Our previous in vitro studies showed that mutant RAD51 proteins, in which Tyr-315 is replaced with Asp or Glu to mimic phosphorylation, were proficient in double-strand DNA (dsDNA) binding, but were defective in dsDNA unwinding, probably due to defect in filament formation on dsDNA [18]. Consistent with these findings, fluorescence spectroscopic analysis of the RAD51-Y315W mutant suggested that Tyr-315 is closed to the subunit–subunit interacting site [19] (see also Supplementary Fig. 1). Thus, it is of interest to study whether c-ABL has any effects on in vivo behaviors of self-association-defective RAD51 mutants. Since RAD51filament formation in vivo is a complex cellular process, especially in higher
vitamin D binding protein precrusor (208-21 cool cells, requiring transport to and assembly in chromatin, before RAD51 nucleoprotein filament formation on damaged DNA [14] and [20], we examined effects of c-ABL on nuclear transport or chromatin association of RAD51. HA-tagged RAD51-WT or RAD51-R167G was transiently co-expressed with Flag-tagged active (WT) or kinase-dead (kd) c-ABL in 293T cells, and cells were fractionated into cytoplasmic, nuclear and chromatin-associated fractions. Anti-human RAD51
antibodies were used to detect endogenous RAD51 as well as transiently expressed HA-tagged RAD51, and α-tubulin and Histone H3 were used as specific makers for the soluble cytoplasmic and insoluble chromatin-associated fractions, respectively. As overall distributions of RAD51-WT, RAD51-R167G, endogenous RAD51 and c-ABL were comparable in the cytoplasmic and nuclear fractions (data not shown), data for protein levels in the nuclear fraction was not shown for simplicity.
