PEN1 catalyses RNA primer removal during plastid DNA replication in maize

The plastid DNA (ptDNA) replication is initiated by primases, which synthesize RNA primers; following the synthesis of DNA fragments, primers must be removed before ligation. However, the enzymes and mechanisms underlying this process are poorly understood. Here we cloned a gene from maize that encodes a plastid-localized and Mn²⁺-dependent 5'-3' exonuclease (designated PEN1) responsible for this process. The pen1 seeds show development and filling defects that intensify across generations. PEN1 cleaves the RNA primers, allowing for the complete excision of ribonucleotides. We reconstituted the plastid RNA primer removal processes in vitro. We also determined the crystal structure of the PEN1-dsDNA binary complex and explained the structural mechanism of the 5' to 3' exonuclease activity. Mutation of Pen1 resulted in the accumulation of ptDNA breaks, thereby compromising plastid function. These studies fill a critical gap that has long existed in the understanding of ptDNA replication.