Proteolytic cleavage of HsRad51 during apoptosis.
The Rad51 gene of Saccharomyces cerevisiae is required for genetic recombination and recombinational repair of DNA strand breaks. In higher eukaryotes Rad51 is essential for embryonic development, and is involved in cell proliferation and DNA repair. Here we show that human Rad51 (HsRad51) is proteolytically cleaved during apoptosis in two T-lymphocyte cell lines, Jurkat and PFI-285. Apoptosis was induced by camptothecin or anti-Fas monoclonal antibody (anti-Fas mAb). HsRad51 was cleaved with similar kinetics as human poly(ADP-ribose) polymerase (HsPARP) after treatment with either agent. The time course of cleavage coincided with internucleosomal DNA fragmentation. The HsRad51 fragments observed in apoptotic cells were identical to those generated from in vitro translated (IVT) HsRad51 exposed to activated Jurkat S-100 extract in a cell-free system. In each case, cleavage of HsRad51 was abolished by acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO). However, cleavage of IVT HsRad51 could not be demonstrated using purified caspase-2, -3 or -6 to -10, and the identity of the responsible protease thus remains to be determined. In summary, we have shown that HsRad51 belongs to a group of repair proteins, including PARP and DNA-dependent protein kinase, which are specifically cleaved during the execution phase of apoptosis.
Flygare
J
J
Department of Biosciences, CNT, Novum, Karolinska Institutet, Huddinge, Sweden. jenny.flygare@cnt.ki.se
Armstrong
R C
RC
Wennborg
A
A
Orsan
S
S
Hellgren
D
D
D000911:Antibodies, Monoclonal; D017209:Apoptosis / Q000502:physiology*; D002166:Camptothecin / Q000494:pharmacology; D002461:Cell Line, Transformed; D002474:Cell-Free System; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004264:DNA Topoisomerases, Type I / Q000494:pharmacology; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells / Q000201:enzymology; D009687:Nuclear Proteins; D009842:Oligopeptides / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051135:Rad51 Recombinase; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013601:T-Lymphocytes / Q000201:enzymology*; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology; D019014:fas Receptor / Q000502:physiology
D000911:Antibodies, Monoclonal; D015853:Cysteine Proteinase Inhibitors; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009842:Oligopeptides; D015842:Serine Proteinase Inhibitors; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; D019014:fas Receptor; C096429:L 709049; D014108:Tosylphenylalanyl Chloromethyl Ketone; D011065:Poly(ADP-ribose) Polymerases; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin