The C-terminal motif of Mph1p for interaction with RPA has a critical role for GCR-promoting activity. (A) Patch test of an mph1Δ mutation that no longer produced colonies resistant to canavanine and 5-FOA that reflected the absence of GCR. (B) Schematic demonstration of a mutation that did not show GCR enhancement when it was overexpressed. It was named Mph1-CΔ because it translates C-terminus–truncated Mph1p. (C) Mph1p interacts with RPA through its C-terminal motif. Immunoprecipitation of Mph1p through its Flag tag pulled down RPA that was detected by GFP tag at its C terminus (top) using ATCC201388 with different plasmids (MATa his3Δ1leu2Δ0 met15Δ0 ura3Δ0 RFA1-GFP). Immunoprecipitation of RPA pulled down the full-length Mph1p (bottom). Ctrl, control plasmid; o/e, overexpression; WT wild-type plasmid. (D) The mph1Δ mutation reduced the number of cells with spontaneous RPA foci that are independent of Rad51p. (top) Examples of GFP-RPA cells, ATCC201388: wild type, mph1Δ, and mph1Δ strain complemented by a plasmid expressing Mph1 (mph1 + pMph1). (bottom) A graphic presentation of percentage of cells having spontaneous RPA foci from 100 cells from each strain counted. (E) Excess Mph1p enhanced RPA accumulation to DSB. ChIP of RPA at DSB with α-Rpa1p antibody was performed as described in Materials and methods. (F) Mph1p accumulated at DSB. ChIP of Mph1p was performed with α-HA antibody that recognizes the tag of Mph1p. Error bars represent standard deviation.