Ays a role in DSB repair upon the completion of meiotic recombination. To explore this in more detail, we utilised immunoprecipitation (IP) as a tool to determine endogenous GGN1-interacting partners in the mouse testis. In order to enrich the spermatocyte population and to avoid the presence of postmeiotic germ cells, postnatal day 20 Title Loaded From File testis was chosen for IPs. Equal amounts of testis extract were loaded onto a GGN1 Ig column and a control goat IgG column. Columns were prepared and 1655472 used in an identical manner and equal quantities of eluate were loaded onto the SDS-PAGE gels prior to immunoblotting. We began by confirming that endogenous GGN1 bound to FANCL in the testis (Figure 1A), consistent with previous yeasttwo-hybrid and over-expression coupled with pull-down studies [7]. This result identifies for the first time that GGN binds to FANCL under normal physiological conditions in vitro. FANCL is a member of the FA core complex that composed of at least 8 proteins including FANCA, -B, -C, -E, -F, -G, -L and M, with FANCL serving as catalytic subunit [11,15]. In somatic cells, a primary function of the core complex is to ubiquitinate FANCD2 and FANCI, which in turn leads to a nuclear translocation of the ubiquitinated FANCD2/FANCI complex and the recruitment of several proteins to the sites of DNA damage including proteins within the BRCA pathway. In germ cells, the components of the FA and BRCA pathways and their regulation are largely unidentified. As such, we next investigated if GGN1 also interacted with other components of the FA and BRCA pathways. Elutes from GGN1 testis immunopreciptations were probed with additional antibodies against proteins within the FA and BRCA pathways including FANCA, FANCD2, FANCI, BRCA1 and BRCC36. In addition to FANCL, the major component of the FA pathway, FANCD2, and the deubiquitinating enzyme within the BRCA pathway, BRCC36 [16] (Figure 1A) were co-immunoprecipitated with GGN1 while FANCA, FANCI and BRCA1 were not. The specificity of the interaction was further confirmed by reciprocal IPs. GGN1 was co-immunoprecipitated with BRCC36 (Figure 1B).Figure 1. GGN1 is a binding partner of FANCL, FANCD2 and BRCC36 in the mouse testis. (A) GGN1 interacted with FANCL, FANCD2 and BRCC36 in the mouse testis as determined by immunoprecipitation using spermatocyte-enriched postnatal day 20 testis lysate. (B) GGN1 co-immunoprecipitated with BRCC36 as determined by reciprocal pull down. doi:10.1371/journal.pone.0056955.gIn HeLa cells BRCC36 plays a role in DSB repair in response to ionizing radiation and the G2/M cell cycle Title Loaded From File checkpoint [17,18]. As a binding partner of FANCL, FANCD2 and BRCC36, we propose that GGN1 plays a role in DNA repair in the testis via its connection with the FA and BRCA pathways.Loss of GGN Leads to Pre-implantation Embryonic Lethality in the MouseWhile the expression of GGN highlighted its potential role in male germ cell development, the in vivo function of Ggn has not been defined. As such we generated a Ggn null mouse line (Figure 2A). The strategy used to generate the Ggn knockout mice was to delete the protein-coding region of Ggn gene in all cell types. Targeted 129Sv ES clones were verified by Southern blotting using 59 and 39external probes (Figure 2B). Although the heterozygous knockout mice (Ggn+/2) appeared grossly normal compared to wild-type littermates (Ggn+/+), homozygous knockout mice (Ggn2/2) were never found at weaning age (Table 1). To assess the potential for embryonic.Ays a role in DSB repair upon the completion of meiotic recombination. To explore this in more detail, we utilised immunoprecipitation (IP) as a tool to determine endogenous GGN1-interacting partners in the mouse testis. In order to enrich the spermatocyte population and to avoid the presence of postmeiotic germ cells, postnatal day 20 testis was chosen for IPs. Equal amounts of testis extract were loaded onto a GGN1 Ig column and a control goat IgG column. Columns were prepared and 1655472 used in an identical manner and equal quantities of eluate were loaded onto the SDS-PAGE gels prior to immunoblotting. We began by confirming that endogenous GGN1 bound to FANCL in the testis (Figure 1A), consistent with previous yeasttwo-hybrid and over-expression coupled with pull-down studies [7]. This result identifies for the first time that GGN binds to FANCL under normal physiological conditions in vitro. FANCL is a member of the FA core complex that composed of at least 8 proteins including FANCA, -B, -C, -E, -F, -G, -L and M, with FANCL serving as catalytic subunit [11,15]. In somatic cells, a primary function of the core complex is to ubiquitinate FANCD2 and FANCI, which in turn leads to a nuclear translocation of the ubiquitinated FANCD2/FANCI complex and the recruitment of several proteins to the sites of DNA damage including proteins within the BRCA pathway. In germ cells, the components of the FA and BRCA pathways and their regulation are largely unidentified. As such, we next investigated if GGN1 also interacted with other components of the FA and BRCA pathways. Elutes from GGN1 testis immunopreciptations were probed with additional antibodies against proteins within the FA and BRCA pathways including FANCA, FANCD2, FANCI, BRCA1 and BRCC36. In addition to FANCL, the major component of the FA pathway, FANCD2, and the deubiquitinating enzyme within the BRCA pathway, BRCC36 [16] (Figure 1A) were co-immunoprecipitated with GGN1 while FANCA, FANCI and BRCA1 were not. The specificity of the interaction was further confirmed by reciprocal IPs. GGN1 was co-immunoprecipitated with BRCC36 (Figure 1B).Figure 1. GGN1 is a binding partner of FANCL, FANCD2 and BRCC36 in the mouse testis. (A) GGN1 interacted with FANCL, FANCD2 and BRCC36 in the mouse testis as determined by immunoprecipitation using spermatocyte-enriched postnatal day 20 testis lysate. (B) GGN1 co-immunoprecipitated with BRCC36 as determined by reciprocal pull down. doi:10.1371/journal.pone.0056955.gIn HeLa cells BRCC36 plays a role in DSB repair in response to ionizing radiation and the G2/M cell cycle checkpoint [17,18]. As a binding partner of FANCL, FANCD2 and BRCC36, we propose that GGN1 plays a role in DNA repair in the testis via its connection with the FA and BRCA pathways.Loss of GGN Leads to Pre-implantation Embryonic Lethality in the MouseWhile the expression of GGN highlighted its potential role in male germ cell development, the in vivo function of Ggn has not been defined. As such we generated a Ggn null mouse line (Figure 2A). The strategy used to generate the Ggn knockout mice was to delete the protein-coding region of Ggn gene in all cell types. Targeted 129Sv ES clones were verified by Southern blotting using 59 and 39external probes (Figure 2B). Although the heterozygous knockout mice (Ggn+/2) appeared grossly normal compared to wild-type littermates (Ggn+/+), homozygous knockout mice (Ggn2/2) were never found at weaning age (Table 1). To assess the potential for embryonic.
