Structural mechanism of ATP-dependent DNA binding and resection by the Mre11-Rad50 complex

Abstract

The MRN complex composed of Mre11, Rad50, and Nbs1 (Xrs2 in S.cerevisiae) is a sophisticated protein machine which orchestrates molecular choreography of a series of significant biological events including double strand break detection, ATM activation, DNA end tethering and processing. During the past decades, a plethora of structures of individual and complex of MRN have greatly illuminated the understanding of mechanism and architecture of this multifunctional complex. Structures of prokaryotic MR catalytic domain (MRcd) revealed that Mre11 dimer and the two Rad50 nucleotide-binding domains (NBDs) assemble into a M2R2 heterotetramer in which two Ra50 ATPase head domains are engaged together with the sandwiched nucleotide to adopt a closed conformation and located on top of the nuclease domain of Mre11. In contrast to this closed form, crystal structures of the catalytic domains of bacterial MR in the absence of ATP exhibited an “open” structure with the ATPase head domains being widely separated from each other. Based on structural and biochemical data a model has been established to connect the two different conformations of MR complex with their distinct biological functions. It is proposed that the ATP bound closed MR complex involves in DNA binding and tethering thus facilitates NHEJ while the ATP unbound MR complex with an open conformation stimulates DNA resection, and DSB repair by homologous recombination. Although this model illuminated the roles of ATP in driving the MR complex to switch between different functional conformations, it has a lot of limitations due to the fact that it is established based on structural analysis of truncated MR complex with more than half of the coiled-coil region being deleted. Considering that the intact coiled-coil region may inhibit or at least pose some restriction to the movement of ATPase head domain of Rad50, it remains doubtable whether the full length MR will also adopt a widely opened conformation, similar to MRcd complex, in the absence of ATP. Besides, it remains enigmatic that how the MR complex undergoes conformational rearrangement from the closed state to make the Mre11’s occluded nuclease site become accessible for DNA, and how the DNA is recognized and cleaved by MR complex. Moreover, the structural basis for the roles of ATP in DNA binding and processing remains unknown. This work aimed to determine the crystal structure of the DNA bound Methanococcus jannaschii Mre11/Rad50 (MjMR) complex in the presence of ATPγS and to uncover the mechanism of DNA binding and processing by the MR complex. The structure reveal symmetrical binding of DNA binding by the MR complex. DNA is positioned at a central groove formed at the dimeric interface of two Rad50 catalytic domains engaged together by ATPγS which shows the significance of ATP-dependent DNA binding by the MR complex. Structural analysis indicated that conformational changes induced in MR by ATP hydrolysis may trigger DNA unwinding, and subsequent biochemical tests indeed lend credence to this assumption. DNA binding analysis revealed that the ATPase head domains of full length MR adopts a conformation different from that of MRcd (MR catalytic domain) in the absence of ATP, indicating that the hook and coiled coil domain affect the MR-DNA binding pattern. Nuclease assay using both the intact and unwound DNA demonstrated that the ATP dependent DNA binding and unwinding of Rad50 are essential for the efficient endonucleolytic cleavage of DNA by the MR complex. In vitro and in vivo functional analysis validated the DNA-MR complex interacting motifs observed in the structure, and demonstrated that binding of DNA to Rad50 is important for double strand break repair in vivo. The significance of this thesis lies in several aspects. Firstly, by determining the crystal structure of DNA bound MjMR in the presence of ATPγS, this thesis study provided the first structural basis for ATP dependent DNA binding by the MR complex. Secondly, this study revealed the roles of ATP in DNA binding and unwinding by Rad50, and elucidated that ATP is an essential determinant in coupling the functions of Rad50 and Mre11 together. Thirdly, it demonstrated the important regulatory roles of coiled coil and hook domain in DNA binding and processing by revealing that truncated MR and full length MR complex adopt different DNA binding fashions. Fourthly, this study manifested that interaction between DNA and Rad50 is critical for the double strand break repair in vivo, thus validated the significance of MR complex as a double strand break repair protein machinery. Taken together, by using the multidisciplinary approach, this thesis study proposed a model whereby ATP-dependent DNA binding and unwinding activity of Rad50 is coupled to the endonuclease activity of Mre11. This study not only contributed to help filling the fundamental gap in understanding of the roles of ATP in the MR complex, but also elucidated a mechanistic framework for better understanding the interaction between DNA and MR complex.