Sequence and properties of the human KB cell and mouse L cell D-loop regions of mitochondrial DNA

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.H uman mitochondrial DNA (mtDNA) is most frequently organized as 16.5-kb circles of duplex DNA, whose two strands are called heavy (H) and light (L), based on their nucleotide composition. The human mitochondrial genome is gene dense, with only one substantial noncoding region (NCR) of approximately 1 kb (1). Based on electron microscopy (2), 5′ end mapping of DNA ends (3-5) and later 2D agarose gel electrophoresis (2D-AGE) studies (6, 7) it was inferred that replication of mammalian mtDNA is frequently unidirectional, commencing within the NCR.In mammalian mitochondria, initiation of DNA synthesis occurs preferentially at or near two specific sites. On the leading strand, initiation has been assigned to a prominent free 5′ end of DNA, designated as Ori-H, located at nucleotide position 16,034 on the map of the mouse mitochondrial genome. Ori-H lies downstream of the light-strand promoter (LSP), and RNA species spanning from LSP to approximately Ori-H have been detected in mammalian mitochondria, albeit not covalently linked with DNA (8). LSP has been robustly mapped both in vivo (9) and in vitro (10) and is assumed to be used by mitochondrial RNA polymerase (POLRMT) to synthesize polycistronic transcripts of the light strand and to generate much shorter products terminating in the NCR, which serve as primers for leading (H-)strand DNA synthesis. POLRMT is also implicated in the synthesis of a primer at a short spacer DNA amid a cluster of five tRNA genes (termed Ori-L) that is a major site of initiation of lagging-strand DNA synthesis (11,12). A role for encoding ribonuclease H1 (RNase H1) in generating, processing, or removing primers at Ori-H and Ori-L has been inferred but has not been experimentally tested.Other data indicate that the initiation of mtDNA replication is more complex, because the NCR contains a second putative origin of replication, termed cluster II, or Ori-b (7, 13). Moreover, other sites of second-strand DNA synthesis than Ori-L have been inferred both by atomic force microscopy (14) and neutral 2D agarose gel electrophoresis (7, 13). Mitochondrial DNA rep...

Section: Resultssupporting

confidence: 68%

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

Holmes

Akman²,

Wood

et al. 2015

“…These mice had 3Ј PstI-independent deletion breakpoints at bp 15368-15439 (28), overlapping with our breakpoints. The hotspot region in human and mouse mtDNA is located between tRNA Pro and the D-loop and contains the most highly divergent primary sequence of mtDNA (29). It seems evident that the general mechanism causing multiple deletions with breakpoints at the specific site is not due to the primary sequence itself but its secondary structure or functional location.…”

Section: Discussionmentioning

confidence: 99%

Mutant mitochondrial helicase Twinkle causes multiple mtDNA deletions and a late-onset mitochondrial disease in mice

Tyynismaa

Mjøsund

Wanrooij

et al. 2005

302

285

Defects of mitochondrial DNA (mtDNA) maintenance have recently been associated with inherited neurodegenerative and muscle diseases and the aging process. Twinkle is a nuclear-encoded mtDNA helicase, dominant mutations of which cause adult-onset progressive external ophthalmoplegia (PEO) with multiple mtDNA deletions. We have generated transgenic mice expressing mouse Twinkle with PEO patient mutations. Multiple mtDNA deletions accumulate in the tissues of these mice, resulting in progressive respiratory dysfunction and chronic late-onset mitochondrial disease starting at 1 year of age. The muscles of the mice faithfully replicate all of the key histological, genetic, and biochemical features of PEO patients. Furthermore, the mice have progressive deficiency of cytochrome c oxidase in distinct neuronal populations. These ''deletor'' mice do not, however, show premature aging, indicating that subtle accumulation of mtDNA deletions and progressive respiratory chain dysfunction are not sufficient to accelerate aging. This model is a valuable tool for therapy development and testing for adult-onset mitochondrial disorders. mouse model ͉ progressive external ophthalmoplegia ͉ mitochondrial DNA replication

“…2 and SI Appendix, Table S1), suggesting that the LSP is the ultimate terminus for R-loop synthesis. Of the 41 R-loops sequenced, the eight longest (with respect to the inferred 5′ end) began at np 15,476 ± 2 nt, within a region (np 15,423-15,483) (19) predicted to form a secondary structure (20) known as the "termination-associated sequence" (TAS) because of its proximity to the 3′ end of the 7S DNAs (8). Seventeen of the other 5′ ends were clustered around np 15,600, within 25 nt of the second origin of replication in the CR, Ori-b (15).…”

mentioning

confidence: 99%

Pathological ribonuclease H1 causes R-loop depletion and aberrant DNA segregation in mitochondria

Akman

Desai

Bailey

et al. 2016

The genetic information in mammalian mitochondrial DNA is densely packed; there are no introns and only one sizeable noncoding, or control, region containing key cis-elements for its replication and expression. Many molecules of mitochondrial DNA bear a third strand of DNA, known as "7S DNA," which forms a displacement (D-) loop in the control region. Here we show that many other molecules contain RNA as a third strand. The RNA of these R-loops maps to the control region of the mitochondrial DNA and is complementary to 7S DNA. Ribonuclease H1 is essential for mitochondrial DNA replication; it degrades RNA hybridized to DNA, so the R-loop is a potential substrate. In cells with a pathological variant of ribonuclease H1 associated with mitochondrial disease, R-loops are of low abundance, and there is mitochondrial DNA aggregation. These findings implicate ribonuclease H1 and RNA in the physical segregation of mitochondrial DNA, perturbation of which represents a previously unidentified disease mechanism.RNase H1 | R-loop | mitochondrial DNA | DNA segregation | mitochondrial disease