A cryptic targeting signal creates a mitochondrial FEN1 isoform with tailed R-Loop binding properties.

TitleA cryptic targeting signal creates a mitochondrial FEN1 isoform with tailed R-Loop binding properties.
Publication TypeJournal Article
Year of Publication2013
AuthorsKazak, L, Reyes, A, He, J, Wood, SR, Brea-Calvo, G, Holen, TT, Holt, IJ
JournalPLoS One
Volume8
Issue5
Paginatione62340
Date Published2013
ISSN1932-6203
KeywordsCell Line, Tumor, Cell Nucleus, DNA, Ethidium, Flap Endonucleases, Gene Expression Regulation, HEK293 Cells, HeLa Cells, Humans, Isoenzymes, Mitochondria, Nucleic Acid Conformation, Peptide Chain Initiation, Translational, Protein Binding, Protein Sorting Signals, Protein Transport, RNA, Signal Transduction
Abstract

A growing number of DNA transacting proteins is found in the nucleus and in mitochondria, including the DNA repair and replication protein Flap endonuclease 1, FEN1. Here we show a truncated FEN1 isoform is generated by alternative translation initiation, exposing a mitochondrial targeting signal. The shortened form of FEN1, which we term FENMIT, localizes to mitochondria, based on import into isolated organelles, immunocytochemistry and subcellular fractionation. In vitro FENMIT binds to flap structures containing a 5' RNA flap, and prefers such substrates to single-stranded RNA. FENMIT can also bind to R-loops, and to a lesser extent to D-loops. Exposing human cells to ethidium bromide results in the generation of RNA/DNA hybrids near the origin of mitochondrial DNA replication. FENMIT is recruited to the DNA under these conditions, and is released by RNase treatment. Moreover, high levels of recombinant FENMIT expression inhibit mtDNA replication, following ethidium bromide treatment. These findings suggest FENMIT interacts with RNA/DNA hybrids in mitochondrial DNA, such as those found at the origin of replication.

DOI10.1371/journal.pone.0062340
Alternate JournalPLoS ONE
Citation Key10.1371/journal.pone.0062340
PubMed ID23675412
PubMed Central IDPMC3652857
Grant List / / Medical Research Council / United Kingdom