摘要
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 various mechanisms.Our previous work indicated that the formation of unsaturated Rec A nucleofilaments 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 further investigated the assembly patterns of four Rec A mutants that display differential DSB repair ability and ATPase activity.We observed that the mutants(G204S and S69G) possessing both ATP hydrolysis and DSB repair activities form unsaturated nucleofilaments similar to that formed by the wild type Rec A,whereas the other two ATP hydrolysis-deficient mutants(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 Rec A nucleofilaments.This linkage provides a potential 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.
基金
supported by the National Natural Science Foundation of China (Nos.21927807 and 91743201)
the Ministry of Science and Technology of China (Nos.2018YFC1005003 and Y9L10301)。
