Light-strand bias and enriched zones of embedded ribonucleotides are associated with DNA replication and transcription in the human-mitochondrial genome.

Abundant ribonucleoside-triphosphate (rNTP) incorporation into DNA by DNA polymerases in the form of ribonucleoside monophosphates (rNMPs) is a widespread phenomenon in nature, resulting in DNA-structural change and genome instability. The rNMP distribution, characteristics, hotspots and association with DNA metabolic processes in human mitochondrial DNA (hmtDNA) remain mostly unknown. Here, we utilize the ribose-seq technique to capture embedded rNMPs in hmtDNA of six different cell types. In most cell types, the rNMPs are preferentially embedded on the light strand of hmtDNA with a strong bias towards rCMPs; while in the liver-tissue cells, the rNMPs are predominately found on the heavy strand. We uncover common rNMP hotspots and conserved rNMP-enriched zones across the entire hmtDNA, including in the control region, which links the rNMP presence to the frequent hmtDNA replication-failure events. We show a strong correlation between coding-sequence size and rNMP-embedment frequency per nucleotide on the non-template, light strand in all cell types, supporting the presence of transient RNA-DNA hybrids preceding light-strand replication. Moreover, we detect rNMP-embedment patterns that are only partly conserved across the different cell types and are distinct from those found in yeast mtDNA. The study opens new research directions to understand the biology of hmtDNA and genomic rNMPs.

DSBplot: Indels in DNA Double-strand Break Repair Experiments.

Double-strand breaks (DSBs) in DNA are naturally occurring destructive events in all organisms that may lead to genome instability. Cells employ various repair methods known as non-homologous end joining (NHEJ), microhomology mediated end joining (MMEJ), and homology-directed recombination (HDR). These repair processes may lead to DNA sequence variations (e.g., nucleotide insertions, deletions, and substitutions) at the location of the break. Studying DNA DSB repair processes often involves the use of high throughput sequencing assays to precisely quantify the sequence variations near the break with software tools. Often methods of assessing and visualizing these data have not taken into account the full complexity of the sequencing data, such as the frequency, type, and position of the sequence variations in a single comprehensive representation. Here we present a method that allows visualization of the overall variation pattern as well as comparison of these patterns among experimental setups.

RNA-mediated double-strand break repair by end-joining mechanisms.

Double-strand breaks (DSBs) in DNA are challenging to repair. Cells employ at least three DSB-repair mechanisms, with a preference for non-homologous end joining (NHEJ) over homologous recombination (HR) and microhomology-mediated end joining (MMEJ). While most eukaryotic DNA is transcribed into RNA, providing complementary genetic information, much remains unknown about the direct impact of RNA on DSB-repair outcomes and its role in DSB-repair via end joining. Here, we show that both sense and antisense-transcript RNAs impact DSB repair in a sequence-specific manner in wild-type human and yeast cells. Depending on its sequence complementarity with the broken DNA ends, a transcript RNA can promote repair of a DSB or a double-strand gap in its DNA gene via NHEJ or MMEJ, independently from DNA synthesis. The results demonstrate a role of transcript RNA in directing the way DSBs are repaired in DNA, suggesting that RNA may directly modulate genome stability and evolution.

Disproportionate presence of adenosine in mitochondrial and chloroplast DNA of Chlamydomonas reinhardtii.

Ribonucleoside monophosphates (rNMPs) represent the most common non-standard nucleotides found in the genome of cells. The distribution of rNMPs in DNA has been studied only in limited genomes. Using the ribose-seq protocol and the Ribose-Map bioinformatics toolkit, we reveal the distribution of rNMPs incorporated into the whole genome of a photosynthetic unicellular green alga, Chlamydomonas reinhardtii. We discovered a disproportionate incorporation of adenosine in the mitochondrial and chloroplast DNA, in contrast to the nuclear DNA, relative to the corresponding nucleotide content of these C. reinhardtii organelle genomes. Our results demonstrate that the rNMP content in the DNA of the algal organelles reflects an elevated ATP level present in the algal cells. We reveal specific biases and patterns in rNMP distributions in the algal mitochondrial, chloroplast, and nuclear DNA. Moreover, we identified the C. reinhardtii orthologous genes for all three subunits of the RNase H2 enzyme using GeneMark-EP + gene finder.