Abstract
Ribonucleotide reductase (RnR) is a key enzyme synthesizing deoxyribonucleotides for DNA replication and repair. In mammals, the R1 catalytic subunit forms an active complex with either one of the two small subunits R2 and p53R2. Expression of R2 is S phase-specific and required for DNA replication. The p53R2 protein is expressed throughout the cell cycle and in quiescent cells where it provides dNTPs for mitochondrial DNA synthesis. Participation of R2 and p53R2 in DNA repair has also been suggested. In this study, we investigated the fate of the RnR subunits during apoptosis. The p53R2 protein was cleaved in a caspase-dependent manner in K-562 cells treated with inhibitors of the Bcr-Abl oncogenic kinase and in HeLa 229 cells incubated with TNF-α and cycloheximide. The cleavage site was mapped between Asp(342) and Asn(343). Caspase attack released a C-terminal p53R2 peptide of nine residues containing the conserved heptapeptide essential for R1 binding. As a consequence, the cleaved p53R2 protein was inactive. In vitro, purified caspase-3 and -8 could release the C-terminal tail of p53R2. Knocking down these caspases, but not caspase-2, -7, and -10, also inhibited p53R2 cleavage in cells committed to die via the extrinsic death receptor pathway. The R2 subunit was subjected to caspase- and proteasome-dependent proteolysis, which was prevented by siRNA targeting caspase-8. Knocking down caspase-3 was ineffective. Protein R1 was not subjected to degradation. Adding deoxyribonucleosides to restore dNTP pools transiently protected cells from apoptosis. These data identify RnR activity as a prosurvival function inactivated by proteolysis during apoptosis.
Highlights
Proteins involved in DNA replication and repair are cleaved by caspases during apoptosis
The p53R2 protein is expressed throughout the cell cycle and in quiescent cells where it provides dNTPs for mitochondrial DNA synthesis
The p53R2 protein was cleaved in a caspase-dependent manner in K-562 cells treated with inhibitors of the Bcr-Abl oncogenic kinase and in HeLa 229 cells incubated with TNF-␣ and cycloheximide
Summary
Proteins involved in DNA replication and repair are cleaved by caspases during apoptosis. Results: Caspase-dependent degradation of human ribonucleotide reductase subunits R2 and p53R2 occurs in apoptotic cells. Ribonucleotide reductase (RnR) is a key enzyme synthesizing deoxyribonucleotides for DNA replication and repair. Adding deoxyribonucleosides to restore dNTP pools transiently protected cells from apoptosis These data identify RnR activity as a prosurvival function inactivated by proteolysis during apoptosis. Caspase-dependent Cleavage of R2 and p53R2 during Apoptosis key players in signaling transduction pathways; cell cycle regulators; and DNA-binding proteins, leads to characteristic morphological changes, chromatin condensation, and nucleus fragmentation. RnR supplies dNTPs for DNA synthesis and repair, but the fate of the enzyme subunits during apoptosis is currently unknown. R1, which is a long lived protein present in resting and proliferating cells like p53R2, is not subjected to proteolysis during apoptosis
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