Rice RAD51 Paralogs Play Essential Roles In Somatic Homologous Recombination In DNA Repair

DNA double strand breaks(DSB)are highly cytotoxic DNA damage and will severely threaten the physical and genetic integrity of genome if not properly repaired.Inappropriate repair of broken DNA ends may result in the generation of harmful genomic rearrangements.The accumulation of DSBs could lead to apoptosis or development of cancer.In order to minimize the catastrophic consequence,the eukaryotic cells are equipped with 2 major mechanisms to deal with DSB.One is nonhomologous DNA end-joining(NHEJ)and the other one is homologous recombination(HR).NHEJ can directly rejoin two DNA ends and functions throughout the cell cycle,however,is often error-prone.On the contrary,HR is restricted in S and G2 phases and can utilize the homologous strand as template to recover the damage strand,ensuring high precision repair.Synthesis-dependent strand annealing(SDSA)and single-strand annealing(SSA)are the two main homologous recombination(HR)pathways in double-strand break(DSB)repair.RAD51 paralogs proteins mainly has five members:RAD51B,RAD51C,RAD51D,XRCC2,and XRCC3.The involvement of rice RAD51 paralogs in HR is well known in meiosis,although their molecular mechanisms in somatic HR remain obscure.And their action mechanisms presented in Arabidopsis display difference with that in human cells.In the present study,we show here that loss-of-function mutants of rad51 paralogs present increased sensitivity to the DSB-inducer bleomycin,which results in greatly compromised somatic recombination efficiencies(xrcc3 in SDSA,rad51b and xrcc2 in SSA,rad51c and rad5 1d in both).Using immunostaining,we found that mutations in RAD51 paralogs(XRCC3,RAD51C,or RAD51D)lead to tremendous impairment in RAD51 focus formation at DSBs.Intriguingly,mutation of RAD51C has a strong impact on the protein loading of its partners(XRCC3 and RAD51B)at DSBs,which is similar to the phenomenon observed in the case of blocking PI3K-like kinases in wild-type plant.We conclude that the rice CDX3 complex acts in the SDSA recombination while the BCDX2 complex acts in the SSA recombination in somatic DSB repair.Importantly,the RAD51C is not only required for the recruitment of RAD51 proteins(RAD51A1 and RAD51A2),it also serves as a fulcrum for the local recruitment of its partners(XRCC3 for SDSA and RAD51B for SSA)positively modulated by PI3K-like kinases,to facilitate both the SDSA and SSA pathways in RAD51 paralog-dependent somatic HR.