Dual functions for the ssDNA-binding protein RPA in meiotic recombination

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.

SKP1 drives the prophase I to metaphase I transition during male meiosis

“…Replication protein A (RPA) is a single-strand DNA binding heterotrimeric complex of RPA1, RPA2, and RPA3. RPA is essential for meiotic recombination (15). We examined the localization of RPA2 as a representative of the RPA complex.…”

“…Cells were suspended in 100 mM sucrose and were then spread on a thin layer of paraformaldehyde solution containing Triton X-100. The following primary antibodies were used for immunofluorescence analyses: rabbit anti-SYCP1 (1:100, catalog number ab15090, Abcam), mouse anti-SYCP1 (1:200; a gift from C. Hoog), guinea pig anti-SYCP2 (43), mouse anti-SYCP3 (1:200; catalog number ab97672, Abcam), rabbit anti-SYCP3 (1:200; 23024-1-AP), rabbit anti-SKP1 (1:50; catalog number ab10546, Abcam), rabbit anti-HORMAD1 (12), rabbit anti-HORMAD1 (1:200; 13917-1-AP, Proteintech Group), rabbit anti-HORMAD2 (10), mouse anti-H2AX (1:500; catalog number 16-202A, clone JBW301, Millipore), guinea pig anti-H1T (1:500; a gift from M. A. Handel) (4), rabbit anti-RPA2 (1:200; UP2436) (15), mouse anti-MLH1 (1:50; catalog number 550838, clone G168-15, BD Biosciences), human anti-CREST (1:100; 15-234, Antibodies Incorporated), mouse anti-CCNB1 (1:200; ab72, Abcam), rabbit anti-pHH3 (1:300; 9701S, Cell Signaling Technology), rabbit anti-PLK1 (1:50; UP2456; this study), mouse anti-BUB1 (1:50; a gift from Y. Watanabe) (44,45), and rabbit anti-CENP-C (1:500; a gift from Y. Watanabe) (46).…”

A missense in HSF2BP causing Primary Ovarian Insufficiency affects meiotic recombination by its novel interactor C19ORF57/MIDAP

“…In meiosis, RPA is forming a heterodimer with the essential meiotic players MEIOB (homologue of RPA) and SPATA22. However, the loading of the complex formed by MEIOB/SPATA22 to DSBs is RPA-independent (Shi et al, 2019). It is postulated that RPA functions in meiosis at two different stages; i) during the early recombination stages when the DSBs ends are resected by the MRN complex and ii) during the strand invasion into the homologue duplex that is carried out by RAD51 and DMC1, ssDNA is generated at the displacement loops (Shi et al, 2019).…”

“…However, the loading of the complex formed by MEIOB/SPATA22 to DSBs is RPA-independent (Shi et al, 2019). It is postulated that RPA functions in meiosis at two different stages; i) during the early recombination stages when the DSBs ends are resected by the MRN complex and ii) during the strand invasion into the homologue duplex that is carried out by RAD51 and DMC1, ssDNA is generated at the displacement loops (Shi et al, 2019). The accumulation of RPA observed in the Hsf2bp -/and Midap -/zygonema-like and in the Hfs2bp S167L/S167L spermatocytes is likely to occur at the early stages of recombination, because SPATA22 loading to the DSBs is also increased in the HSF2BP and MIDAP KOs and also to a lesser extent in the Hsf2bp S167L/S167L .…”

RPA complexes inCaenorhabditis elegansmeiosis; unique roles in replication, meiotic recombination and apoptosis

“…RPA is required for efficient RAD-51 recruitment to ssDNA, an essential step in the repair by HR (3,49,50). Therefore, if RPA loading is impaired, we would not expect RAD-51 to form foci in pachytene nuclei.…”