Abstract
MEIOB and SPATA22 are gonad-specific proteins that function in meiosis recombination. Mutations in these two proteins cause oligospermia or azoospermia in human males. It has been reported that the heterodimer composed of MEIOB and SPATA22 recognizes and binds to the single-strand DNA (ssDNA) protected by the replication protein A (RPA) complex to promote DNA damage repair during homologous recombination. However, the amino acid sequences of the two proteins are inconsistent in humans and rodents, which leads to functional differences in meiosis. In this study, human-derived MEIOB (hMEIOB) and SPATA22 (hSPATA22) are expressed and purified for electrophoretic mobility shift assay (EMSA), magnetic tweezer (MT) assay and bio-layer interferometry (BLI) assay to analyze the ssDNA binding patterns. The results show that hMEIOB has low ssDNA-binding affinity and stability alone, but hSPATA22 binds to ssDNA faster and more stably and promotes ssDNA condensation. Strong binding affinity and stability to ssDNA are present when the hMEIOB-hSPATA22 heterodimer is formed. Moreover, we find that multiple hMEIOB-hSPATA22 heterodimers spontaneously aggregate in vitro. hRPA complex weakens the binding affinity of hMEIOB, hSPATA22 and hMEIOB-hSPATA22 heterodimer to ssDNA, and it can also bind to hSPATA22 and hMEIOB-hSPATA22 heterodimer in vitro, which might be related to the proven function of RPA complex to protect ssDNA and recruit proteins related to DNA damage repair during meiosis. Overall, this study is the first time to elucidate the binding patterns of the hMEIOB and hSPATA22 to ssDNA in vitro, and to verify the relationship between the RPA complex and meiosis-related proteins, MEIOB and SPATA22, from single-molecule perspective.