Novel Synaptonemal Complex Protein SYCE3 Figure 10. SYCE3 interacts with SYCE1 and SYCE2. Co-immunoprecipitation analysis was carried out by using transfected COS-7 cells. Cells were transfected with myc-SYCE3 and the respective EGFP fusion constructs of SYCE1, SYCE2, Tex12, SYCP1 1–200 and SYCP1 820–997. Protein- complexes were immunoprecipitated overnight with either an anti-myc antibody (A, C, E, F and G, bait: myc-SYCE3) or an anti-EGFP antibody (B, bait: EGFP-SYCE1; D, bait: EGFP-SYCE2) and analyzed by Western blot analysis. As a negative control, we performed an immunoprecipitation using the same antibody but cells that were solely transfected with the ‘‘prey’’ protein in parallel to each experiment. The input sample contained 5% of the total proteins used for the immunoprecipitation. Upper panels in each sub-figure show the detection of prey protein. Arrowheads indicate specific co-immunoprecipitated proteins. The lower part of each panel displays detection of the bait protein. Asterisks mark heavy chains of immunoglobulins (anti-myc or anti-EGFP antibody) utilized for immunoprecipitation. (A) myc-SYCE3 co-precipitates EGFP-SYCE1 highlighted by the arrowhead marking the SYCE1 specific band. (B) ‘‘Reverse’’ co-immunoprecipitation showing that EGFP-SYCE1 precipitates myc-SYCE3 (arrowhead marks myc-SYCE3). It can be seen in (C) that myc-SYCE3 pulls down EGFP-SYCE2 (arrowhead). (D) ‘‘Reverse’’ co-immunoprecipitation with EGFP-SYCE2 pulling down myc-SYCE3. (E–G) myc-SYCE3 does not bind to SYCP1 1-200-EGFP (E), EGFP-SYCP1 820–997 (F) or Tex12-EGFP (G). doi:10.1371/journal.pgen.1002088.g010 of SYCE3 requires SYCP1. On the other hand, in our co- transfection/immunoprecipitation studies we obtained no evi- dence for an interaction between SYCP1 and SYCE3 (Figure 10). One possibility might be that interaction between these two proteins requires higher order structures of SYCP1 (for example dimerization or N-terminal association of dimers) that cannot occur under the conditions of the experimental assays. However, the existence of additional, yet undiscovered CE proteins that would mediate this binding cannot be ruled out a priori. Homologous Recombination Fails to Complete in the Absence of SYCE3 In early spermatocytes of Syce3-deficient mice cH2AX is distributed as in wild-type animals suggesting that homologous recombination is initiated in a wild-type manner. However, in later stages of prophase I cH2AX shows altered dynamics in spermatocytes and oocytes. In these cells it remains associated with chromosomes (Figure 8 and Figure 9). Thus we suggest that induced DSBs are repaired inefficiently in Syce3 knockout meiocytes. This assumption is further supported by the analysis of components of the recombination machinery that assemble at the sites of DSBs as a further step during the process of meiotic recombination [41,43]. In the absence of SYCE3, both Syce32/2 spermatocytes and oocytes reveal differences to the wild-type: RAD51 - as well as RPA foci - stay associated with chromosomal cores and virtually no MLH1 signal is detectable (Figure 8 and Figure 9). The observed defects indicate that the exchange of homologous DNA strands (catalyzed by RAD51) and the formation of early recombination intermediates can take place in the absence of SYCE3, but further processing of the recombination sites fails to occur. As a consequence, no crossovers are formed in Syce32/2 meiocytes (see also [41,43]). Interestingly, disruption of the CE by eliminating either Syce1, Syce2 or Tex12 results in a similar PLoS Genetics | www.plosgenetics.org 11 May 2011 | Volume 7 | Issue 5 | e1002088