title: Caspase-3 mediated cleavage of HsRad51 at an unconventional site.
abstract: 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 downward arrow 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 downward arrow 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 downward arrow 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 downward arrow N HsRad51 cleavage product. In Jurkat cell extracts, the AQVD274 downward arrow G and DVLD187 downward arrow 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.
authors.affiliation: Department of Biosciences, CNT, Novum, Karolinska Institute, Huddinge, Sweden. jenny.flygare@cnt.ki.se
authors.forename: J
authors.initials: J
authors.lastname: Flygare
authors.forename: D
authors.initials: D
authors.lastname: Hellgren
authors.forename: A
authors.initials: A
authors.lastname: Wennborg
mesh_terms: D000230:Adenocarcinoma / Q000201:enzymology / Q000473:pathology; D000595:Amino Acid Sequence; D017209:Apoptosis / Q000502:physiology; D001943:Breast Neoplasms / Q000201:enzymology / Q000473:pathology; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D019169:Jurkat Cells / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D051135:Rad51 Recombinase; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured / Q000378:metabolism
chemical_list: D061945:Caspase Inhibitors; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D019311:Staurosporine