Caspase‐3 mediated cleavage of HsRad51 at an unconventional site

The human recombinase HsRad51 is cleaved during apoptosis. We have earlier observed cleavage of the 41‐kDa full‐length protein into a 33‐kDa product in apoptotic Jurkat cells and in in vitro translated HsRad51 after treatment with activated S‐100 extract. In this study, site‐directed mutagenesis was used for mapping of the cleavage site to AQVD274 ↓ G, which does not correspond to a conventional caspase cleavage site. The absence of HsRad51 cleavage in staurosporine‐treated apoptotic MCF‐7 cells, which lack caspase‐3, indicates that caspase‐3 is essential for HsRad51 cleavage in vivo . Cleavage into the 33‐kDa fragment was generated by recombinant caspase‐3 and ‐7 in in vitro translated wild type HsRad51, but not in the HsRad51 AQVE274 ↓ G mutant. Similarly, HsRad51 of Jurkat cell extracts was cleaved into the 33‐kDa product by recombinant caspase‐3, whereas caspase‐7 failed to cleave endogenous HsRad51. The cleavage of in vitro translated wild type and AQVE274 ↓ G mutant HsRad51 as well as of endogenous HsRad51 also gave rise to a smaller fragment, which corresponds in size to a recently reported DVLD187 ↓ N HsRad51 cleavage product. In Jurkat cell extracts, the AQVD274 ↓ G and DVLD187 ↓ N cleavage products of HsRad51 appeared at equal concentrations of caspase‐3. Moreover both fragments were generated by induction of apoptosis in MDA‐MB 157 cells with staurosporine and in Jurkat cells with camptothecin. Thus, two sites in the HsRad51 sequence are targets for caspase cleavage both in vitro and in vivo .