Meiosis-specific proteins MEIOB and SPATA22 cooperatively associate with the single-stranded DNA-binding replication protein A complex and DNA double-strand breaks

Abstract

Meiotic recombination ensures faithful segregation of homologous chromosomes during meiosis and generates genetic diversity in gametes. MEIOB (meiosis specific with OB domains), a meiosis-specific single-stranded DNA-binding homolog of replication protein A1 (RPA1), is essential for meiotic recombination. Here, we investigated the molecular mechanisms of MEIOB by characterizing its binding partners spermatogenesis associated 22 (SPATA22) and RPA. We find that MEIOB and SPATA22 form an obligate complex and contain defined interaction domains. The interaction between these two proteins is unusual in that nearly any deletion in the binding domains abolishes the interaction. Strikingly, a single residue D383 in MEIOB is indispensable for the interaction. The MEIOB/SPATA22 complex interacts with the RPA heterotrimeric complex in a collaborative manner. Furthermore, MEIOB and SPATA22 are recruited to induced DNA double-strand breaks (DSBs) together but not alone. These results demonstrate the cooperative property of the MEIOB-SPATA22 complex in its interaction with RPA and recruitment to DSBs.

Keywords: DNA double-strand breaks; MEIOB; RPA; SPATA22; meiosis.

Figure 1.

MEIOB forms an obligate complex with SPATA22. (A) Western blot analysis of MEIOB and SPATA22 protein levels in cells transfected with indicated plasmids. β-actin serves as a loading control and GFP serves as a transfection control. The numbers below the bands indicate normalized fold increases of MEIOB and SPATA22 protein levels. **, P < 0.01. (B) Co-IP analysis of MEIOB deletions and SPATA22. (C) Schematic diagram of MEIOB deletions and results of interactions with SPATA22 or RPA1, respectively. +, positive co-IP; –, negative co-IP. Western blots (WB) of the first five MEIOB deletions are shown in panel B and the remaining 11 deletions are shown in Supplementary Figure S1A and S1B. The OB fold (aa 167–272) is also shown. Western blots concerning RPA1 interaction are shown in Figure 4A. (D) Modeling of mouse MEIOB structure based on the fungus RPA heterotrimer structure by Phyre2 algorithm [19,20]. Residues 10–468 of MEIOB were modeled. Defined interaction domains and various regions are color-coded. The critical Asp383 residue is shown in blue.

Figure 2.

D383A mutation in MEIOB abolishes its interaction with SPATA22 but not with RPA1. (A) Mutations of D383 in MEIOB disrupt interaction with SPATA22. MEIOB WT serves as a positive co-IP control. *, antibody heavy chain. (B) Left, schematic of the co-IP analysis using separate MEIOB- or SPATA22-containing cell lysates. Right, co-IP analysis of the interaction between SPATA22 and WT MEIOB or mutant MEIOB (D383A) by mixing of separate protein lysates. *, antibody heavy chain. (C) Co-IP analysis of the interaction between RPA1 and WT MEIOB or mutant MEIOB (D383A). The numbers indicate a normalized RPA1/MEIOB ratio from the IP blots. NS, not significant.

Figure 3.

Identification of the MEIOB-binding domain in SPATA22. (A) Schematic diagram of SPATA22 deletions and co-IP results of interactions with MEIOB. +, positive co-IP; –, negative co-IP. (B) Western blots of co-IP analysis of the first five SPATA22 deletions. The remaining eight deletions are shown in Supplementary Figure S2A and S2B. *, antibody heavy chain. Co-IP results of SPATA22 interaction with RPA1 are shown in panel A but relevant western blots are shown in Supplementary Figure S3A.

Figure 4.

MEIOB and SPATA22 cooperatively interact with RPA. (A) Mapping of the RPA1-binding domain in MEIOB. Co-IP analysis of RPA1 and MEIOB deletions. (B) Co-IP analysis of MEIOB and RPA2 interaction in the presence or absence of SPATA22. (C) Co-IP analysis of MEIOB and RPA3 interaction in the presence or absence of SPATA22. *, antibody heavy chain. (D) Diagram of interactions among MEIOB, SPATA22, and the three RPA subunits. Solid lines indicate strong interactions as assayed by co-IP. Dashed lines indicate interactions only in the presence of both MEIOB and SPATA22. The interactions among RPA1, RPA2, and RPA3 are not shown. RPA1/RPA2/RPA3 or RPA2/RPA3 form soluble complexes when coexpressed in bacteria; however, RPA1/RPA2 or RPA1/RPA3 do not form soluble complexes in bacteria [43].

Figure 5.

DSB recruitment of MEIOB and SPATA22 depends on their coexpression and interaction. (A) Schematic of the inducible DSB reporter in U2OS cells. ER, estrogen receptor; DD, destabilization domain; 4-OHT, 4-hydroxytamoxifen; Shield-1, stabilizing ligand of DD-tagged protein. (B) Representative images of the colocalization between GFP-tagged proteins and induced DSBs (red). Reporter U2OS cells were transfected with indicated plasmids before administration of Shield-1 and 4-OHT. Scale bar, 10 μm. (C) Percentage of the indicated GFP-tagged proteins that colocalize with DSBs. More than 30 GFP and DSB double-positive cells were counted per experiment. The experiments were performed three times. ***, P < 0.005; N.S., not significant.