Determination of structure of Mre11-Rad50 complex bound to DNA double strand break by NMR spectroscopy.
Our genomic DNA is exposed to a number of stress conditions such as reactive oxygen species, ionizing radiation, chemical agents and ultraviolet light which can result in DNA double strand breaks (DSBs). Inefficiency of the proteins involved in the DNA damage response to repair the damage might have tumorogenic potential. The initial complex involved in DNA DSB repair is Mre11-Rad50-Nbs1 (MRN). It is thought to be involved in binding to and unwinding the damaged DNA double helix, processing the DNA ends and recruiting other downstream proteins to repair the damage. Advances in solution state NMR spectroscopy have enabled us to selectively label and study large protein complexes like MR (~200 kDa). We utilize side chain methyl labelling of Pyrococcus furiosus (Pf) Mre11 and Rad50 to study the structure and dynamics of the individual proteins and their complexes. Methyl groups are isotopically ILVM-labeled at isoleucine(Cδ1), leucine(Cδ), valine(Cγ) and methionine(Cε) for HMQC and NOESY experiments. The existing X-ray crystal structure of the Pf Mre11 nuclease and capping domains was used to help assign the labeled methyl groups in the NMR spectra leading to more than 80% assignments. We have made MR complex using ILVM-labeled Mre11 and deuterated Rad50 and have added a 15 bp DNA with a 2 nucleotide 3’ overhang, mimicking a DNA DSB substrate, to form the complete MR-DNA complex. HMQC spectra show significant changes in chemical shifts of various Mre11 methyl groups upon DNA binding, including M109 and M146, which is in agreement with the published Mre11-DNA crystal structure. We have looked at different DNA substrates bound to MR, such as ssDNA and a branched DNA to mimic a stalled replication fork. Distance restraints obtained from Paramagnetic Relaxation Enhancement (PRE) experiments and Chemical Shift Perturbations (CSPs) observed after DNA binding were used to dock the dsDNA molecules into MR structure. Together this data reveals the changes in the structure of the MR complex when it binds to DNA DSBs which will further enable us to study its role in DNA damage repair.