In vitro reconstitution reveals a key role of human mitochondrial EXOG in RNA primer processing

Anna Karlowicz, Andrzej B. Dubiel, Jolanta Czerwinska, Adela Bledea, Piotr Purzycki, Marta Grzelewska, Ryan J. McAuley, Roman J. Szczesny, Gabriela Brzuska, Ewelina Krol, Bartosz Szczesny, Michal R. Szymanski

Research output: Contribution to journalArticlepeer-review


The removal of RNA primers is essential for mitochondrial DNA (mtDNA) replication. Several nucleases have been implicated in RNA primer removal in human mitochondria, however, no conclusive mechanism has been elucidated. Here, we reconstituted minimal in vitro system capable of processing RNA primers into ligatable DNA ends. We show that human 5′-3′ exonuclease, EXOG, plays a fundamental role in removal of the RNA primer. EXOG cleaves short and long RNA-containing flaps but also in cooperation with RNase H1, processes non-flap RNA-containing intermediates. Our data indicate that the enzymatic activity of both enzymes is necessary to process non-flap RNA-containing intermediates and that regardless of the pathway, EXOG-mediated RNA cleavage is necessary prior to ligation by DNA Ligase III. We also show that upregulation of EXOG levels in mitochondria increases ligation efficiency of RNA-containing substrates and discover physical interactions, both in vitro and in cellulo, between RNase H1 and EXOG, Pol γA, Pol γB and Lig III but not FEN1, which we demonstrate to be absent from mitochondria of human lung epithelial cells. Together, using human mtDNA replication enzymes, we reconstitute for the first time RNA primer removal reaction and propose a novel model for RNA primer processing in human mitochondria.

Original languageEnglish (US)
Pages (from-to)7991-8007
Number of pages17
JournalNucleic acids research
Issue number14
StatePublished - Aug 12 2022
Externally publishedYes

ASJC Scopus subject areas

  • Genetics


Dive into the research topics of 'In vitro reconstitution reveals a key role of human mitochondrial EXOG in RNA primer processing'. Together they form a unique fingerprint.

Cite this