The role of the S. pombe MRN complex in the removal of

covalently bound protein from the DNA

Edgar HartsuikerKenichi MizunoTony Carr

The Mre11 Rad50 Nbs1 (MRN) complex

Plays a central role in the early response to DNA double strand breaks: sensing, processing and repair

Meiosis as a model system to study MRN function in DSB repair

Scott Keeney, Sloan Kettering Institute

Rad50 has been implicated in DSB formation and Spo11 removal in S.

cerevisiae meiosis

rad50S isolated as a separation of function mutation: strong meiotic phenotype, only slight defect in vegetative cells

meiotic DSB's formed but not repaired in rad50S, Spo11 not removed from the DNA

Which activity is responsible for Spo11 removal?

Role of Rad50 and Rad32 in removal of covalently bound proteins in meiosis:

Rec12 (Spo11)

WT

rad5

0Δ

rad5

0S

rec1

2Δ

rec1

2Δ ra

d50Δ

rec1

2Δ ra

d50S W

T

rad5

0Δ

rad5

0S

rec1

2Δ

rec1

2Δ ra

d50Δ

rec1

2Δ ra

d50S

25 °C 34 °C

100

50

40

30

20

10

60

70

80

90

0

Rel

ativ

e sp

ore

via

bili

ty (

%)

100

0.1

14.6

23.5

16.3

25.4

100

0.3 0.6

22.7

9.8

20.4

S. pombe rad50S is TS for meiotic spore viability

0 1 2 3 4 5 6 128

25 °C 34 °C

10

0 1 2 3 4 5 6 8 100

10

20

30

40

50

60

70Meiotic progression

rad50S 25 ˚Crad50S 34 ˚C

Time (hrs)

% o

f ce

lls ≥

2 n

ucl

ei

0 1 2 3 4 5 6 128 10Time (hrs)

I

II

III

DSB

S. pombe rad50S is TS for meiotic DSB repair

WT ra

d50S

rad5

0Δ

WT ra

d50S

rad5

0Δ

0 hrs 6 hrs

Rec12 is covalently bound to the DNA in rad50Δ/rad50S meiosis

10 x diluted

S. pombe rad50S is a temperature sensitive separation of function mutation

Defective in removing Rec12 from DSB ends

Proficient in meiotic recombination, meiosis specific chromatinremodeling and linear element formation

Is the Mre11/Rad32 endonuclease acitvityresponsible for the removal of Rec12?

rad32-D25A mutation is defective for nuclease activity, still forms complex

Is rad32-D25A epistatic with rad50S or rad50Δ?Is rad32-D25A defective for Rec12 removal?

rad32 nuclease dead mutant shows reduced spore viability, epistatic with rad50Δ and rad50S

100

0.12

0.48

0.04

0.11

0.03

WT

rad50Δ

rad50S

rad32-D25A

rad32-D25A rad50Δ

rad32-D25A rad50S

0.0

0.1

0.2

0.3

0.4

0.5100

0.11

9.78

0.01

0.07

0.01

Spore viability 25 ºC

WT

rad50Δ

rad50S

rad32-D25A

rad32-D25A rad50Δ

rad32-D25A rad50S

Rela

tive m

eio

tic

spore

via

bili

ty (

%)

Spore viability 34 ºC

6 hrs into meiosis0 hrs into meiosis

WT

rad50Δ

rad50S

rad32-D25A

rad32-D25A rad50Δ

rad32-D25A rad50S

Bottom Top Bottom Top

rad32 nuclease dead mutant is defective for Rec12 removal

Main conclusions MRN role in meiosis

rad50S is specifically defective in removal of covalently bound Rec12 from the DSB

rad50S is proficient for other Rad50 dependent recombination related functions

Rad32 endonuclease activity is responsible for removal of covalently bound Rec12

Role of Rad50 and Rad32 in removal of covalently bound proteins in vegetative cells:

Topoisomerase I and Topoisomerase II

1 μM CPT

WT

rad50Δ

rad50S

0 μM CPT

WT

rad50Δ

rad50S

WT

rad50Δ

rad50S

0 % MMS

105 104 103 102 10 105 104 103 102 10

WT

rad50Δ

rad50S

400 Gy

WT

rad50Δ

rad50S

0 Gy

WT

rad50Δ

rad50S

0.002 % MMS

25 ºC 34 ºC

rad50S is not sensitive to MMS

rad50S is not sensitive to

rad50S is temperature sensitive to CPT

rad32 nuclease dead mutant is epistatic with rad50/rad50S for CPT

WT

rad50Δ

rad50S

rad32-D25A

rad50Δ rad32-D25A

rad50S rad32-D25A

WT

rad50Δ

rad50S

rad32-D25A

rad50Δ rad32-D25A

rad50S rad32-D25A

Camptothecin 0 µM 0.1 µM 0.3 µM

25 ºC

36 ºC

0.4 µM

0 2 4 8 16TOP-53(µg/ml)

WT

rad50Δ

rad50S

rad32-D25A

rad32-D25A rad50Δ

rad32-D25A rad50S

WT

rad50Δ

rad50S

rad32-D25A

rad32-D25A rad50Δ

rad32-D25A rad50S

rad50S is temperature sensitive for the Topoisomerase II inhibitor TOP-53 and epistatic with rad32-D25A

Main conclusions

The MRN complex is involved in the removal of Rec12 and probably Topoisomerase I and Topoisomerase II from the DNA

Rad32 endonuclease activity is probably responsible for Rec12/Top1/Top2 removal

Acknowledgements

Kenichi MizunoTony Carr