Copy-choice recombination during mitochondrial L-strand synthesis causes DNA deletions.

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.

Regulation of DNA replication at the end of the mitochondrial D-loop involves the helicase TWINKLE and a conserved sequence element.

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.

In vivo occupancy of mitochondrial single-stranded DNA binding protein supports the strand displacement mode of DNA replication.

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.

Increased RNA polymerase availability directs resources towards growth at the expense of maintenance.

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.

Metabolic control of the Escherichia coli universal stress protein response through fructose-6-phosphate.

The universal stress protein (Usp) superfamily encompasses a conserved group of proteins involved in stress resistance, adaptation to energy deficiency, cell motility and adhesion, and is found in all kingdoms of life. The paradigm usp gene, uspA, of Escherichia coli is transcriptionally activated by a large variety of stresses, and the alarmone ppGpp is required for this activation. Here, we show that the uspA gene is also regulated by an intermediate of the glycolytic/gluconeogenic pathways. Specifically, mutations and conditions resulting in fructose-6-phosphate (F-6-P) accumulation elicit superinduction of uspA upon carbon starvation, whereas genetic manipulations reducing the pool size of F-6-P have the opposite effect. This metabolic control of uspA does not act via ppGpp. Other, but not all, usp genes of the usp superfamily are similarly affected by alterations in F-6-P levels. We suggest that alterations in the pool size of phosphorylated sugars of the upper glycolytic pathway may ensure accumulation of required survival proteins preceding the complete depletion of the external carbon source. Indeed, we show that uspA is, in fact, induced before the carbon source is depleted from the medium.

Decline in ribosomal fidelity contributes to the accumulation and stabilization of the master stress response regulator sigmaS upon carbon starvation.

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.