Bootstrapping DNA replication with ribonucleotide reductase in a minimal cell-free system

All living organisms must maintain a balanced pool of DNA building blocks to replicate and propagate their genetic information. In living cells, ribonucleotide reductase (RNR) is the central enzyme responsible for dNTP synthesis. In contrast, minimal protein-based cell-free systems, are powerful platforms for reconstituting biological processes in vitro, but lack the endogenous metabolic pathways required to autonomously replicate their DNA and rely entirely on externally supplied dNTPs. This dependency prevents cell-free systems from achieving the metabolic autonomy required for self-sufficient genetic replication. In this work, we successfully integrated RNR’s redox activity and complex allosteric regulation for in situ synthesis of all dNTPs from endogenous NTP pools. We combined RNR’s activity to DNA synthesis and propagation of the genetic information encoding a self-contained minimal DNA replication machinery. The replication products retain the genetic information and enable re-booting of self-encoded RNR-dependent DNA synthesis. Using this strategy, cell-free systems with self-sufficient dNTP metabolism may open new avenues toward completely autonomous synthetic cells.