zinc hook domain of RAD50 is for DNA binding and facilitates the formation of dimers
• Protein-protein interaction domains of NBS1 facilitate downstream functions

Question 12

What are the yeast versions of the MRN complex? (2)

Answer 12

• NBS1 is called Xrs2
• MRN complex is called MRX complex

Question 13

How was the timing of Mre11 binding to DSB sites determined? (2)

Answer 13

• Fluorescently labelled Mre11 and induced DSBs in cells using laser micro-irradiation
• Live cell image analysis showed Mre11 recruitment to DSB sites very early on (within 1 minute whereas the whole repair process takes 30min-several hours)

Question 14

What polarity is the exonuclease activity of Mre11?

Answer 14

3’-5’ (strange because would expect 5’-3’ for 5’ end resection)

Question 15

What experiment showed the polarity of MRN complex exonuclease activity? (3)

Answer 15

• Incubate MRN complex with 2 DNA substrates: 5’ end radiolabel or 3’ end radiolabel (P32)
• Radiation of 3’ end labelled substrate decreased over time but the 5’ radiation remained stable
• Suggests that MRN exonuclease activity unexpectedly occurs in a 3’-5’ direction

Question 16

How was the endonuclease activity of the MRN complex discovered? (2)

Answer 16

• Labelled circular DNA substrate was incubated with MRN complex and the signal decreased over time showing that it was being degraded by MRN
• Because the DNA doesn’t have a 3’ or 5’ free end it can’t be digested by an exonuclease which shows that MRN also has endonuclease activity

Question 17

What is the mechanism of 5’-3’ DNA end resection by Mre11? (2)

Answer 17

• Mre11 nicks DNA using its endonuclease activity
• Mre11 degrades the DNA in the 3’-5’ direction towards the DSB end using its 3’-5’ exonuclease activity resulting in a 3’ ssDNA tail (sequential activity)

Question 18

Which protein is required to regulate the sequential endo and exonuclease activity of Mre11?

Answer 18

CtIP (humans)

Question 19

What is the yeast version of CtIP?

Answer 19

Sae2

Question 20

What is the effect of CtIP on Mre11 nuclease activity? (2)

Answer 20

• CtIP interacts with the Mre11 complex and stimulates Mre11’s endonuclease activity but has no effect on its exonuclease activity (initiation of resection)
• BRCA1 accelerates CtIP stimulation of Mre11-mediated DNA resection

Question 21

What is caused by CtIP mutations in humans?

Answer 21

CtIP is required for Mre11-mediated DNA resection so mutation causes Seckel and Jawad Syndrome which belongs to a group of genome instability disorders/DNA repair syndromes

Question 22

How does Mre11 complex initialise DNA resection? (3)

Answer 22

• CtIP (+ BRCA1) stimulates Mre11’s endonuclease activity to form DNA nicks
• Mre11 complex degrades DNA in the 3’-5’ direction using its exonuclease activity
• Produces 100-300nt of 3’ ssDNA

Question 23

Why isn’t DNA resection simply performed by 5’-3’ digestion instead of such a complex pathway? (2)

Answer 23

• 5’ to 3’ nucleolytic degradation requires an accessible 5’ end to an exonuclease
• Accessibility is often blocked because break sites aren’t ‘clean’

Question 24

What can block accessibility to the 5’ end at a DSB site? (2)

Answer 24

• Covalently bound proteins
• Secondary structures

Question 25

What proteins may be covalently bound at a 5' DSB site? (2)

Answer 25

- Top2 - Spo11 (meiosis)

Question 26

What secondary structures may form at the end of a DSB?

Answer 26

Inverted repeats form a cruciform structure which is cleaved by resolvases resulting in a hairpin-capped DSB so there isn't a free 5' end for enzyme binding

Question 27

What is the main function of Mre11 in DNA resection?

Answer 27

INITIALISE DNA resection by cleaning the DSB ends

Question 28

What is the limitation of DNA end resection by Mre11 complex and CtIP? (2)

Answer 28

- Slow and limited to only 100-300nt so not efficient for HR - Resection can be processed up to 3.5kb in human cells suggesting there are other more efficient resection pathways

Question 29

Which redundant resection pathways take over resection after it has been initialised by Mre11? (2)

Answer 29

- Exo1 - Sgs1 (BLM helicase)/Dna2

Question 30

What is 2-step DNA resection? (5)

Answer 30

- Mre11 complex binds DSB - CtIP stimulates Mre11 endonuclease activity which nicks DNA - Mre11 resects DNA by 3'-5' exonuclease activity - RPA binds the resected ends to protect them - RPA is displaced by Exo1 or BLM/Dna2 for further resection up to multiple kb

Question 31

How does resection by BLM/Dna2 occur?

Answer 31

BLM helicase unwinds the duplex, creating a 5' flap which is cleaved off by Dna2 endonuclease

Question 32

How is resection regulated? (5)

Answer 32

- CtIP protein levels are low in G1 phase and high in S, G2 and M phase - CDK2 phosphorylates CtIP at S347 which promotes its association with BRCA1 and the MRN complex - Additional phosphorylation of CtIP at T847 by CDK2 could facilitate CtIP function - Mechanism of CtIP function in DNA resection remains to be determined - CtIP modification or binding to MRN could influence MRN enzymatic activity and CtIP may also promote limited DNA resection in G1 in the absence of CDK2 or BRCA1 which would promote NHEJ over HR

Question 33

What are the other functions of the MRN complex? (6)

Answer 33

- Telomere maintenance - Activation of the DNA damage response (ATM) and checkpoint signalling pathways - Genome surveillance - HR - Meiosis - NHEJ

Question 34

How does the MRN complex interact with ATM?

Answer 34

Involved in ATM activation which phosphorylates downstream targets including p53 during the DNA damage response