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Double-strand breaks(DSBs),one class of the most harmful DNA damage forms that bring elevated health risks,need to be repaired timely and effectively.However,an increasing number of environmental pollutants have been identified to impair DSB repair from vari-ous mechanisms.Our previous work indicated that the formation of unsaturated RecA nu-cleofilaments plays an essential role in homology recombination(HR)pathway which can accurately repair DSBs.In this study,by developing a benzonase cutting protection assay and combining it with traditional electrophoretic mobility shift assay(EMSA)analysis,we fur-ther investigated the assembly patterns of four RecA mutants that display differential DSB repair ability and ATPase activity.We observed that the mutants(G204S and S69G)possess-ing both ATP hydrolysis and DSB repair activities form unsaturated nucleofilaments similar to that formed by the wild type RecA,whereas the other two ATP hydrolysis-deficient mu-tants(K72R and E96D)that fail to mediate HR form more compacted nucleofilaments in the presence of ATP.These results establish a coupling of ATPase activity and effective DSB repair ability via the assembly status of RecA nucleofilaments.This linkage provides a po-tential target for environmental factors to disturb the essential HR pathway for DSB repair by suppressing the ATPase activity and altering the assembly pattern of nucleofilaments.