posted on 2023-06-09, 23:32authored byStephen Hallett, Pascale Schellenberger, Lihong Zhou, Fabienne Beuron, Ed Morris, Jo Murray, Antony OliverAntony Oliver
The multi-component Smc5/6 complex plays a critical role in the resolution of recombination intermediates formed during mitosis and meiosis, and in the cellular response to replication stress. Using recombinant proteins, we have reconstituted a series of defined Saccharomyces cerevisiae Smc5/6 complexes, visualised them by negative stain electron microscopy, and tested their ability to function as an ATPase. We find that only the six protein ‘holo-complex’ is capable of turning over ATP and that its activity is significantly increased by the addition of double-stranded DNA to reaction mixes. Furthermore, stimulation is wholly dependent on functional ATP-binding pockets in both Smc5 and Smc6. Importantly, we demonstrate that budding yeast Nse5/6 acts as a negative regulator of Smc5/6 ATPase activity, binding to the head-end of the complex to suppress turnover, irrespective of the DNA-bound status of the complex.
Funding
How do Smc5/6 interactions with DNA coordinate replication and recombination?; G2119; MRC-MEDICAL RESEARCH COUNCIL; MR/P018955/1