Date Published: February 4, 2019
Publisher: Public Library of Science
Author(s): Baolu Shi, Jiangyang Xue, Hao Yin, Rui Guo, Mengcheng Luo, Lan Ye, Qinghua Shi, Xiaoyan Huang, Mingxi Liu, Jiahao Sha, P. Jeremy Wang, Paula E. Cohen
Abstract: Meiotic recombination permits exchange of genetic material between homologous chromosomes. The replication protein A (RPA) complex, the predominant ssDNA-binding complex, is required for nearly all aspects of DNA metabolism, but its role in mammalian meiotic recombination remains unknown due to the embryonic lethality of RPA mutant mice. RPA is a heterotrimer of RPA1, RPA2, and RPA3. We find that loss of RPA1, the largest subunit, leads to disappearance of RPA2 and RPA3, resulting in the absence of the RPA complex. Using an inducible germline-specific inactivation strategy, we find that loss of RPA completely abrogates loading of RAD51/DMC1 recombinases to programmed meiotic DNA double strand breaks, thus blocking strand invasion required for chromosome pairing and synapsis. Surprisingly, loading of MEIOB, SPATA22, and ATR to DNA double strand breaks is RPA-independent and does not promote RAD51/DMC1 recruitment in the absence of RPA. Finally, inactivation of RPA reduces crossover formation. Our results demonstrate that RPA plays two distinct roles in meiotic recombination: an essential role in recombinase recruitment at early stages and an important role in promoting crossover formation at later stages.
Partial Text: During sexual reproduction, meiotic recombination permits reciprocal exchange of genetic materials between homologous chromosomes and ensures faithful chromosome segregation [1, 2]. Abnormalities in meiotic recombination are a leading cause of aneuploidy, infertility, and pregnancy loss in humans . Meiotic recombination is initiated by the formation of programmed DNA double strand breaks (DSBs) in germ cells and involves a large number of single-stranded DNA (ssDNA)-binding proteins . These DSBs undergo end resection to generate 3’ ssDNA overhangs; subsequent loading of RAD51 and DMC1 recombinases and other proteins enables strand invasion into duplex DNA for homologue pairing and recombination intermediate formation [4–7]. All meiotic DSBs are repaired but only a subset lead to crossovers, which are critical for proper segregation of homologous chromosomes during the first meiotic cell division.
RPA is often included in in vitro recombination reactions but its specific requirement is unknown. Recombinases RAD51 and DMC1 form helical nucleoprotein filaments on ssDNA [7, 32] and RPA is necessary for efficient RAD51 filament formation . The DNA strand exchange activities catalysed by RAD51 or DMC1 nucleoprotein filaments are rather inefficient but strongly stimulated by RPA [6, 7, 32]. These biochemical studies support RPA as an important accessary factor in recombination. While studies of yeast hypomorphic Rpa1 mutants also support a role for RPA in meiotic recombination , these studies did not address whether RPA is required for pre-synaptic filament formation. Using loss of function Rpa1 mouse mutants, we demonstrate that RPA loading to meiotic DSBs is a pre-requisite for RAD51/DMC1 loading in vivo (Fig 5C). One possible explanation is that RPA loading prevents formation of ssDNA secondary structure and recruits RAD51/DMC1 to these sites of recombination. An alternative but less likely explanation is that RAD51/DMC1 nucleation on ssDNA is independent of RPA, but that their localization to DSBs is stabilized by RPA. Finally, the fact that MEIOB/SPATA22 still localizes as foci in Rpa1-null spermatocytes suggests that the requirement of RPA in RAD51/DMC1 loading is unique and cannot be compensated for by other ssDNA-binding complexes such as MEIOB/SPATA22.