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Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo
Published on: September 18, 2014
Mechanism of DNA End Sensing and Processing by the Mre11-Rad50 Complex
Lisa Käshammer1, Jan-Hinnerk Saathoff1, Katja Lammens1
1Department of Biochemistry, Ludwig-Maximilians-Universität, 81377 Munich, Germany; Gene Center, Ludwig-Maximilians-Universität, 81377 Munich, Germany.
Abstract:
DNA double-strand breaks (DSBs) threaten genome stability throughout life and are linked to tumorigenesis in humans. To initiate DSB repair by end joining or homologous recombination, the Mre11-nuclease Rad50-ATPase complex detects and processes diverse and obstructed DNA ends, but a structural mechanism is still lacking. Here we report cryo-EM structures of the E. coli Mre11-Rad50 homolog SbcCD in resting and DNA-bound cutting states. In the resting state, Mre11's nuclease is blocked by ATP-Rad50, and the Rad50 coiled coils appear flexible. Upon DNA binding, the two coiled coils zip up into a rod and, together with the Rad50 nucleotide-binding domains, form a clamp around dsDNA. Mre11 moves to the side of Rad50, binds the DNA end, and assembles a DNA cutting channel for the nuclease reactions. The structures reveal how Mre11-Rad50 can detect and process diverse DNA ends and uncover a clamping and gating function for the coiled coils.
Insights
The Mre11-Rad50 complex detects and processes DNA double-strand breaks (DSBs) to maintain genome stability. Structural insights reveal a clamping mechanism involving Rad50 coiled coils that gates Mre11 nuclease activity.
Area of Science:
- Molecular Biology
- Structural Biology
- Genetics
Background:
- DNA double-strand breaks (DSBs) are critical DNA lesions impacting genome stability and human tumorigenesis.
- The Mre11-Rad50 complex is essential for DSB repair pathways like end joining and homologous recombination.
- A detailed structural understanding of Mre11-Rad50's DNA end processing mechanism is currently lacking.
Purpose of the Study:
- To elucidate the structural mechanism by which the Mre11-Rad50 homolog, SbcCD, detects and processes diverse DNA ends.
- To provide high-resolution structural insights into the resting and DNA-bound states of the SbcCD complex.
Main Methods:
- Cryo-electron microscopy (cryo-EM) was employed to determine the structures of E. coli SbcCD.
- Structures were resolved for both the resting state and DNA-bound cutting states of the complex.
Main Results:
- The resting state shows Mre11 nuclease activity blocked by ATP-bound Rad50 with flexible Rad50 coiled coils.
- Upon DNA binding, Rad50 coiled coils form a rigid rod, creating a clamp around double-stranded DNA (dsDNA).
- Mre11 relocates to bind the DNA end, forming a channel for nuclease activity, revealing a clamping and gating function.
Conclusions:
- The study reveals the structural basis for Mre11-Rad50's ability to detect and process various DNA ends.
- The findings uncover a novel clamping and gating mechanism mediated by Rad50 coiled coils, crucial for Mre11 nuclease function in DNA repair.
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