Envisioning the dynamics and flexibility of Mre11-Rad50-Nbs1 complex to decipher its roles in DNA replication and repair

Abstract

The Mre11-Rad50-Nbs1 (MRN) complex is a dynamic macromolecular machine that acts in the first steps of DNA double strand break repair, and each of its components has intrinsic dynamics and flexibility properties that are directly linked with their functions. As a result, deciphering the functional structural biology of the MRN complex is driving novel and integrated technologies to define the dynamic structural biology of protein machinery interacting with DNA. Rad50 promotes dramatic long-range allostery through its coiled-coil and zinc-hook domains. Its ATPase activity drives dynamic transitions between monomeric and dimeric forms that can be modulated with mutants modifying the ATPase rate to control end joining versus resection activities. The biological functions of Mre11's dual endo- and exonuclease activities in repair pathway choice were enigmatic until recently, when they were unveiled by the development of specific nuclease inhibitors. Mre11 dimer flexibility, which may be regulated in cells to control MRN function, suggests new inhibitor design strategies for cancer intervention. Nbs1 has FHA and BRCT domains to bind multiple interaction partners that further regulate MRN. One of them, CtIP, modulates the Mre11 excision activity for homologous recombination repair. Overall, these combined properties suggest novel therapeutic strategies. Furthermore, they collectively help to explain how MRN regulates DNA repair pathway choice with implications for improving the design and analysis of cancer clinical trials that employ DNA damaging agents or target the DNA damage response.

Keywords: Allostery; Conformational change; CtIP; Double strand break repair; Dynamics; Mre11-Rad50-Nbs1.

Figure 1. Architecture of the Mre11-Rad50-Nbs1 complex with its partner CtIP

The Rad50 dimer is in orange and yellow (for each monomer) and the coiled-coils are represented as helices extending towards the zinc-hook. The Mre11 dimer is shown in blue and dark blue and interacts with Nbs1, shown in green (different shades represent the different domains). Nbs1 interacts with CtIP through its FHA domain (dark green). Interaction with ATM is shown by a black arrow. The dotted lines represent disordered protein linkers.

Figure 2. Model of MRN’s functions in DSB repair

(A) MRN first detects and interacts with the DNA end. (B) If the process goes into HRR, MRN moves away from the break (shown with an arrow) and recruits CtIP. (C) CtIP induces changes in MRN leading to an open complex capable of nuclease activities. (D) After an initial endonuclease cut, MRN resects from 3′ to 5′ towards the DSB, with its exonuclease activity, while EXO1/BLM or DNA2/BLM resects from 5′ to 3′. (E) In NHEJ or HRR, the DNA tethering functions of MRN can be used. Color-coding is the same as in Figure 1.

Figure 3. Long-range allostery is a crucial feature of Rad50

Top: ATP binding induces rotation of the C-lobe relative to the N-lobe of Rad50’s head domain. This is communicated to the zinc-hook domain through the coiled-coils, possibly by helix sliding. Bottom: schematic of Rad50’s sequence with arrows depicting segments of flexibility and dynamics.

Figure 4. Rad50 ATP binding and hydrolysis controls its conformational state and pathway choice

(A) Crystal structures showing the Mre11-Rad50 head without nucleotide (top, PDB 3QG5) and bound to nucleotide (bottom, PDB 3AV0). Mre11 (surface representation) and Rad50 (cartoon representation) are colored by subdomains as indicated (the color of the font matches the color of the domain). In the nucleotide-free state, Rad50 monomers are flexible and conformationally sample the closed state, as indicated by dashed-arrows. (B) Top: Crystal structures of P. furiosus Rad50 without nucleotide (PDB 3QKS) show a core cavity (green surface) adjacent to the signature motif (magenta) that remodels upon ATP-binding. The L802W mutation (PDB 4NCH) partially fills the core cavity, destabilizing the ATP-bound closed state. In contrast, the R805E variant (PDB 4NCI) destabilizes the Rad50 monomer resulting in a stabilization of the ATP-bound closed state. Bottom: The effects of the mutations compared to wildtype Rad50 on Mre11-Rad50 conformational states and activities are depicted.

Figure 5. Flexibility and dynamics are part of Mre11 and Nbs1’s structures

Top: Structure of MRN highlighting flexible segments (arrows). Color-coding is the same as Figure 1, except Rad50 is gray and the capping domain of Mre11 is cyan. Bottom: schematic of Mre11 and Nbs1’s sequences with arrows depicting segments of flexibility and dynamics. Dashed lines and arrows represent intrinsically disordered segments.