A unique exonuclease ExoG cleaves between RNA and DNA in mitochondrial DNA replication.

Chyuan-Chuan Wu,Hanna S Yuan,Jason L J Lin,Hsin-Fang Yang-Yen

doi:10.1093/nar/gkz241

Chyuan-Chuan Wu, Hanna S Yuan + Show 2 more

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https://doi.org/10.1093/nar/gkz241

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Abstract

Replication of sufficient mitochondrial DNA (mtDNA) is essential for maintaining mitochondrial functions in mammalian cells. During mtDNA replication, RNA primers must be removed before the nascent circular DNA strands rejoin. This process involves mitochondrial RNase H1, which removes most of the RNA primers but leaves two ribonucleotides attached to the 5′ end of nascent DNA. A subsequent 5′-exonuclease is required to remove the residual ribonucleotides, however, it remains unknown if any mitochondrial 5′-exonuclease could remove two RNA nucleotides from a hybrid duplex DNA. Here, we report that human mitochondrial Exonuclease G (ExoG) may participate in this particular process by efficiently cleaving at RNA–DNA junctions to remove the 5′-end RNA dinucleotide in an RNA/DNA hybrid duplex. Crystal structures of human ExoG bound respectively with DNA, RNA/DNA hybrid and RNA–DNA chimeric duplexes uncover the underlying structural mechanism of how ExoG specifically recognizes and cleaves at RNA–DNA junctions of a hybrid duplex with an A-form conformation. This study hence establishes the molecular basis of ExoG functioning as a unique 5′-exonuclease to mediate the flap-independent RNA primer removal process during mtDNA replication to maintain mitochondrial genome integrity.

Highlights

Mitochondria are the power plants of a cell, providing cellular energy in the form of ATP through oxidative phosphorylation (OXPHOS)
Little is known regarding Exonuclease G (ExoG)’s RNA-degrading activity, so we firstly examined if ExoG could process RNA in an RNA/DNA hybrid duplex
ExoG directly removes two 5 -end nucleotides from a gap DNA substrate without a flap region [24,30], so it removes the 5 -blocking moiety and generates optimal substrates with gaps of at least two nucleotides for subsequent gap-filling synthesis by Pol␥, which is catalytically ineffective at a single-nucleotide gap [26]

Summary

Introduction

Mitochondria are the power plants of a cell, providing cellular energy in the form of ATP through oxidative phosphorylation (OXPHOS). According to the strand-displacement model of mtDNA replication, both strands are synthesized continuously, each with only one priming event [1] This process initiates at the light strand promoter (LSP) where mitochondrial RNA polymerase (POLRMT) starts to transcribe RNA until reaching conserved sequence block 2 (CSB2). The replication of L-strand occurs when the H-strand replicating machinery reaches the L-strand origin (OriL), where POLRMT makes RNA primers of about 25 nucleotides [1,7] This short RNA primer on L-strand and the long RNA primer on H-strand, as well as ∼100 DNA nucleotides downstream from CSB2 to the H-strand replication origin (OriH), have to be removed before the circular mtDNA can be rejoined by mitochondrial DNA ligase (ligase III) [8]. It remains largely unknown how these RNA primers and their downstream DNA are processed to generate the mature DNA 5 ends for DNA ligation, allowing restoration of mitochondrial genome integrity

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