Previous work has demonstrated the presence of ribonucleotides in human mitochondrial DNA (mtDNA) and in the present study we use a genome-wide approach to precisely map the location of these. We find that ribonucleotides are distributed evenly between the heavy- and light-strand of mtDNA. The relative levels of incorporated ribonucleotides reflect that DNA polymerase γ discriminates the four ribonucleotides differentially during DNA synthesis. The observed pattern is also dependent on the mitochondrial deoxyribonucleotide (dNTP) pools and disease-causing mutations that change these pools alter both the absolute and relative levels of incorporated ribonucleotides. Our analyses strongly suggest that DNA polymerase γ-dependent incorporation is the main source of ribonucleotides in mtDNA and argues against the existence of a mitochondrial ribonucleotide excision repair pathway in human cells. Furthermore, we clearly demonstrate that when dNTP pools are limiting, ribonucleotides serve as a source of building blocks to maintain DNA replication. Increased levels of embedded ribonucleotides in patient cells with disturbed nucleotide pools may contribute to a pathogenic mechanism that affects mtDNA stability and impair new rounds of mtDNA replication.

Inherited optic neuropathies include complex phenotypes, mostly driven by mitochondrial dysfunction. We report an optic atrophy spectrum disorder, including retinal macular dystrophy and kidney insufficiency leading to transplantation, associated with mitochondrial DNA (mtDNA) depletion without accumulation of multiple deletions. By whole-exome sequencing, we identified mutations affecting the mitochondrial single-strand binding protein (SSBP1) in 4 families with dominant and 1 with recessive inheritance. We show that SSBP1 mutations in patient-derived fibroblasts variably affect the amount of SSBP1 protein and alter multimer formation, but not the binding to ssDNA. SSBP1 mutations impaired mtDNA, nucleoids, and 7S-DNA amounts as well as mtDNA replication, affecting replisome machinery. The variable mtDNA depletion in cells was reflected in severity of mitochondrial dysfunction, including respiratory efficiency, OXPHOS subunits, and complex amount and assembly. mtDNA depletion and cytochrome c oxidase-negative cells were found ex vivo in biopsies of affected tissues, such as kidney and skeletal muscle. Reduced efficiency of mtDNA replication was also reproduced in vitro, confirming the pathogenic mechanism. Furthermore, ssbp1 suppression in zebrafish induced signs of nephropathy and reduced optic nerve size, the latter phenotype complemented by WT mRNA but not by SSBP1 mutant transcripts. This previously unrecognized disease of mtDNA maintenance implicates SSBP1 mutations as a cause of human pathology.

The fission yeast Mcs6 -Mcs2-Pmh1 complex, homologous to metazoan Cdk7-cyclin H-Mat1, has dual functions in cell division and transcription: as a partially redundant cyclin-dependent kinase (CDK)-activating kinase (CAK) that phosphorylates the major cell cycle CDK, Cdc2, on Thr-167; and as the RNA polymerase (Pol) II carboxyl-terminal domain (CTD) kinase associated with transcription factor (TF) IIH. We analyzed conditional mutants of mcs6 and pmh1, which activate Cdc2 normally but cannot complete cell division at restrictive temperature and arrest with decreased CTD phosphorylation. Transcriptional profiling by microarray hybridization revealed only modest effects on global gene expression: a one-third reduction in a severe mcs6 mutant after prolonged incubation at 36°C. In contrast, a small subset of transcripts (ϳ5%) decreased by more than twofold after Mcs6 complex function was compromised. The signature of repressed genes overlapped significantly with those of cell separation mutants sep10 and sep15. Sep10, a component of the Pol II Mediator complex, becomes essential in mcs6 or pmh1 mutant backgrounds. Moreover, transcripts dependent on the forkhead transcription factor Sep1, which are expressed coordinately during mitosis, were repressed in Mcs6 complex mutants, and Mcs6 also interacts genetically with Sep1. Thus, the Mcs6 complex, a direct activator of Cdc2, also influences the cell cycle transcriptional program, possibly through its TFIIH-associated kinase function. INTRODUCTIONCyclin-dependent kinases (CDKs) play a central role in driving cell division in eukaryotic organisms and also perform essential, conserved functions in the transcription cycle of RNA polymerase (Pol) II (reviewed by Morgan, 1997). Whereas most CDKs can be classified as either cell cycle or transcriptional regulators based on a preeminent physiological function, the metazoan Cdk7 complex is essential in both cell division and gene expression, as the CDK-activating kinase (CAK) and as a component of the general transcription factor IIH (TFIIH) (reviewed by Harper and Elledge, 1998). To execute its dual functions in vivo, Cdk7 has evolved distinct substrate specificities for the activation segment (T-loop) of CDKs and the carboxy-terminal domain (CTD) of the Pol II large subunit, and mechanisms to allow their independent regulation (Garrett et al., 2001;Larochelle et al., 2001). Less clear is how (or whether) the Cdk7 complex serves to coordinate cell division with gene expression.To address this question genetically, we turned to the fission yeast Schizosaccharomyces pombe, which also relies, in part, on a dual-function CDK complex to activate CDKs and phosphorylate Pol II (Buck et al., 1995;Damagnez et al., 1995;Hermand et al., 1998;Lee et al., 1999;Saiz and Fisher, 2002). The orthologue of Cdk7 in S. pombe is Mcs6, which associates with the cyclin Mcs2 and the RING-finger protein Pmh1. Both mcs6 and mcs2 were identified in genetic screens for positive regulators of Cdc2 (S. pombe Cdk1), the major cell cycle CDK (Molz et al., 1989...

Temporal changes in transcription programs are coupled to control of cell growth and division. We here report that Mediator, a conserved coregulator of eukaryotic transcription, is part of a regulatory pathway that controls mitotic entry in fission yeast. The Mediator subunit cyclin-dependent kinase 8 (Cdk8) phosphorylates the forkhead 2 (Fkh2) protein in a periodic manner that coincides with gene activation during mitosis. Phosphorylation prevents degradation of the Fkh2 transcription factor by the proteasome, thus ensuring cell cycle-dependent variations in Fkh2 levels. Interestingly, Cdk8-dependent phosphorylation of Fkh2 controls mitotic entry, and mitotic entry is delayed by inactivation of the Cdk8 kinase activity or mutations replacing the phosphorylated serine residues of Fkh2. In addition, mutations in Fkh2, which mimic protein phosphorylation, lead to premature mitotic entry. Therefore, Fkh2 regulates not only the onset of mitotic transcription but also the correct timing of mitotic entry via effects on the Wee1 kinase. Our findings thus establish a new pathway linking the Mediator complex to control of mitotic transcription and regulation of mitotic entry in fission yeast. Signaling pathways can control the activation of gene expression programs and thereby regulate cell fate determination. In embryonic stem cells, certain gene expression programs allow the cells to self-renew whereas other programs trigger differentiation into specific cell types as a response to developmental signaling (58). Elucidation of how temporal changes in transcription programs are coupled to control of cell growth and division is therefore of fundamental importance for our understanding of developmental processes.Global gene transcription analysis in yeasts and higher eukaryotes has revealed that a significant proportion of the genome is transcribed in a periodic manner during cell cycle progression (5,15,34,49,55). Correct periodic regulation is believed to play a critical role in normal cell proliferation, and the genes are often deregulated in different forms of cancer (6). Depending on the organism, the number of periodically expressed genes ranges from ϳ400 to more than 1,000 (5, 6, 56). These include genes with well-established roles in cell cycle progression, such as those encoding cyclins, transcription factors and protein kinases.A cluster named CLB2 in budding yeast (35 genes) or cluster 1 in fission yeast (87 genes) is periodically expressed and activated at mitosis and repressed in G 1 of the next cell cycle (4,5,34,56). In budding yeast, transcription of the CLB2 cluster is controlled by the forkhead proteins Fkh1 and Fkh2, which cooperate with Mcm1 (a MADS box protein) and the Ndd1 coactivator (27, 28). In fission yeast, forkhead proteins Sep1 and Fkh2 and the MADS box protein Mbx1 regulate mitotic transcription (12,13,49,53). Deletion of the sep1 gene results in reduced transcription, whereas overexpression of sep1 induces expression of the same genes. In contrast, deletion of fkh2 causes elevated levels of g...

The level of malondialdehyde, a stable end product of lipid peroxidation induced by reactive oxygen intermediates and the activity of two potent antioxidant enzymes, superoxide dismutase and glutathione peroxidase, was investigated in tissue homogenates of 22 surgical periapical granuloma specimens. Malondialdehyde levels were significantly higher and glutathione peroxidase activity was significantly lower in periapical granuloma samples than in healthy gingival tissue homogenates, which were used as controls. The activity of superoxide dismutase was similar in periapical granuloma and in control samples. Our results indicate an altered balance between the production and the elimination of toxic oxygen metabolites in chronic apical periodontitis. We hypothesize that reactive oxygen intermediates, which are being produced by activated phagocytic cells abundantly present in periapical granulomas, can contribute to periapical tissue injury and bone loss in this disease.