ARTEMIS nuclease facilitates apoptotic chromatin cleavage.
One hallmark of apoptosis is DNA degradation that first appears as high molecular weight fragments followed by extensive internucleosomal fragmentation. During apoptosis, the DNA-dependent protein kinase (DNA-PK) is activated. DNA-PK is involved in the repair of DNA double-strand breaks (DSB) and its catalytic subunit is associated with the nuclease ARTEMIS. Here, we report that, on initiation of apoptosis in human cells by agents causing DNA DSB or by staurosporine or other agents, ARTEMIS binds to apoptotic chromatin together with DNA-PK and other DSB repair proteins. ARTEMIS recruitment to chromatin showed a time and dose dependency. It required DNA-PK protein kinase activity and was blocked by antagonizing the onset of apoptosis with a pan-caspase inhibitor or on overexpression of the antiapoptotic BCL2 protein. In the absence of ARTEMIS, no defect in caspase-3, poly(ADP-ribose) polymerase-1, and XRCC4 cleavage or in H2AX phosphorylation was observed and DNA-PK catalytic subunit was still phosphorylated on S2056 in response to staurosporine. However, DNA fragmentation including high molecular weight fragmentation was delayed in ARTEMIS-deficient cells compared with cells expressing ARTEMIS. In addition, ARTEMIS enhanced the kinetics of MLL gene cleavage at a breakage cluster breakpoint that is frequently translocated in acute or therapy-related leukemias. These results show a facilitating role for ARTEMIS at least in early, site-specific chromosome breakage during apoptosis.
Britton
Sébastien
S
CNRS, Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, UPS, Institut de Pharmacologie et de Biologie Structurale, Toulouse, France.
Frit
Philippe
P
Biard
Denis
D
Salles
Bernard
B
Calsou
Patrick
P
D017209:Apoptosis; D015139:Blotting, Southern; D053148:Caspase 3 / Q000378:metabolism; D002478:Cells, Cultured; D002843:Chromatin / Q000235:genetics / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004720:Endonucleases; D005347:Fibroblasts / Q000201:enzymology*; D011495:Histone-Lysine N-Methyltransferase; D006657:Histones / Q000378:metabolism; D006801:Humans; D051788:Myeloid-Lymphoid Leukemia Protein / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology
D002843:Chromatin; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; C497422:KMT2A protein, human; D009687:Nuclear Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; C101467:XRCC4 protein, human; D051788:Myeloid-Lymphoid Leukemia Protein; D011495:Histone-Lysine N-Methyltransferase; D011065:Poly(ADP-ribose) Polymerases; D051747:DNA-Activated Protein Kinase; C426344:DCLRE1C protein, human; D004720:Endonucleases; D053148:Caspase 3; D019311:Staurosporine