No abstract

Summary Kinetoplast DNA (kDNA), the trypanosome mitochondrial DNA, contains thousands of minicircles and dozens of maxicircles interlocked in a giant network. Remarkably, Trypanosoma brucei's genome encodes eight PIF1-like helicases, six of which are mitochondrial. We now show that TbPIF2 is essential for maxicircle replication. Maxicircle abundance is controlled by TbPIF2 level, as RNAi of this helicase caused maxicircle loss and its overexpression caused a 3- to 6-fold increase in maxicircle abundance. This regulation of maxicircle level is mediated by the TbHslVU protease. Previous experiments demonstrated that RNAi knockdown of TbHslVU dramatically increased abundance of minicircles and maxicircles, presumably because a positive regulator of their synthesis escaped proteolysis and allowed synthesis to continue. Here we found that TbPIF2 level increases following RNAi of the protease. Therefore this helicase is a TbHslVU substrate and the first example of a positive regulator, thus providing a molecular mechanism for controlling maxicircle replication.

Trypanosomes have an unusual mitochondrial genome, called kinetoplast DNA, that is a giant network containing thousands of interlocked minicircles. During kinetoplast DNA synthesis, minicircles are released from the network for replication as -structures, and then the free minicircle progeny reattach to the network. We report that a mitochondrial protein, which we term p38, functions in kinetoplast DNA replication. RNA interference (RNAi) of p38 resulted in loss of kinetoplast DNA and accumulation of a novel free minicircle species named fraction S. Fraction S minicircles are so underwound that on isolation they become highly negatively supertwisted and develop a region of Z-DNA. p38 binds to minicircle sequences within the replication origin. We conclude that cells with RNAi-induced loss of p38 cannot initiate minicircle replication, although they can extensively unwind free minicircles.Kinetoplast DNA (kDNA) is the unusual mitochondrial genome of Trypanosoma brucei and related parasites (13,15,34). kDNA contains several thousand minicircles and a few dozen maxicircles catenated into a giant network. Each cell has one network, condensed into a disk-shaped structure, residing in the single mitochondrion. Maxicircles encode rRNAs and mRNAs for mitochondrial proteins, such as subunits of respiratory complexes, and minicircles encode guide RNAs that are templates for editing of maxicircle transcripts (15, 37).The network structure of kDNA requires a complex and unusual replication mechanism. One important feature of this mechanism is that many of the replication proteins are localized in discrete positions within or near the kDNA disk (13). Another key feature is that replication occurs during a distinct phase of the cell cycle, nearly concurrent with the nuclear S phase (44). The current working model of minicircle replication is outlined in the following paragraph.When replication begins, individual covalently closed minicircles are released from the network, by a topoisomerase, into a region of the mitochondrial matrix between the kDNA disk and the mitochondrial membrane near the flagellar basal body (3). Within this region, called the kinetoflagellar zone, the minicircles encounter proteins such as the origin recognition protein (universal minicircle sequence binding protein [UMSBP]) (1, 39), primase (12), and two DNA polymerases (11). Interaction of these and other proteins with the minicircle promotes unidirectional replication as -structures (26). The progeny minicircles are thought to segregate in the kinetoflagellar zone and then migrate to two antipodal sites, protein assemblies flanking the kDNA disk and positioned 180°apart (7). Within the antipodal sites, the next steps of replication occur. These include removal of RNA primers by structurespecific endonuclease-1 (6), filling in most (but not all) of the gaps between Okazaki fragments by DNA polymerase ␤ (38), and sealing the resulting nicks by DNA ligase k␤ (2). Then a topoisomerase II reattaches the progeny minicircles, still containing at least one ...

Introduced in the 1950s, ethidium bromide (EB) is still used as an anti-trypanosomal drug for African cattle although its mechanism of killing has been unclear and controversial. EB has long been known to cause loss of the mitochondrial genome, named kinetoplast DNA (kDNA), a giant network of interlocked minicircles and maxicircles. However, the existence of viable parasites lacking kDNA (dyskinetoplastic) led many to think that kDNA loss could not be the mechanism of killing. When recent studies indicated that kDNA is indeed essential in bloodstream trypanosomes and that dyskinetoplastic cells survive only if they have a compensating mutation in the nuclear genome, we investigated the effect of EB on kDNA and its replication. We here report some remarkable effects of EB. Using EM and other techniques, we found that binding of EB to network minicircles is low, probably because of their association with proteins that prevent helix unwinding. In contrast, covalently-closed minicircles that had been released from the network for replication bind EB extensively, causing them, after isolation, to become highly supertwisted and to develop regions of left-handed Z-DNA (without EB, these circles are fully relaxed). In vivo, EB causes helix distortion of free minicircles, preventing replication initiation and resulting in kDNA loss and cell death. Unexpectedly, EB also kills dyskinetoplastic trypanosomes, lacking kDNA, by inhibiting nuclear replication. Since the effect on kDNA occurs at a >10-fold lower EB concentration than that on nuclear DNA, we conclude that minicircle replication initiation is likely EB's most vulnerable target, but the effect on nuclear replication may also contribute to cell killing.

IMPORTANCE There are limited data on which factors affect the critical and complex decision to withdraw life-supporting treatment (LST) in patients with severe traumatic brain injury (sTBI).OBJECTIVE To determine demographic and clinical factors associated with the decision to withdraw LST in patients with sTBI. DESIGN, SETTING, AND PARTICIPANTSThis retrospective analysis of inpatient data from more than 825 trauma centers across the US in the American College of Surgeons Trauma Quality Improvement Program database from January 2013 to December 2015 included adult patients with sTBI and documentation of a decision regarding withdrawal of LST (WLST). Data analysis was conducted in September 2019. MAIN OUTCOMES AND MEASURES Factors associated with WLST in sTBI.RESULTS A total of 37931 patients (9817 women [25.9%]) were included in the multivariable analysis; 7864 (20.7%) had WLST. Black patients (4806 [13.2%]; odds ratio [OR], 0.66; 95% CI, 0.59-0.72; P < .001) and patients of other race (4798 [13.2%]; OR, 0.83; 95% CI, 0.76-0.91; P < .001) were less likely than white patients (26 864 [73.7%]) to have WLST. Patients from hospitals in the Midwest (OR, 1.12; 95% CI, 1.04-1.20; P = .002) or Northeast (OR, 1.23; 95% CI, 1.13-1.34; P < .001) were more likely to have WLST than patients from hospitals in the South. Patients with Medicare (OR, 1.55; 95% CI, 1.43-1.69; P < .001) and self-pay patients (OR, 1.36; 95% CI, 1.25-1.47; P < .001) were more likely to have WLST than patients with private insurance. Older patients and those with lower Glasgow Coma Scale scores, higher Injury Severity Scores, or craniotomy were generally more likely to have WLST. Withdrawal of LST was more likely for patients with functionally dependent health status (

The transcription factor E2F1 is a key regulator of proliferation and apoptosis but the molecular mechanisms that mediate these cell fate decisions remain unclear. Here, we identify FOXO transcription factors as E2F1 target genes that act in a feed-forward regulatory loop to reinforce gene induction of multiple apoptotic genes. We found that E2F1 forms a complex with FOXO1 and FOXO3. RNAi-mediated silencing of FOXO impaired E2F1 binding to the promoters of cooperative targets genes. A FOXO3 mutant insensitive to inactivation by survival kinases rescued the inhibitory effect of growth factor signaling on E2F1-mediated transcription and apoptosis. The E2F1/FOXO axis is frequently blocked in cancer, as evidenced by the specific down regulation of the FOXO-dependent E2F1 transcriptional program in multiple cancer types and by the association of a reduced E2F1/FOXO transcriptional program with poor prognosis. HDAC and PI3K inhibitors were identified as specific activators of E2F1/FOXO transcription, acting to enhance E2F1-induced apoptosis in a FOXO3-dependent manner. Notably, combining the HDAC inhibitor vorinostat with a PI3K inhibitor led to enhanced FOXO-dependent apoptosis. Collectively, our results identify E2F1/FOXO cooperation as a regulatory mechanism that places E2F1 apoptotic activity under the control of survival signaling. Therapeutic reactivation of this tumor suppressive mechanism may offer a novel broad-acting therapy for cancer.

Kinetoplast DNA, the trypanosome mitochondrial genome, is a network of interlocked DNA rings including several thousand minicircles and a few dozen maxicircles. Minicircles replicate after release from the network, and their progeny reattach. Remarkably, trypanosomes have six mitochondrial DNA helicases related to yeast PIF1 helicase. Here we report that one of the six, TbPIF1, functions in minicircle replication. RNA interference (RNAi) of TbPIF1 causes a growth defect and kinetoplast DNA loss. Minicircle replication intermediates decrease during RNAi, and there is an accumulation of multiply interlocked, covalently closed minicircle dimers (fraction U). In studying the significance of fraction U, we found that this species also accumulates during RNAi of mitochondrial topoisomerase II. These data indicate that one function of TbPIF1 is an involvement, together with topoisomerase II, in the segregation of minicircle progeny.Trypanosomes and related parasites have a giant DNA network, called kinetoplast DNA (kDNA), 3 residing in their single mitochondrion (1, 2). The network is composed of several thousand minicircles (1 kb) and a few dozen maxicircles (23 kb), all of which are interlocked. The network is condensed into a disk-shaped structure that resides in the mitochondrial matrix near the flagellar basal body that is in the cytoplasm. The condensed network is actually linked to the basal body by a transmembrane filament system called the tripartite attachment complex (3). Maxicircles encode rRNAs and mRNAs for a few proteins such as subunits of respiratory complexes. Some maxicircle transcripts are edited by insertion or deletion of uridylate residues at specific sites, thereby creating an open reading frame. Minicircles encode guide RNAs used as templates for editing maxicircle transcripts (4, 5). Thus mitochondrial gene expression is a joint endeavor of both maxicircles and minicircles.It is not surprising that the unusual kDNA structure has an unconventional mechanism for replication. We will focus here on minicircle synthesis, and the first step is the release by a topoisomerase II of monomeric covalently closed minicircles into an intramitochondrial region between the kDNA network and the membrane near the flagellar basal body. This region, known as the kinetoflagellar zone (KFZ), is the site of replication of the free minicircles. Proteins involved in replication include the universal minicircle sequence-binding protein (UMSBP) and p38 that bind the replication origin (6, 7), primase (8), two DNA polymerases (9), and a type IA topoisomerase (10). These and other proteins not yet identified (such as a DNA helicase) replicate minicircles unidirectionally via -type structures. After replication, the progeny are thought to segregate in the KFZ and then migrate to two antipodal sites positioned on opposite sides of the kDNA disk. Within the antipodal sites are enzymes that remove primers (11) and a DNA polymerase ␤ and ligase k␤ that repair most but not all of the resulting gaps (12, 13). Finally, the minic...

Background: E2F7 is a transcription factor that controls cell cycle by repressing the expression of G 1 /S genes in late S phase. Results: E2F7 forms a heterodimer with E2F1, and it recruits the co-repressor CtBP to repress G 1 /S transcription. Conclusion: E2F7 represses gene transcription by interacting with E2F1 and co-repressor CtBP. Significance: These findings suggest a mechanism for the repression of transcription by E2F7.