Beginning at the end: DNA replication within the telomere

Using single molecule analysis of replicated DNA (SMARD), Drosopoulos et al. (2015; J. Cell Biol. http://dx.doi.org/10.1083/jcb.201410061) report that DNA replication initiates at measurable frequency within the telomere of mouse chromosome arm 14q. They demonstrate that resolution of G4 structures on the G-rich template strand of the telomere requires some overlapping functions of BLM and WRN helicase for leading strand synthesis.Double-strand breaks in DNA can wreak havoc in cells if not repaired. Therefore, it was proposed that the ends of chromosomes may be specialized cap structures that are not recognized as double-strand breaks, thus preventing cell cycle arrest, degradation, and recombinational fusion (Muller, 1938; McClintock, 1939). We now know that telomeres comprise the ends of chromosomes and are essential for genome stability. Telomeres are composed of tandem head-to-tail repeats of a short G-rich sequence; for example, human telomeres are 2–20 kb of (TTAGGG)_n repeats. The chromosome ends are not blunt, and the 3′ end (G-rich strand) overhangs in a single strand that can invade the interior of the telomere to displace the internal G-rich sequence and form a T-loop structure (Griffith et al., 1999; Cesare et al., 2003; Doksani et al., 2013), thus protecting the chromosome ends from being recognized by the cell as double-strand breaks, in addition to protection by proteins that bind the telomere.Eukaryotic chromosomes are duplicated via semiconservative replication with a leading (continuous synthesis for net growth at the 3′ end of the nascent leading strand) and lagging (discontinuous Okazaki fragment synthesis for net growth at the 5′ end of the nascent lagging strand) elongating strand as shown in Fig. 1. In chromosomal semiconservative replication, the short 5′ RNA primer is removed from the nascent strand and the gap is filled in by DNA that is ligated to the adjacent nascent DNA. However, at the end of the chromosome, the gap after removal of the 5′ terminal RNA primer on the lagging strand cannot be filled in, and the chromosome may become shorter with each ensuing round of replication. This has been termed the end-replication problem (Watson, 1972; Olovnikov, 1973), and telomerase helps to solve this problem (Greider and Blackburn, 1987; Soudet et al., 2014).Open in a separate windowFigure 1.DNA replication at the end of chromosomes. (A) DNA replication can initiate within the subtelomeric region with replication forks (green arrows) progressing bidirectionally away from the origin. Telomere DNA is replicated by a replication fork that passes through this region. In each panel, leading nascent strand synthesis is indicated by a blue line with a single arrowhead; lagging nascent strand synthesis is indicated by a blue line with multiple arrowheads. At the top of each panel, the red line indicates the signal seen by microscopy of replication that initiated and continued during administration of the first pulse (IdU, red), and the dotted green line indicates the signal seen for replication extension during the second pulse (CldU, green). (B) On some DNA molecules from mouse chromosome 14q, DNA replication initiates within the telomere itself. In practice, the second (green) pulse was often not observed in the telomere. (C) Partially overlapping functions of BLM and WRN helicases are used to resolve G-quadruplex (G4) DNA (blue structure) that can form on the G-rich parental strand of the telomeres. In cells deficient of BLM and/or WRN helicase, progression of the nascent leading strand in the telomere is impaired; the slowed replication forks are indicated by red arrows. The resulting replication stress is accompanied by activation of dormant replication origins in the subtelomere. The cartoon is not drawn to scale, and the infrequently used subtelomeric replication origin in C is closer to the telomere than the subtelomeric origin in A.Semiconservative replication occurs before the action of telomerase. Previously it was thought that DNA replication began at an origin in chromosomal DNA adjacent to the telomere repeats, with the replication forks moving bidirectionally away from the subtelomeric origin (Fig. 1 A), thus replicating the telomere. However, the question remained whether DNA replication might initiate with some frequency within the telomere itself (Fig. 1 B). This question has now been answered in the affirmative in this issue by Drosopoulos et al., who used single molecule analysis of replicated DNA (SMARD; Norio and Schildkraut, 2001). In this approach, replicating cells are sequentially labeled by two different nucleotide analogues that are subsequently identified by immunofluorescence. For example, in bidirectional replication, red signals from the first pulse will be flanked at each end by green signals from the second pulse. Earlier reports using SMARD had concluded that most replication initiates at subtelomeric regions in the mouse and human genome and rarely in the telomeres themselves (Sfeir et al., 2009; Drosopoulos et al., 2012). In the recent study by Drosopoulos et al. (2015), fluorescence in situ hybridization (FISH) using probes from the telomere region allowed the replication pattern to be analyzed for a 320 kb genomic segment from the end of mouse chromosome arm 14q. Due to the long time (4 h) for the first (red) pulse, usually only red tracts of signal within the telomere were seen, but since many such molecules did not have the red signal extend into the subtelomeric region, it can be comfortably concluded that replication must have initiated within the telomere (Fig. 1 B). Moreover, some molecules did have red signal in the telomere flanked by green signal, supporting this conclusion. Although in these cases there was chromosome-proximal green signal, chromosome-distal green signal was rarely seen. Thus, although there was limited evidence for bidirectional replication originating in the telomere, it is very clear that a replication origin can exist within the telomere proper with a replication fork that extends over time into the subtelomere. It remains to be investigated whether replication initiates at a relatively high frequency in the telomeres of chromosomes other than 14q.These findings raise the question of whether the origin for DNA replication coincides with the simple sequence repeat found in telomeres or instead if it coincides with some other sequence that might be interspersed within the telomere. The former is suggested by a study with Xenopus cell-free extracts that could assemble the pre-replication complex and undergo some DNA replication on exogenous DNA containing exclusively telomeric repeats (Kurth and Gautier, 2010). Similar conclusions that DNA replication can initiate in the simple DNA repeats found in centromeres where replication bubbles have been observed in Drosophila virilis by electron microscopy have been reached (Zakian, 1976), and a recent study suggests that DNA replication initiates within human alpha-satellite DNA (Erliandri et al., 2014).Replications forks move slowly through telomeric DNA (Ivessa et al., 2002; Makovets et al., 2004; Miller et al., 2006; Sfeir et al., 2009) due to the high thermal stability of GC-rich telomeric DNA as well as its propensity to form stable secondary structures, such as G-quadruplex (G4) DNA, which can pose problems for DNA replication (Lopes et al., 2011; Paeschke et al., 2011). Various helicases help solve this problem; for example, Pif1 helicase helps to unwind G4 (Paeschke et al., 2013). Bloom syndrome helicase (BLM) and the Werner syndrome helicase (WRN) have also been implicated in assisting telomere replication: BLM suppresses replication-dependent fragile telomeres (Sfeir et al., 2009), and WRN suppresses defects in telomere lagging strand synthesis (Crabbe et al., 2004). Drosopoulos et al. (2015) now report that leading strand synthesis that initiates within the telomere has a slower rate of progression into the subtelomere in BLM-deficient cells as visualized by SMARD. Moreover, there was a higher frequency of replication initiation in the 14q subtelomere of the BLM-deficient cells, originating closer to the telomere than in BLM-proficient cells. These observations suggest that dormant replication origins in the 14q subtelomere can be activated when fork progression is impeded in BLM-deficient cells (Fig. 1 C). Drosopoulos et al. (2015) also found an increase in subtelomeric replication initiation when replication fork progression from the telomere was hindered by aphidicolin, as an alternate means to activate dormant origins by replication stress. When cells were treated with the G4 stabilizer PhenDC3, 14q subtelomeric origin firing increased further in BLM-deficient cells. Collectively, the data suggest a slowdown of progression of leading strand synthesis from an origin in the 14q telomere (using the G-rich parental strand as the template) when G4 structures cannot be resolved in BLM-deficient cells. As further support for a role of BLM helicase to remove G4 structures, there was increased staining in BLM-deficient cells by the BG4 antibody (Biffi et al., 2013) against G4 in the whole genome and especially in telomeres.WRN helicase can unwind G4 in vitro (Fry and Loeb, 1999; Mohaghegh et al., 2001). When Drosopoulos et al. (2015) used SMARD to analyze replication in cells doubly deficient of both BLM and WRN, they found a marked decrease of red replication signal in 14q telomeres, suggesting some functional overlap between BLM and WRN with regard to leading strand synthesis off the G-rich strand of telomeres. Supporting this conclusion, there was more G4 staining by the BG4 antibody in cells doubly deficient of both BLM and WRN than in cells deficient of just BLM or just WRN. This is the first direct demonstration in vivo of a contribution of BLM and WRN helicases in the resolution of G4 structures, which is especially needed for progression of leading strand synthesis that initiates in telomeres and is copied from the G-rich strand.     

T-helper 17 cell cytokines and interferon type I: partners in crime in systemic lupus erythematosus?

Introduction

A hallmark of systemic autoimmune diseases like systemic lupus erythematosus (SLE) is the increased expression of interferon (IFN) type I inducible genes, so-called IFN type I signature. Recently, T-helper 17 subset (Th17 cells), which produces IL-17A, IL-17F, IL-21, and IL-22, has been implicated in SLE. As CCR6 enriches for Th17 cells, we used this approach to investigate whether CCR6⁺ memory T-helper cells producing IL-17A, IL-17F, IL-21, and/or IL-22 are increased in SLE patients and whether this increase is related to the presence of IFN type I signature.

Methods

In total, 25 SLE patients and 15 healthy controls (HCs) were included. SLE patients were divided into IFN type I signature-positive (IFN⁺) (n = 16) and negative (IFN^-) (n = 9) patients, as assessed by mRNA expression of IFN-inducible genes (IFIGs) in monocytes. Expression of IL-17A, IL-17F, IL-21, and IL-22 by CD4⁺CD45RO⁺CCR6⁺ T cells (CCR6⁺ cells) was measured with flow cytometry and compared between IFN⁺, IFN^- patients and HCs.

Results

Increased percentages of IL-17A and IL-17A/IL-17F double-producing CCR6⁺ cells were observed in IFN⁺ patients compared with IFN^- patients and HCs. IL-17A and IL-17F expression within CCR6⁺ cells correlated significantly with IFIG expression. In addition, we found significant correlation between B-cell activating factor of the tumor necrosis family (BAFF)–a factor strongly correlating with IFN type I - and IL-21 producing CCR6⁺ cells.

Conclusions

We show for the first time higher percentages of IL-17A and IL-17A/IL-17F double-producing CCR6⁺ memory T-helper cells in IFN⁺ SLE patients, supporting the hypothesis that IFN type I co-acts with Th17 cytokines in SLE pathogenesis.     

The nucleolus: a site of ribonucleoprotein maturation

The nucleolus is the site of ribosomal RNA synthesis, processing and ribosome maturation. Various small ribonucleoproteins also undergo maturation in the nucleolus, involving RNA modification and RNA-protein assembly. Such steps and other activities of small ribonucleoproteins also take place in Cajal (coiled) bodies. Events of ribosome biogenesis are found solely in the nucleolus, which is the final destination of small nucleolar RNAs after their traffic through Cajal bodies. However, nucleoli are just a stopping point in the intricate cellular traffic for small nuclear RNAs and other ribonucleoproteins.     

Origin recognition complex binding to a metazoan replication origin

The initiation of DNA replication in eukaryotic cells at the onset of S phase requires the origin recognition complex (ORC) [1]. This six-subunit complex, first isolated in Saccharomyces cerevisiae [2], is evolutionarily conserved [1]. ORC participates in the formation of the prereplicative complex [3], which is necessary to establish replication competence. The ORC-DNA interaction is well established for autonomously replicating sequence (ARS) elements in yeast in which the ARS consensus sequence [4] (ACS) constitutes part of the ORC binding site [2, 5]. Little is known about the ORC-DNA interaction in metazoa. For the Drosophila chorion locus, it has been suggested that ORC binding is dispersed [6]. We have analyzed the amplification origin (ori) II/9A of the fly, Sciara coprophila. We identified a distinct 80-base pair (bp) ORC binding site and mapped the replication start site located adjacent to it. The binding of ORC to this 80-bp core region is ATP dependent and is necessary to establish further interaction with an additional 65-bp of DNA. This is the first time that both the ORC binding site and the replication start site have been identified in a metazoan amplification origin. Thus, our findings extend the paradigm from yeast ARS1 to multicellular eukaryotes, implicating ORC as a determinant of the position of replication initiation.     

Pedigree with frontotemporal lobar degeneration – motor neuron disease and Tar DNA binding protein-43 positive neuropathology: genetic linkage to chromosome 9

Background

Frontotemporal lobar degeneration (FTLD) represents a clinically, pathologically and genetically heterogenous neurodegenerative disorder, often complicated by neurological signs such as motor neuron-related limb weakness, spasticity and paralysis, parkinsonism and gait disturbances. Linkage to chromosome 9p had been reported for pedigrees with the neurodegenerative disorder, frontotemporal lobar degeneration (FTLD) and motor neuron disease (MND). The objective in this study is to identify the genetic locus in a multi-generational Australian family with FTLD-MND.

Methods

Clinical review and standard neuropathological analysis of brain sections from affected pedigree members. Genome-wide scan using microsatellite markers and single nucleotide polymorphism fine mapping. Examination of candidate genes by direct DNA sequencing.

Results

Neuropathological examination revealed cytoplasmic deposition of the TDP-43 protein in three affected individuals. Moreover, we identify a family member with clinical Alzheimer's disease, and FTLD-Ubiquitin neuropathology. Genetic linkage and haplotype analyses, defined a critical region between markers D9S169 and D9S1845 on chromosome 9p21. Screening of all candidate genes within this region did not reveal any novel genetic alterations that co-segregate with disease haplotype, suggesting that one individual carrying a meiotic recombination may represent a phenocopy. Re-analysis of linkage data using the new affection status revealed a maximal two-point LOD score of 3.24 and a multipoint LOD score of 3.41 at marker D9S1817. This provides the highest reported LOD scores from a single FTLD-MND pedigree.

Conclusion

Our reported increase in the minimal disease region should inform other researchers that the chromosome 9 locus may be more telomeric than predicted by published recombination boundaries. Moreover, the existence of a family member with clinical Alzheimer's disease, and who shares the disease haplotype, highlights the possibility that late-onset AD patients in the other linked pedigrees may be mis-classified as sporadic dementia cases.     

Investigation of the diurnal variation in bone resorption for optimal drug delivery and efficacy in osteoporosis with oral calcitonin

Background  

Bone resorption displays marked diurnal variation. Reversible inhibition of bone resorption may result in best possible efficacy when bone resorption peaks. The aim of the study was to assess the pharmacokinetic (PK) and pharmacodynamic (PD) profiles of 0.8 mg of oral salmon calcitonin (sCT) and the effect of timing of drug intake.     

Human immunodeficiency virus seroconversion presenting with acute inflammatory demyelinating polyneuropathy: a case report

Introduction

Electrocardiogram (ECG) abnormalities in patients with blunt chest trauma are diverse and non-specific, but may be indicative of potentially life-threatening conditions.

Case presentation

We report a rare case of pneumopericardium with extreme ECG abnormalities after blunt chest trauma in a 22-year-old male. The diagnosis was confirmed using computed tomography (CT) scanning. The case is discussed, together with its differential diagnosis and the aetiology of pneumopericardium and tension pneumopericardium.

Conclusion

Pneumopericardium should be distinguished from other pathologies such as myocardial contusion and myocardial infarction because of the possible development of tension pneumopericardium. Early CT scanning is important in the evaluation of blunt chest trauma.