Mph1p promotes gross chromosomal rearrangement through partial inhibition of homologous recombination

Abstract

Gross chromosomal rearrangement (GCR) is a type of genomic instability associated with many cancers. In yeast, multiple pathways cooperate to suppress GCR. In a screen for genes that promote GCR, we identified MPH1, which encodes a 3'-5' DNA helicase. Overexpression of Mph1p in yeast results in decreased efficiency of homologous recombination (HR) as well as delayed Rad51p recruitment to double-strand breaks (DSBs), which suggests that Mph1p promotes GCR by partially suppressing HR. A function for Mph1p in suppression of HR is further supported by the observation that deletion of both mph1 and srs2 synergistically sensitize cells to methyl methanesulfonate-induced DNA damage. The GCR-promoting activity of Mph1p appears to depend on its interaction with replication protein A (RPA). Consistent with this observation, excess Mph1p stabilizes RPA at DSBs. Furthermore, spontaneous RPA foci at DSBs are destabilized by the mph1Delta mutation. Therefore, Mph1p promotes GCR formation by partially suppressing HR, likely through its interaction with RPA.

Figure 1.

A high level of Mph1p enhances GCR. (A) GCR formation caused by excess Mph1p depends on telomerase activity. (B) Defects in HR but not NHEJ enhanced GCR rates synergistically when Mph1p was highly expressed. The yku70Δ mutation decreased the level of GCR enhancement. (C) Inactivation of HR with Mph1p overexpression synergistically enhanced GCRs. o/e, overexpression; WT, wild type. − (gray) and + (black) indicate without and with Mph1p overexpression, respectively. The GCR rates are provided in Table S1 (available at http://www.jcb.org/cgi/content/full/jcb.200711146/DC1). Rates are presented as the mean of two median values with standard deviation.

Figure 2.

Excess Mph1p down-regulates HR. (A) High expression of Mph1p reduced mating type switching frequency using JKM161 with different plasmids (Δho HMLalpha MATa Δhmr:;ADE1 ade1-100 leu2-3,112 lys5 trp1∷hisg ura3-52 ade3∷GAL-HO endonuclease his-). (B) Excess Mph1p reduces the spontaneous recombination rate. (top) A schematic diagram of his3 inverted repeat spontaneous recombination assay using M137-11B with different plasmids (MATa can1-100 his3p∷INV leu2 lys2-128 trp1 ura3). (bottom) A graphic presentation of spontaneous recombination rates of cells carrying control (Ctrl) or Mph1p overexpression (o/e) plasmids. (C) GCR enhancement by excess Mph1p was completely blocked by Rad52p cooverexpression. (D) Excess Mph1p slowed down Rad51p recruitment to DSB. ChIP was performed using JKM161 with different plasmids. (E) Excess Mph1p made cells sensitive to γ irradiation and MMS. (F) Excess Mph1p made the dnl4Δ strain sensitive to MMS. (G) Strains carrying both mph1Δ and srs2Δ mutations showed synergistic sensitivity to MMS compared with strains carrying each single mutation. Rates are presented as the mean of two median values with standard deviation.

Figure 3.

ATPase, DEAH, or helicase motifs of Mph1p are dispensable for GCR-promoting activity and synergistic sensitivity to MMS with the srs2Δ mutation. (A) Locations of mutations used in this study. (B) The overexpression of ATPase, DEAH, or helicase mutant Mph1p proteins still showed strong GCR enhancement similar to what was achieved by the overexpression of wild-type Mph1p. The GCR rates are provided in Table S2 (available at http://www.jcb.org/cgi/content/full/jcb.200711146/DC1). (C) γ ray and MMS sensitivities caused by excess Mph1p remain when mutant Mph1p proteins were overexpressed. (D) The synergistic MMS sensitivity by the mph1Δ mutation in the srs2Δ strain was rescued by the Mph1ps carrying a mutation in the helicase or DEAH motifs. Rates are presented as the mean of two median values with standard deviation.

Figure 4.

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.