| 1. |
- Fredriksson, Åsa, 1968, et al.
(författare)
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Decline in ribosomal fidelity contributes to the accumulation and stabilization of the master stress response regulator sigma S upon carbon starvation
- 2007
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Ingår i: Genes & Development. - : Cold Spring Harbor Laboratory. - 0890-9369 .- 1549-5477. ; 21:7, s. 862-874
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Tidskriftsartikel (refereegranskat)abstract
- The {sigma}S subunit of RNA polymerase is a master regulator of Escherichia coli that retards cellular senescence and bestows cells with general stress protective functions during growth arrest. We show that mutations and drugs triggering translational errors elevate {sigma}S levels and stability. Furthermore, mutations enhancing translational fidelity attenuate induction of the rpoS regulon and prevent stabilization of {sigma}S upon carbon starvation. Destabilization of {sigma}S by increased proofreading requires the presence of the {sigma}S recognition factor SprE (RssB) and the ClpXP protease. The data further suggest that {sigma}S becomes stabilized upon starvation as a result of ClpP sequestration and this sequestration is enhanced by oxidative modifications of aberrant proteins produced by erroneous translation. ClpP overproduction counteracted starvation-induced stabilization of {sigma}S, whereas overproduction of a ClpXP substrate (ssrA-tagged GFP) stabilized {sigma}S in exponentially growing cells. We present a model for the sequence of events leading to the accumulation and activation of {sigma}S upon carbon starvation, which are linked to alterations in both ribosomal fidelity and efficiency.
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| 2. |
- Fusté, Javier Miralles, et al.
(författare)
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In Vivo Occupancy of Mitochondrial Single-Stranded DNA Binding Protein Supports the Strand Displacement Mode of DNA Replication
- 2014
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Ingår i: Plos Genetics. - : Public Library of Science (PLoS). - 1553-7390 .- 1553-7404. ; 10:12
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Tidskriftsartikel (refereegranskat)abstract
- Mitochondrial DNA (mtDNA) encodes for proteins required for oxidative phosphorylation, and mutations affecting the genome have been linked to a number of diseases as well as the natural ageing process in mammals. Human mtDNA is replicated by a molecular machinery that is distinct from the nuclear replisome, but there is still no consensus on the exact mode of mtDNA replication. We here demonstrate that the mitochondrial single-stranded DNA binding protein (mtSSB) directs origin specific initiation of mtDNA replication. MtSSB covers the parental heavy strand, which is displaced during mtDNA replication. MtSSB blocks primer synthesis on the displaced strand and restricts initiation of light-strand mtDNA synthesis to the specific origin of light-strand DNA synthesis (OriL). The in vivo occupancy profile of mtSSB displays a distinct pattern, with the highest levels of mtSSB close to the mitochondrial control region and with a gradual decline towards OriL. The pattern correlates with the replication products expected for the strand displacement mode of mtDNA synthesis, lending strong in vivo support for this debated model for mitochondrial DNA replication.
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| 3. |
- Gummesson, Bertil, 1977, et al.
(författare)
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Increased RNA polymerase availability directs resources towards growth at the expense of maintenance. : RNAP overproduction
- 2009
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Ingår i: The EMBO journal. - : Wiley. - 1460-2075 .- 0261-4189. ; 28:15, s. 2209-2219
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Tidskriftsartikel (refereegranskat)abstract
- Nutritionally induced changes in RNA polymerase availability have been hypothesized to be an evolutionary primeval mechanism for regulation of gene expression and several contrasting models have been proposed to explain how such 'passive' regulation might occur. We demonstrate here that ectopically elevating Escherichia coli RNA polymerase (Esigma(70)) levels causes an increased expression and promoter occupancy of ribosomal genes at the expense of stress-defense genes and amino acid biosynthetic operons. Phenotypically, cells overproducing Esigma(70) favours growth and reproduction at the expense of motility and damage protection; a response reminiscent of cells with no or diminished levels of the alarmone guanosine tetraphosphate (ppGpp). Consistently, we show that cells lacking ppGpp displayed markedly elevated levels of free Esigma(70) compared with wild-type cells and that the repression of ribosomal RNA expression and reduced growth rate of mutants with constitutively elevated levels of ppGpp can be suppressed by overproducing Esigma(70). We conclude that ppGpp modulates the levels of free Esigma(70) and that this is an integral part of the alarmone's means of regulating a trade-off between growth and maintenance.
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| 4. |
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| 5. |
- Jemt, Elisabeth, et al.
(författare)
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Regulation of DNA replication at the end of the mitochondrial D-loop involves the helicase TWINKLE and a conserved sequence element
- 2015
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 43:19, s. 9262-9275
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Tidskriftsartikel (refereegranskat)abstract
- The majority of mitochondrial DNA replication events are terminated prematurely. The nascent DNA remains stably associated with the template, forming a triple-stranded displacement loop (D-loop) structure. However, the function of the D-loop region of the mitochondrial genome remains poorly understood. Using a comparative genomics approach we here identify two closely related 15 nt sequence motifs of the D-loop, strongly conserved among vertebrates. One motif is at the D-loop 5'-end and is part of the conserved sequence block 1 (CSB1). The other motif, here denoted coreTAS, is at the D-loop 3'-end. Both these sequences may prevent transcription across the D-loop region, since light and heavy strand transcription is terminated at CSB1 and coreTAS, respectively. Interestingly, the replication of the nascent D-loop strand, occurring in a direction opposite to that of heavy strand transcription, is also terminated at coreTAS, suggesting that coreTAS is involved in termination of both transcription and replication. Finally, we demonstrate that the loading of the helicase TWINKLE at coreTAS is reversible, implying that this site is a crucial component of a switch between D-loop formation and full-length mitochondrial DNA replication.
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| 6. |
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| 7. |
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| 8. |
- Persson, Örjan, 1974, et al.
(författare)
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Copy-choice recombination during mitochondrial L-strand synthesis causes DNA deletions
- 2019
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Mitochondrial DNA (mtDNA) deletions are associated with mitochondrial disease, and also accumulate during normal human ageing. The mechanisms underlying mtDNA deletions remain unknown although several models have been proposed. Here we use deep sequencing to characterize abundant mtDNA deletions in patients with mutations in mitochondrial DNA replication factors, and show that these have distinct directionality and repeat characteristics. Furthermore, we recreate the deletion formation process in vitro using only purified mitochondrial proteins and defined DNA templates. Based on our in vivo and in vitro findings, we conclude that mtDNA deletion formation involves copy-choice recombination during replication of the mtDNA light strand.
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| 9. |
- Persson, Örjan, 1974
(författare)
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General stress proteins: Novel function and signals for induction of stationary phase genes in E.coli
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Survival during conditions when nutrients become scarce requires adaptation and expression of genes for maintenance in order for the cell to survive. Among the numerous proteins involved in adaptation and regulation under these conditions, the stationary phase sigma factor, σS, and the Universal stress proteins contribute to survival and bestow the cell with general stress protective functions during growth arrest. In this work we found new mechanisms for the cell to prepare and sense the intracellular environment and respond accordingly. The usp genes and the rpoS gene (encoding σS) were found to be positively regulated by metabolic intermediates of the glycolysis in the central metabolic pathway. Specifically, mutations and conditions resulting in fructose-6-phosphate accumulation elicit superinduction of these genes upon carbon starvation, whereas genetic manipulations reducing the pool size of fructose-6-phosphate have the opposite effect. Under carbon starvation, transcription of the usp and the rpoS genes require and are modulated by the alarmone ppGpp. The observed positive transcriptional regulation by fructose-6-phosphate is not via alterations of the levels of ppGpp. None of the known regulators examined were found to be required for the superinduction. We suggest a novel regulatory mechanism involving the phosphorylated intermediates as a signal molecule for monitoring and subsequent regulation of stress defense genes. Based on mutational studies we also suggest that this signaling mechanism secures accumulation of required survival proteins preceding the complete depletion of the external carbon source. Entry into stationary phase promotes a dramatic stabilization on the sigma factor σS. Mistranslated and oxidized proteins were shown to contribute to elevated levels of σS and transcription of its regulon. Furthermore, ribosomal alleles with enhanced translational accuracy attenuate induction of the RpoS regulon and prevent stabilization of σS. Destabilization of σS is governed by the ClpXP protease, for which aberrant proteins also are substrates. Mechanistically, generation of mistranslated proteins by starvation, or other means, competes for the common enzyme for degradation, and thereby sequesters the pool in favor of σS stabilization. A growing body of evidence shows that there is an intimate connection between proteins required for genome stability and stationary phase survival. We show that the integral membrane protein UspB, a member of the RpoS regulon, is required for proper DNA repair as mutants lacking uspB are sensitive to several DNA damaging conditions. Genetic and biochemical studies demonstrate that UspB acts in the RuvABC recombination repair pathway and removing uspB creates a phenocopy of the DNA resolvase mutant, ruvC, which includes a reduced efficiency in resolving Holliday junctions. Further, we show that the uspB mutant phenotype can be suppressed by ectopic overproduction of RuvC and that both ruvC and uspB mutants can be suppressed by inactivating recD. The fact that RuvABC-dependent repair requires UspB for proper activity suggests that the σS-regulon works together with DNA repair pathways under stress conditions to defend the cell against genotoxic stress.
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| 10. |
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