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<p>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 Mn<sup>2+</sup>-dependent 5′–3′ exonuclease (designated PEN1) responsible for this process. The <i>pen1</i> 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 <i>Pen1</i> 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.</p>

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PEN1 catalyses RNA primer removal during plastid DNA replication in maize

  • 期刊论文

  • 发表日期：

    2025年6月25日

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  • Xing Huang,
  • Guolong Shi,
  • Qiao Xiao,
  • Jiaojiao Feng,
  • Yongcai Huang,
  • Hai Shi,
  • Qiong Wang,
  • Yu Su,
  • Jiechen Wang,
  • Xingguo Wu,
  • Yuwei Cao,
  • Haihai Wang,
  • Wenqin Wang,
  • Yu Zhang,
  • Yongrui Wu

摘要

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.