The role of the S. pombe MRN complex in the removal of covalently bound protein from the DNA Edgar Hartsuiker Kenichi Mizuno Tony 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 rad50Δrad50S rec12Δ rec12Δ rad50Δ rec12Δ rad50S WT rad50Δ rad50S rec12Δ rec12Δ rad50Δ rec12Δ rad50S 25 °C34 °C Relative spore viability (%) S. pombe rad50S is TS for meiotic spore viability

°C34 °C Meiotic progression rad50S 25 ˚C rad50S 34 ˚C Time (hrs) % of cells ≥ 2 nuclei Time (hrs) I II III DSB S. pombe rad50S is TS for meiotic DSB repair

WT rad50S rad50Δ WT rad50S rad50Δ 0 hrs6 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 chromatin remodeling and linear element formation

Is the Mre11/Rad32 endonuclease acitvity responsible 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 WT rad50Δ rad50S rad32-D25A rad32-D25A rad50Δ rad32-D25A rad50S Spore viability 25 ºC WT rad50Δ rad50S rad32-D25A rad32-D25A rad50Δ rad32-D25A rad50S Relative meiotic spore viability (%) Spore viability 34 ºC

6 hrs into meiosis0 hrs into meiosis WT rad50Δ rad50S rad32-D25A rad32-D25A rad50Δ rad32-D25A rad50S BottomTopBottomTop 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 WT rad50Δ rad50S 400 Gy WT rad50Δ rad50S 0 Gy WT rad50Δ rad50S % MMS 25 ºC34 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 Camptothecin0 µM0.1 µM0.3 µM 25 ºC 36 ºC 0.4 µM

TOP-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 Mizuno Tony Carr