Fig 10. Rif2 enhances MRX ATPase activity.

(A–C) The ATPase activity of wild-type MRX, MR^V1269MX and MR complexes was determined in the absence or presence of Rif2. Plotted values are the mean value with error bars denoting s.d. (n = 3). (D) Exponentially growing cultures were serially diluted (1:10) and each dilution was spotted out onto YEPD plates with or without CPT, phleomycin, or MMS. (E) Model for the roles of Tel1 and Rif2 in controlling MRX function at DSBs. In wild-type cells (left), Tel1 enhances MRX function by promoting MRX association to DSBs (black arrows). Proper MRX accumulation to DNA increases the number of productive intercomplex interactions between hook domains to maintain DNA strands in close proximity. Rif2 inhibits MRX accumulation at DSBs by counteracting MRX-Tel1 interaction (black bar-headed line) and enhances MRX ATPase activity (red arrows). In tel1Δ rad50-V1269M cells (middle), the reduced amount of MR^V1269MX bound at the DSB severely impairs end-tethering, HR and NHEJ. In rif2Δ tel1Δ rad50-V1269M cells (right), the lack of Rif2 restores end-tethering, HR and NHEJ by increasing the time spent by MRX in the ATP-bound state. The MR^V1269MX complex is indicated in grey.