Evidence for a role of FEN1 in maintaining mitochondrial DNA integrity

Kalifa, Lidza; Beutner, Gisela; Phadnis, Naina; Sheu, Shey‐Shing; Sia, Elaine A.

doi:10.1016/j.dnarep.2009.07.008

Cited by 65 publications

(85 citation statements)

References 67 publications

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“…However, RNase H1 cannot remove every last ribonucleotide of an RNA/ DNA hybrid, and therefore other factors should also be required, such as those implicated in Okazaki fragment processing in the nucleus, that have also been found in mitochondria (25)(26)(27), and, possibly, MGME1 (28).…”

Section: Resultsmentioning

confidence: 99%

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

Holmes

Akman²,

Wood

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Resultsmentioning

confidence: 99%

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

Holmes

Akman²,

Wood

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Media used in this study were previously described (Kalifa et al 2009). S. cerevisiae strains used in this study are listed in Table 1 and are isogenic with DFS188 except NPY66, which is derived from DFS160 (Sia et al 2000;Phadnis et al 2005).…”

Section: Media and Strainsmentioning

confidence: 99%

“…Rates of nuclear and mitochondrial DRMD using the 96-bp repeat reporters were determined as described previously (Kalifa et al 2009) with minor modifications. Briefly, cells containing both intact reporters were selected on SD 2Arg 2Ura media, and independent colonies were isolated on YPD to release from selection.…”

Section: Fluctuation Analysismentioning

confidence: 99%

“…Strains containing two or more gene replacements were constructed by mating isogenic haploid strains, dissecting diploids, and screening for the desired genotypes by PCR. The DRMD reporter (LKY196) was constructed previously (Kalifa et al 2009). Gene deletions in this strain were constructed by mating, followed by sporulation and dissection and screening for the appropriate genotype.…”

Section: Media and Strainsmentioning

confidence: 99%

“…We previously described the construction and use of yeast strains with reporters to simultaneously measure nuclear and mitochondrial DRMDs. Using these reporter strains we can monitor the mutagenic effect of DNA repair protein disruption on both mitochondrial and nuclear DNA in the same population of cells (Kalifa et al 2009). In this study, we assessed mutagenic consequences of loss of the NHEJ pathway.…”

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Mitochondrial Genome Maintenance: Roles for Nuclear Nonhomologous End-Joining Proteins inSaccharomyces cerevisiae

et al. 2012

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Mitochondrial DNA (mtDNA) deletions are associated with sporadic and inherited diseases and age-associated neurodegenerative disorders. Approximately 85% of mtDNA deletions identified in humans are flanked by short directly repeated sequences; however, mechanisms by which these deletions arise are unknown. A limitation in deciphering these mechanisms is the essential nature of the mitochondrial genome in most living cells. One exception is budding yeast, which are facultative anaerobes and one of the few organisms for which directed mtDNA manipulation is possible. Using this model system, we have developed a system to simultaneously monitor spontaneous direct-repeat-mediated deletions (DRMDs) in the nuclear and mitochondrial genomes. In addition, the mitochondrial DRMD reporter contains a unique KpnI restriction endonuclease recognition site that is not present in otherwise wild-type (WT) mtDNA. We have expressed KpnI fused to a mitochondrial localization signal to induce a specific mitochondrial doublestrand break (mtDSB). Here we report that loss of the MRX (Mre11p, Rad50p, Xrs2p) and Ku70/80 (Ku70p, Ku80p) complexes significantly impacts the rate of spontaneous deletion events in mtDNA, and these proteins contribute to the repair of induced mtDSBs. Furthermore, our data support homologous recombination (HR) as the predominant pathway by which mtDNA deletions arise in yeast, and suggest that the MRX and Ku70/80 complexes are partially redundant in mitochondria.

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DNA repair in mammalian mitochondria: Much more than we thought?

Liu

Demple

2010

Environ and Mol Mutagen

154

127

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For many years, the repair of most damage in mitochondrial DNA (mtDNA) was thought limited to short-patch base excision repair (SP-BER), which replaces a single nucleotide by the sequential action of DNA glycosylases, an apurinic/apyrimidinic (AP) endonuclease, the mitochondrial DNA polymerase gamma, an abasic lyase activity, and mitochondrial DNA ligase. However, the likely array of lesions inflicted on mtDNA by oxygen radicals and the possibility of replication errors and disruptions indicated that such a restricted repair repertoire would be inadequate. Recent studies have considerably expanded our knowledge of mtDNA repair to include long-patch base excision repair (LP-BER), mismatch repair, and homologous recombination and nonhomologous end-joining. In addition, elimination of mutagenic 8-oxodeoxyguanosine triphosphate (8-oxodGTP) helps prevent cell death due to the accumulation of this oxidation product in mtDNA. Although it was suspected for many years that irreparably damaged mtDNA might be targeted for degradation, only recently was clear evidence provided for this hypothesis. Therefore, multiple DNA repair pathways and controlled degradation of mtDNA function together to maintain the integrity of mitochondrial genome.

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Evidence for a role of FEN1 in maintaining mitochondrial DNA integrity

Cited by 65 publications

References 67 publications

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

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

Mitochondrial Genome Maintenance: Roles for Nuclear Nonhomologous End-Joining Proteins inSaccharomyces cerevisiae

DNA repair in mammalian mitochondria: Much more than we thought?

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