Journal Article

Primer retention owing to the absence of RNase H1 is catastrophic for mitochondrial DNA replication

Stuart R. Wood, Susana M. Cerritelli, Howard T. Jacobs, Ian J. Holt, Kiran Sakhuja, Mark R. Bowmaker, Gokhan Akman, Chloe Moss, Robert J. Crouch, Holmes, J. Bradley

2015

Overview

Encoding ribonuclease H1 (RNase H1) degrades RNA hybridized to DNA, and its function is essential for mitochondrial DNA maintenance in the developing mouse. Here we define the role of RNase H1 in mitochondrial DNA replication. Analysis of replicating mitochondrial DNA in embryonic fibroblasts lacking RNase H1 reveals retention of three primers in the major noncoding region (NCR) and one at the prominent lagging-strand initiation site termed Ori-L. Primer retention does not lead immediately to depletion, as the persistent RNA is fully incorporated in mitochondrial DNA. However, the retained primers present an obstacle to the mitochondrial DNA polymerase γ in subsequent rounds of replication and lead to the catastrophic generation of a double-strand break at the origin when the resulting gapped molecules are copied. Hence, the essential role of RNase H1 in mitochondrial DNA replication is the removal of primers at the origin of replication. Cellular energy production is a function of the abundance of the small circular DNA molecules in mitochondria. Mitochondrial DNA is replicated in both dividing and nondividing cells, and encoding ribonuclease H1 (RNase H1) is essential to this process. Here, we define its mechanistic role: the removal of the RNA primers used for mitochondrial DNA replication. In the absence of RNase H1, primers are fixed in both template strands of mitochondrial DNA. The retained primers are a major impediment to mitochondrial DNA polymerase γ, leading to the formation of persistent DNA gaps that are catastrophic for subsequent rounds of replication. Moreover, primer retention provides unambiguous identification of RNA-DNA transition sites in the control region of mitochondrial DNA, thereby defining two major origins of replication.

• Publisher: National Academy of Sciences,National Acad Sciences
• Subject: Animals / Biological Sciences / Cell Line / circular DNA / DNA - chemistry / DNA Primers - chemistry / DNA Replication / DNA, Mitochondrial - chemistry / DNA-directed DNA polymerase / energy / Exons / Fibroblasts - metabolism / Genotype / Homozygote / Mice / Mice, Knockout / mitochondria / Mitochondria - metabolism / Mitochondrial DNA / Molecules / Nucleotides - chemistry / origin of replication / production functions / Replication Origin / replication priming / Retention / Ribonuclease H - chemistry / ribonucleases / RNA / RNA - chemistry / RNA, Mitochondrial / RNase H / Rodents