New feature of hMEIOB and hSPATA22 binding to ssDNA from a single-molecule perspective

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.

1. Introduction

The integrity and fidelity of meiotic recombination are prerequisites for the formation of mature gametes during sexual reproduction [1,2]. The formation and repair of DNA double-strand breaks (DSBs) are indispensable for meiosis in mammals [3–6]. The SPO11-TopoVIBL complex cleaves the DNA double strands to form DSBs, DSB ends are further resected by the endonucleases MRE11 and exonuclease EXO1 to generate single strand 3′ overhangs [7]. This single-strand DNA (ssDNA) is coated and protected by RPA complex. Subsequently, the RecA-like protein RAD51 and its meiosis-specific homolog DMC1 are recruited by a variety of proteins, including BRCA2-MEILB2-BRME1 complex and MEIOB-SPATA22 complex, to replace the RPA complex, mediate strand-invasion and promote the formation of displacement loop (D-loop) [6,7]. After D-loop formation, DSBs are repaired via the synthesis-dependent strand annealing pathway or the double Holliday junction pathway [8,9]. Notably, the gonad-specific expression of the DSB repair-related proteins MEIOB-SPATA22 heterodimers found in rodents guide ssDNA strand invasion in the form of the heterodimer, and the subsequent MEIOB may be involved in the second-strand capture process during meiosis [10–12]. Deficiency of Meiob or Spata22 leads to meiosis failure and sterility in both sexes of rodents [12,13]. In addition, truncation of the C-terminus of MEIOB reportedly prevents MEIOB from interacting with SPATA22, resulting in primary ovarian insufficiency in women [14,15]. Mutations in Meiob have also been found in patients with azoospermia and testicular cancer [16–18].

Considering the importance of MEIOB-SPATA22 heterodimers in spermatogenesis, the underlying molecular mechanism has been explored. During strand invasion, the RPA complex, including RPA1, RPA2 and RPA3, first binds to ssDNA to protect it from digestion and recruits the MEIOB-SPATA22 heterodimer to promote strand invasion [19–21]. Recently, new evidence has shown that the C-terminal domains of MEIOB and SPATA22 play a decisive role in heterodimer formation and affinity for the RPA complex [14,22]. Although the MEIOB-SPATA22 heterodimer is conserved between humans and rodents, it is not known whether there are mechanistic differences.