Support for a meiotic recombination initiation complex: interactions among Rec102p, Rec104p, and Spo11p

Abstract

Initiation of meiotic recombination in the yeast Saccharomyces cerevisiae requires at least 10 gene products. The initiation event creates double-strand breaks, which are then processed by other recombination enzymes. A variety of classical observations, such as the existence of recombination nodules, have suggested that the proteins catalyzing recombination form a complex. A variety of lines of evidence indicate that Rad50p, Mre11p, and Xrs2p interact, and genetic data suggesting interactions between Rec102p and Rec104p have been reported. It has recently been shown that Spo11p coimmunoprecipitates with Rec102p in meiosis as well. In this paper, we provide genetic and biochemical evidence that the meiosis-specific proteins Rec102p, Rec104p, and Spo11p all interact with each other in meiosis. Furthermore, we demonstrate that the interaction between Rec102p and Spo11p does not require Rec104p. Likewise, the interaction between Rec104p and Rec102p does not require Spo11p, although Spo11p may stabilize that association. The interactions suggest that Spo11p, Rec102p, and Rec104p may form a trimeric complex during the initiation of recombination.

FIG. 1.

Replica plating assay for suppression of various rec102 alleles by high-copy-number REC104. The assay detected TRP5 recombinants after sporulation at a nonpermissive temperature. V, high-copy-number vector; hc104, high-copy-number REC104.

FIG. 2.

Replica plating assay for suppression of rec104-8 by specific alleles of REC102. Meiotic recombination was measured by replica plating the LS5-1 diploid containing various forms of REC102 on Trp dropout medium. All other diagnostic media showed similar responses.

FIG. 3.

Specific point mutations in REC102 suppress the rec104-8 mutation. (A) Western blot assay measuring the amount of Rec102-HA (v, vector alone (pRS316); h, high-copy-number Rec102-HA; 48, Rec102-48-HA [in the CEN vector pRS316]; c, CEN Rec102-HA [in the vector pRS316]). (B) Amounts of CEN-Rec102-48-HA relative to CEN-Rec102-HA. Quantitation was done with ImageQuant software on a Molecular Dynamics Phosphorimager.

FIG. 4.

Coimmunoprecipitation (IP) of Rec102-HAp and FLAG-Rec104p. All proteins were isolated from an LS8-1 (rec104-Δ1/rec104-Δ1 rec102Δ/rec102Δ) diploid containing pLS37 (FLAG-Rec104p) and/or pJK108 (REC102-HA) after 6 h in sporulation medium. The antibody (Ab) used in the Western blot assay was anti-HA (α-HA). Lane 5 indicates the position of Rec102-HAp in a total protein (0) extract (100 μg loaded). Lane 2 demonstrates that Rec102-HAp can be coprecipitated by antibody to FLAG-Rec104p (α-FLG).

FIG. 5.

Replica plating assay for suppression of rec102 alleles by SPO11. Meiotic recombination was measured by replica plating on Trp dropout medium in JK9-1 diploids (rec102Δ) containing a plasmid with various mutant alleles of REC102 (shown below the replica) and either a vector (V) (pRS426) or a high-copy-number plasmid containing SPO11 (hc11) (pJK64). Tests were cone at the nonpermissive temperature. Four other media diagnostic for recombination showed similar responses.

FIG. 6.

Replica plating assay for suppression of various rec104 alleles by high-copy-number SPO11. Meiotic recombination was measured by replica plating on Trp dropout medium in diploids homozygous for various mutant alleles of REC104 and either a vector (V) (pRS426) or a high-copy-number plasmid containing SPO11 (hc11) (pJK22). Four other media diagnostic for recombination showed similar responses.

FIG. 7.

Spo11p interacts with Rec102p during meiosis. All proteins were isolated from diploid JK12-4 (rec102Δ/rec102Δ spo11Δ/spo11Δ) after 6 h in sporulation medium. The number 0 refers to total yeast protein with no immunoprecipitation (IP). The plus and minus signs above the lanes indicate which components were present. (A) Lanes 1, 3, and 5 are protein from cells containing plasmids pJK108 (REC102-HA) and pEG-KG (a GST expression vector [31]). Lanes 2, 4, and 6 are from cells containing plasmids pJK108 and pJK113 (a GST-SPO11 expression plasmid). Lanes 5 and 6 demonstrate that Rec102-HA coimmunoprecipitates with Gst-Spo11p. Lanes 3 and 4 demonstrate that Rec102-HA copurifies with Gst-Spo11p on a glutathione column. (B) Reciprocal coimmunoprecipitation between Rec102p and Spo11p. All proteins were isolated from diploid JK12-4. Lane 1 contains total protein and shows both Rec102-HAp and GST-Spo11p. Lane 2 demonstrates that GST-Spo11p coimmunoprecipitates with Rec102-HAp. Lane 3 is total protein with only GST present. Lane 4 demonstrates that precipitation of Rec102-Hap does not coprecipitate GST. Lane 5 demonstrates that no protein is precipitated if Rec102-HA is not present. Ab, antibody; IgG, immunoglobulin G.

FIG. 8.

Rec104p is not required for Rec102p-Spo11p association, and Spo11p is not required for Rec102p-Rec104p association. All protein was isolated from cells after 6 h in sporulation medium. The specific antibody (Ab) used to develop the Western blot is shown at the left of the lanes. The plus and minus signs above the lanes indicate which components were present. The number 0 refers to total yeast protein with no immunoprecipitation (IP). (A) Protein was isolated from isogenic rec102Δ spo11Δ REC104 (LS8-1-1A) and rec102Δ spo11Δ rec104-Δ1 (LS8-1-8B) diploids. The REC102-HA gene was carried on plasmid pJK108 and was present in all lanes. The GST-SPO11 construct was carried on plasmid pJK113, and the control GST expression vector was pEG-KG. Total protein (80 μg) was present in lanes 1 to 4 as controls. (B) Protein was isolated from isogenic rec102Δ rec104-Δ1 SPO11 (JK12-4) and rec102Δ rec104-Δ1 spo11Δ (LS8-1-8B) diploids. Lane 1 is total protein from JK12-4. The REC102-HA gene was carried on plasmid pJK108. The FLAG-REC104 gene was carried on pLS37. See Fig. 2 for some controls. (C) The amount of Rec102-HAp present in meiosis is the same in diploid cells that are SPO11 (LS2-8) or spo11Δ (JK12-4) mutants. Lanes: 1, LS2-8; 2, JK12-4. Samples were taken at 6 h, and equal amounts of protein were loaded.

FIG. 9.

Interactions among Rec102p, Rec104p, and Spo11p. (A) Summary of all known interactions among Rec102p, Rec104p, and Spo11p. CoIP, coimmunoprecipitation; Al.S.S, allele-specific suppressor; H.C.S., high-copy-number suppressor. The interaction for which there is the least evidence is that between Rec104p and Spo11p. The data are consistent with the notion that either all three proteins interact together (B) or Rec102p acts as a bridge between Rec104p and Spo11p (C). See the text for a discussion.