Among DNase I family members, only DNase γ causes DNA fragmentation during apoptosis. However, the molecular basis for this functional feature of DNase γ is poorly understood. Here we describe the identification of functional NLSs (nuclear localization signals) in DNase γ and their roles in its apoptotic function. DNase γ contains two NLSs: a classical bipartite-type NLS (NLS1) located in the N-terminal half, and a short basic domain (NLS2) at the C-terminus. No potential NLSs are found in the primary structures of other DNase I family DNases. Inactivation of either NLS1 or NLS2 causes reduced DNA ladder-producing activity in DNase γ. Disruption of NLS2 suppresses ladder formation more effectively than disruption of NLS1. DNase γ doubly mutated in both NLSs is enzymically active, but no longer catalyses apoptotic DNA fragmentation. Although DNase I fails to produce ladder formation during apoptosis, DNase I fused to NLS2 of DNase γ through its C-terminus is able to catalyse DNA fragmentation in apoptotic cells. These results indicate that the presence of either NLS1 or NLS2 is necessary for the apoptotic function of DNase γ, and that the most important domain for this function is NLS2. These findings also explain the lack of apoptotic DNase activity in the other DNase I family DNases.

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