RNase H1 Cooperates with DNA Gyrases to Restrict R-Loops and Maintain Genome Integrity in Arabidopsis Chloroplasts

Abstract

Maintaining organellar genome integrity is essential for eukaryotic cells, and many factors can threaten genome integrity. R-loops are DNA:RNA duplexes produced during transcription, with the nontemplated DNA forming a single-stranded region. R-loops function in the regulation of transcription, DNA replication, and DNA repair, but can also be susceptible to lesions that form double-stranded breaks and thus induce genome instability. From investigating the function of a plant chloroplast-localized R-loop removing enzyme AtRNH1C, we have found that it is responsible for plastid R-loop homeostasis, chloroplast genome instability, and development. Interactome analysis revealed that AtRNH1C associates with multiple chloroplast-localized DNA and RNA metabolism-related proteins, including the core DNA gyrases complex. The interaction between AtRNH1C and AtGyrases was critical for R-loop homeostasis in chloroplast and important to release the transcription-replication conflicts in the highly transcribed and replication originated cp-rDNA regions and thus to reduce the DNA damage. Our results reveal the plastid R-loop accumulation leads to chloroplast DNA instability and provide insight into the maintenance of genome integrity in chloroplasts, in which the evolutionarily conserved RNase H1 and DNA gyrase proteins are involved.

Systematic sequencing of chloroplast transcript termini fromArabidopsis thalianareveals >200 transcription initiation sites and the extensive imprints of RNA-binding proteins and secondary structures

“…Concerning 5' termini, this might include the roles of the sigma factors that specify PEP initiation sites (112) and other PEP-associated proteins (PAPs; 113), a closer view at RNase J specificity, or a better understanding of some of the numerous factors that have been implicated in rRNA maturation (6). Terminome-Seq would be an excellent choice to gain additional insight into poorly-characterized RNases whose targets have only begun to be described (114)(115)(116).…”

Genome-wide mapping reveals conserved and diverged R-loop activities in the unusual genetic landscape of the African trypanosome genome

“…Most prokaryotes and eukaryotes encode two RNase H enzymes (22): one, eukaryotic RNase H1, is monomeric and appears conserved with bacterial RNase HI, while RNase H2 in eukaryotes is trimeric and therefore differs structurally from monomeric bacterial RNase HII. Distinct roles might be predicted by the presence of two RNase H enzymes throughout life, but separate R-loop-associated functions in the eukaryotic nucleus have been hard to identify, though RNase H2 is additionally able to excise ribonucleotides mis-incorporated into DNA (23), while RNase H1 acts on organelle genome R-loops in yeast, mammals and plants (24)(25)(26). Loss of either RNase H is lethal in mammals (25,27), but yeast mutants lacking both enzymes are viable and display increased nuclear R-loop abundance (6,24,28).…”

Structure–Function Analysis Reveals the Singularity of Plant Mitochondrial DNA Replication Components: A Mosaic and Redundant System

“…During metazoan mitochondrial DNA replication, the RNA primers generated by the mitochondrial RNA polymerase are removed by a specific set of nucleases. In humans, five different nucleases participate in this process [47][48][49][50][51][52]. From those enzymes, RNAse H1 plays a predominant role by degrading the RNA primer until it reaches few nucleotides.…”