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.     

Beginning to understand the end of the chromosome

In their 1985 Cell paper, Greider and Blackburn announced the discovery of an enzyme that extended the DNA at chromosome telomeres in the ciliate, Tetrahymena. Since then, there has been an explosion of knowledge about both the RNA and protein subunits of this unusual ribonucleoprotein enzyme in organisms ranging from the ciliates to yeast to humans. The regulation of telomerase is now understood to take place both at the level of synthesis of the enzyme and via the state of its substrate, the telomere itself. The roles of telomerase in both cellular immortality and cancer are vibrant areas of current research.     

Global non-covalent SUMO interaction networks reveal SUMO-dependent stabilization of the non-homologous end joining complex

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SUMO playing tag with ubiquitin

In addition to being structurally related, the protein modifiers ubiquitin and SUMO (small ubiquitin-related modifier), share a multitude of functional interrelations. These include the targeting of the same attachment sites in certain substrates, and SUMO-dependent ubiquitylation in others. Notably, several cellular processes, including the targeting of repair machinery to DNA damage sites, require the sequential sumoylation and ubiquitylation of distinct substrates. Some proteins promote both modifications. By contrast, the activity of some enzymes that control either sumoylation or ubiquitylation is regulated by the respective other modification. In this review, we summarize recent findings regarding intersections between SUMO and ubiquitin that influence genome stability and cell growth and which are relevant in pathogen resistance and cancer treatment.     

SUMO bridges Elg1 and SUMO interactors

Comment on: Parnas O, et al. Cell Cycle 2011; 10:2894-903.     

Suffering at the end of life

In the end‐of‐life context, alleviation of the suffering of a distressed patient is usually seen as a, if not the, central goal for the medical personnel treating her. Yet it has also been argued that suffering should be seen as a part of good dying. More precisely, it has been maintained that alleviating a dying patient’s suffering can make her unable to take care of practical end‐of‐life matters, deprive her of an opportunity to ask questions about and find meaning in and for her existence, and detach her from reality. In this article, I argue that the aims referred to either do not support suffering or are better served by alleviating it. When the aims would be equally well served by enduring suffering and relieving it, the latter appears to be the preferable option, given that the distress a patient experiences has no positive intrinsic value. Indeed, as the suffering can be very distressing, it may not be worth bearing even if that was the best way to achieve the aims: the distress can sometimes be bad enough to outbalance the worth of achieving the goals. Having considered an objection to the effect that a patient can have a self‐regarding moral duty to endure the distress she faces at the end of life, I conclude that the burden of proof is on the side of those who maintain that the suffering experienced at the end of life ought to be endured as a part of dying well.     

SUMO assay with peptide arrays on solid support: insights into SUMO target sites

The modification of proteins by SUMO (small ubiquitin-like modifier) regulates various cellular processes. Sumoylation often occurs on a specific lysine residue within the consensus motif psiKxE/D. However, little is known about the specificity and selectivity of SUMO target sites. We describe here a SUMO assay with peptide array on solid support for the simultaneous characterization of hundreds of different SUMO target sites. This approach was used to characterize known SUMO substrates. The position of the motif within the peptide and the amino acids flanking the acceptor site affected the efficiency of SUMO modification. Interestingly, a sequence of only four amino acids, corresponding to the SUMO consensus motif without flanking amino acids, was a bona fide target site. Analysis of a peptide library for all variants of the psiKxE/D consensus motif revealed that the first and third positions in the tetrapeptide preferably contain aromatic amino acid residues. Furthermore, by adding the SUMO E3 ligase PIAS1 to the reaction mixture, we show specific enhancement of the modification of a PIAS1-dependent SUMO substrate in this system. Overall, our results demonstrate that the sumoylation assay with peptide array on solid support can be used for the high-throughput characterization of SUMO target sites, and provide new insights into the composition, selectivity and specificity of SUMO target sites.     

SUMO meets meiosis: an encounter at the synaptonemal complex: SUMO chains and sumoylated proteins suggest that heterogeneous and complex interactions lie at the centre of the synaptonemal complex

Recent discoveries have identified the small ubiquitin-like modifier (SUMO) as the potential 'missing link' that could explain how the synaptonemal complex (SC) is formed during meiosis. The SC is important for a variety of chromosome interactions during meiosis and appears ladder-like. It is formed when 'axes' of the two homologous chromosomes become connected by the deposition of transverse filaments, forming the steps of the ladder. Although several components of axial and transverse elements have been identified, how the two are connected to form the SC has remained an enigma. Recent discoveries suggest that SUMO modification underlies protein-protein interactions within the SC of budding yeast. The versatility of SUMO in regulating protein-protein interactions adds an exciting new dimension to our understanding of the SC and suggests that SCs are not homogenous structures throughout the nucleus. We propose that this heterogeneity may allow differential regulation of chromosome structure and function.     

Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO

Despite the availability of numerous gene fusion systems, recombinant protein expression in Escherichia coli remains difficult. Establishing the best fusion partner for difficult-to-express proteins remains empirical. To determine which fusion tags are best suited for difficult-to-express proteins, a comparative analysis of the newly described SUMO fusion system with a variety of commonly used fusion systems was completed. For this study, three model proteins, enhanced green fluorescent protein (eGFP), matrix metalloprotease-13 (MMP13), and myostatin (growth differentiating factor-8, GDF8), were fused to the C termini of maltose-binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX), NUS A, ubiquitin (Ub), and SUMO tags. These constructs were expressed in E. coli and evaluated for expression and solubility. As expected, the fusion tags varied in their ability to produce tractable quantities of soluble eGFP, MMP13, and GDF8. SUMO and NUS A fusions enhanced expression and solubility of recombinant proteins most dramatically. The ease at which SUMO and NUS A fusion tags were removed from their partner proteins was then determined. SUMO fusions are cleaved by the natural SUMO protease, while an AcTEV protease site had to be engineered between NUS A and its partner protein. A kinetic analysis showed that the SUMO and AcTEV proteases had similar KM values, but SUMO protease had a 25-fold higher kcat than AcTEV protease, indicating a more catalytically efficient enzyme. Taken together, these results demonstrate that SUMO is superior to commonly used fusion tags in enhancing expression and solubility with the distinction of generating recombinant protein with native sequences.     

Fold-back structures at the distal end influence DNA slippage at the proximal end during mononucleotide repeat expansions.

Polymerase slippage during DNA synthesis by the Klenow fragment of DNA polymerase across A, C, G and T repeats (30 bases) has been studied. Within minutes, duplexes that contain only repeats (30 bp) expand dramatically to several hundred base pairs long. Rate comparisons in a repeat duplex when one strand was expanded as against that when both strands were expanded suggest a model of migrating hairpin loops which in the latter case coalesce into a duplex. Moreover, slippage (at the proximal or 3'-end) is subject to positive and negative effects from the 5'-end (distal) of the same strand. Growing T and G strands generate T.A:T and G-G:C motif fold-back structures at the distal end that hamper slippage at the proximal end. On the other hand, growing tails at the distal end upon annealing with excess complementary template accentuates proximal slippage several-fold.