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

Baolu Shi,Jiahao Sha,Mengcheng Luo,Qinghua Shi,Xiaoyan Huang,Mingxi Liu,Jiangyang Xue,Lan Ye,P Jeremy Wang,Hao Yin,Rui Guo,Paula E Cohen

doi:10.1371/journal.pgen.1007952

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.

Highlights

During sexual reproduction, meiotic recombination permits reciprocal exchange of genetic materials between homologous chromosomes and ensures faithful chromosome segregation [1, 2]
By conventional immunofluorescence microscopy, we previously showed that RPA1
By super-resolution imaging microscopy (SIM), we found that RPA1 foci were present in leptotene, zygotene, early pachytene, and mid pachytene spermatocytes, but absent in late pachytene and diplotene spermatocytes (Fig 1A– 1F)

Summary

Introduction

Meiotic recombination permits reciprocal exchange of genetic materials between homologous chromosomes and ensures faithful chromosome segregation [1, 2]. 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 [2]. 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 [4,5,6,7]. The MEIOB/SPATA22 heterodimer interacts with RPA and could position critical proteins for meiotic recombination [15]. A long-standing question remains as to whether RPA is required for loading of these ssDNA-binding proteins to DSBs in vivo

Methods

Results

Discussion

Conclusion