An essential component of the homologous recombination machinery in eukaryotes, the RAD54 protein is a member of the SWI2/SNF2 family of helicases with dsDNA-dependent ATPase, DNA translocase, DNA supercoiling and chromatin remodelling activities. It is a motor protein that translocates along dsDNA and performs multiple functions in homologous recombination. In particular, RAD54 is an essential cofactor for regulating RAD51 activity. It stabilizes the RAD51 nucleofilament, remodels nucleosomes, and stimulates homology search and strand invasion activity of RAD51. Accordingly, deletion of RAD54 has dramatic consequences on DNA damage repair in mitotic cells. In contrast, its role in meiotic recombination is less clear. RAD54 is essential for meiotic recombination in Drosophila and C. elegans, but plays minor roles in yeast and mammals. We present here characterization of the roles of RAD54 in meiotic recombination in the model plant Arabidopsis thaliana. Absence of RAD54 has no detectable effect on meiotic recombination in otherwise wild-type plants but RAD54 becomes essential for meiotic DSB repair in absence of DMC1. In Arabidopsis, dmc1 mutants have an achiasmate meiosis, in which RAD51 repairs meiotic DSBs. Lack of RAD54 leads to meiotic chromosomal fragmentation in absence of DMC1. The action of RAD54 in meiotic RAD51 activity is thus mainly downstream of the role of RAD51 in supporting the activity of DMC1. Equivalent analyses show no effect on meiosis of combining dmc1 with the mutants of the RAD51-mediators RAD51B, RAD51D and XRCC2. RAD54 is thus required for repair of meiotic DSBs by RAD51 and the absence of meiotic phenotype in rad54 plants is a consequence of RAD51 playing a RAD54-independent supporting role to DMC1 in meiotic recombination.

22An essential component of the homologous recombination machinery in eukaryotes, 23 the RAD54 protein is a member of the SWI2/SNF2 family of helicases with dsDNA-dependent 24 ATPase, DNA translocase, DNA supercoiling and chromatin remodelling activities. It is a motor 25 protein that translocates along dsDNA and performs multiple functions in homologous 26 recombination. In particular, RAD54 is an essential cofactor for regulating RAD51 activity. It 27 stabilizes the RAD51 nucleofilament, remodels nucleosomes, and stimulates homology search 28 and strand invasion activity of RAD51. Accordingly, deletion of RAD54 has dramatic 29 consequences on DNA damage repair in mitotic cells. In contrast, its role in meiotic 30 recombination is less clear. 31RAD54 is essential for meiotic recombination in Drosophila and C. elegans, but plays 32 minor roles in yeast and mammals. We present here characterization of the roles of RAD54 in 33 meiotic recombination in the model plant Arabidopsis thaliana. Absence of RAD54 has no 34 detectable effect on meiotic recombination in otherwise wild-type plants but RAD54 becomes 35 essential for meiotic DSB repair in absence of DMC1. In Arabidopsis, dmc1 mutants have an 36 achiasmate meiosis, in which RAD51 repairs meiotic DSBs. Absence of RAD54 in dmc1 37 mutants leads to meiotic chromosomal fragmentation. The action of RAD54 in meiotic RAD51 38 activity is thus downstream of the role of RAD51 in supporting the activity of DMC1. Equivalent 39 analyses show no effect on meiosis of combining dmc1 with the mutants of the RAD51-40 mediators RAD51B, RAD51D and XRCC2. 41RAD54 is thus required for repair of meiotic DSBs by RAD51 and the absence of 42 meiotic phenotype in rad54 plants is a consequence of RAD51 playing a RAD54-independent 43 supporting role to DMC1 in meiotic recombination. 44 45 46 3 47 Author Summary48 Homologous recombination is a universal pathway which repairs broken DNA molecules 49 through the use of homologous DNA templates. It is both essential for maintenance of genome 50 stability and for the generation of genetic diversity through sexual reproduction. A central step 51 of the homologous recombination process is the search for and invasion of a homologous 52 intact DNA sequence that will be used as template. This key step is catalysed by the RAD51 53 recombinase in somatic cells and RAD51 and DMC1 in meiotic cells, assisted by a number of 54 associated factors. Among these, the chromatin-remodelling protein RAD54 is a required 55 cofactor for RAD51 in mitotic cells. Understanding of its role during meiotic recombination 56 however remains elusive. We show here that RAD54 is required for repair of meiotic double 57 strand breaks by RAD51 in the plant Arabidopsis thaliana, and this function is downstream of 58 the meiotic role of RAD51 in supporting the activity of DMC1. These results provide new 59 insights into the regulation of the central step of homologous recombination in plants and very 60 probably also other multicellular eukaryotes. 61 4 62 Introduction 63 Homolo...