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The fine modulation of mammalian DNA replication in response to endogenous and exogenous stress conditions
Full text linkDNA replication is essential to allow faithful inheritance of the genome. In mammalian cells, many replication origins are grouped within 200-400 kb regions called replication clusters, which are in turn enclosed in large replication domains (Méchali 2010; Cayrou et al. 2011). This hierarchical organisation is required for the temporal and spatial control of DNA replication and it allows modulating origin activation locally within clusters and globally at the level of replication domains (Yekezare et al. 2013).
During G1 several initiation sites are licensed, but only a subset is activated in the following S-phase (Ge et al. 2007; Blow & Ge 2009; Méchali 2010). Furthermore, origins fire stochastically within clusters and in different positions among cells in a population (Hyrien et al. 2003; Gilbert 2007; Méchali 2010). Remarkably, origin redundancy and plasticity are intrinsic features of the mammalian replication process necessary to face changes in chromatin organisation occurring during development and cell differentiation and to overcome replication stress (Cortez 2015; Alver et al. 2014; Palumbo et al. 2013; Zeman & Cimprich 2014). The temporal regulation of origin firing is established at the level of replication domains, which are conserved among subsequent cell cycles and classified as early, mid or late replicating according to their timing of activation throughout the S-phase (Hiratani et al. 2008; Pope & Gilbert 2013; Rivera-Mulia & Gilbert 2016b).
Replication profiles define the replication program in each cell type and are modulated according to the diverse developmental and differentiation cellular stages, as well as in response to replication stress (Palumbo et al. 2013; Courbet et al. 2008; Anglana et al. 2003). Replication profiles are determined by several parameters, including fork rates, origin choice and alternative replication patterns (i.e. unidirectional and asynchronous forks and pause/arrest events), and their regulation is implied by the flexible nature of the mammalian replication process. Hence, not only origin position but the whole replication dynamics must be taken into consideration to better elucidate the complex phenomena associated with mammalian DNA replication (Prioleau & MacAlpine 2016; Hyrien 2015; Palumbo et al. 2013). In this frame, single-molecule techniques represent the most appropriate tool to detect the intrinsic plasticity and cell-to-cell variability of mammalian genomes (Tuduri et al. 2010; Técher et al. 2016; Prioleau & MacAlpine 2016).
Here, I evaluated how replication profiles are regulated in response to endogenous and exogenous replication stress conditions. In particular, I considered the modulation of the mammalian replication program in relation to sequence organisation, cell growth and differentiation. For this purpose, different cellular models were used.
First, I focused on the effects of a trinucleotide repeat (TNR) expansion on DNA replication. TNR are among the most unstable genomic regions and variations of their length are implicated in many human neurodegenerative disorders (McMurray 2010; Lee & McMurray 2014). Expanded repeats are prone to form stable unusual DNA secondary structures (Mirkin & Mirkin 2007; Krasilnikova & Mirkin 2004), which are a well-known source of replication stress (Zeman & Cimprich 2014; Magdalou et al. 2014; León-Ortiz et al. 2014). A replication-based mechanism is largely accepted to be at the origin of expansion and instability of several TNR, including also the GAA-repeat expansion responsible for Friedreich’s ataxia (FRDA). To better understand this mechanism, different experimental systems from yeast to transfected or engineered human cells were employed (Follonier et al. 2013; Chandok et al. 2012; Kim et al. 2008), but none of them displayed the huge amount of repeats observed in patients’ cells. Thus, to understand how replication profiles are modulated in the presence of long repetitive sequences in the endogenous context, I used human lymphoblastoid cell lines derived from FRDA patients carrying an homozygous GAA-repeat expansion within intron 1 of the Frataxin (FXN) gene. In the presence of the GAA-repeat expansion an alteration of the replication timing was observed by interphase FISH and wide changes in the replication profiles were demonstrated. Indeed, in mutant cells, according to the frequency of duplicated FISH spots, the replication of the FXN gene was slowed or delayed during the first half of the S-phase. This effect was normalised in the latter part when both normal and expanded alleles complete their replication. Further, by molecular combing replication dynamics was monitored in a large genomic region harbouring FXN. By this approach I verified that the most relevant effect associated with the presence of the GAA-repeat expansion was the recruitment of additional dormant origins firing downstream the repeat, which can be considered as a rescue mechanism to assure replication of mutant alleles. As a consequence of dormant origin activation, a switch of the prevalent direction by which the expanded GAA-repeat is replicated was observed, indicating that the origin-switch model for TNR instability conform to the case of the GAA-repeat expansion, similarly to what observed at the FMR1 locus (Gerhardt et al. 2014). Same conclusions were reached in parallel in another study (Gerhardt et al. 2016). Remarkably, a strong reduction of unidirectional fork length was observed in mutant alleles with respect to the normal sequence and this may be considered as a second effect of the GAA-repeat expansion. In line with results obtained at the FMR1 locus (Gerhardt et al. 2014), in the present study recurrent paused/arrested forks were recorded in proximity of the short GAA-repeat in one of the normal cell lines analysed, suggesting a possible impact of the non-pathological GAA-repeat on fork progression. Interestingly, it was recently demonstrated an high occurrence of fork pausing at the expanded GAA-repeat in both undifferentiated and differentiated FRDA cells and these events were hypothesised to be caused by a collision between the replication and transcription machineries (Gerhardt et al. 2016). Understanding whether the origin-switch is the cause or instead a consequence of the GAA-repeat expansion will be necessary and future investigations will primarily contribute to clarify this aspect.
Secondly, I evaluated the effects of increased and unbalanced dNTP pools on the replication program. To ensure completion of DNA replication and to avoid genome instability, the balance among dNTP supply, degradation and consumption must be tightly controlled according to the rate of DNA synthesis (Rampazzo et al. 2010; Chabes & Stillman 2007; Chabosseau et al. 2011; Bester et al. 2011). Indeed, the limiting availability of nucleotide precursors has been widely demonstrated to induce replication stress by slowing fork progression and leading to activation of the DNA damage response (Anglana et al. 2003; Courbet et al. 2008). Conversely, the consequences of an increased supply of dNTPs were only partially depicted in yeast mutants, where replication forks move faster upon ribonucleotide reductase overexpression (Poli et al. 2012). Instead, the effects in mammalian cells are still unknown. Thus, here I used human primary fibroblasts and THP1 monocytes with increased and unbalanced dNTP pools due to depletion of SAMHD1, a protein involved in nucleotide metabolism (Franzolin et al. 2013; Miazzi et al. 2014). By this experimental models the replication profiles were analysed genome-wide by molecular combing in comparison to normal cells. My results indicated that independently of the dNTP pool imbalances, DNA replication proceeds mostly undisturbed in mutated and control fibroblasts, both in physiological and perturbed growth conditions. In contrast, an unexpected slow down of replication forks and a consequent increase in origin firing were detected in SAMHD1-depleted THP1 cells with respect to the wildtype cell line. This differential response to the high availability of nucleotide precursors may be ascribed either to a supposed cell type-specific activity of SAMHD1 or to the development of an adaptive phenotype in mutant fibroblasts compensating for the SAMHD1 depletion. Future goals will be to confirm these hypotheses and to evaluate the effects associated with fork stalling and restart.
Finally, I considered how replication dynamics are modulated during cell differentiation. It is well known that replication timing and replication profiles are cell type-specific and regulated according to changes in chromatin organisation occurring during development and cell differentiation (Palumbo et al. 2013; Hiratani et al. 2010; Hiratani et al. 2008). Hence, I assessed whether DNA replication is affected upon forced cell cycle reactivation. In particular, terminally differentiated mouse myotubes forced to re-enter the cell cycle were analysed by molecular combing in comparison to proliferating myoblasts. A significant reduction of fork rates was detected in reactivated myotubes with respect to proliferating muscle cells, resembling the effects seen under replication stress. This result is in line with previous evidence indicating that myotubes fail to complete their replication, as they are not able to properly expand their dNTP pools (Pajalunga et al. 2010; Pajalunga et al. 2017). Accordingly, after addition of nucleotide precursors the reduction of fork rate was partially ameliorated. However, the number of activated origins in each replication cluster was comparable between myotubes dosed with deoxynucleosides and myoblasts, suggesting that some regions along the genome are left under-replicated. Thus, the replication failure detected upon forced cell cycle re-entry may be ascribed not only to the depletion of nucleotide precursors, but also to the inability of these cells to recruit additional origins compensating for the reduction of fork speed. Interestingly, I found an increased proportion of unidirectional forks in reactivated myotubes when compared to myoblasts, in line with previous data obtained in human primary fibroblasts (Palumbo et al. 2013). Thus, unidirectional forks could be viewed as a remnant of the modality by which some replication domains are replicated when cells move toward terminal differentiation. In this perspective, understanding whether replication timing may be implicated in the replication impairment observed upon forced cell cycle reactivation remains an intriguing issue to be further unravelled.
The complexity of mammalian DNA replication may be ascribed to the intrinsic plasticity of the process, and drawing general conclusions from studies based on individual cellular models may be incautious. The results obtained in this study add new evidence for interpret this complexity, and offer insights for future investigations
Altered replication timing of the frataxin gene in the presence of the GAA/TTC repeat expansion
No full textReplication profile of the FXN locus in normal human cells and in cells carrying the allele with GAA/TCC-repeat expansion
No full textREPLICATION PROFILE OF THE FXN LOCUS IN NORMAL HUMAN CELLS AND IN MUTATED CELLS CARRYING THE GAA/TCC-REPEAT EXPANSION
No full textThe Replication of Frataxin Gene Is Assured by Activation of Dormant Origins in the Presence of a GAA-Repeat Expansion
Full text linkIt is well known that DNA replication affects the stability of several trinucleotide repeats, but whether replication profiles of human loci carrying an expanded repeat differ from those of normal alleles is poorly understood in the endogenous context. We investigated this issue using cell lines from Friedreich's ataxia patients, homozygous for a GAA-repeat expansion in intron 1 of the Frataxin gene. By interphase, FISH we found that in comparison to the normal Frataxin sequence the replication of expanded alleles is slowed or delayed. According to molecular combing, origins never fired within the normal Frataxin allele. In contrast, in mutant alleles dormant origins are recruited within the gene, causing a switch of the prevalent fork direction through the expanded repeat. Furthermore, a global modification of the replication profile, involving origin choice and a differential distribution of unidirectional forks, was observed in the surrounding 850 kb region. These data provide a wide-view of the interplay of events occurring during replication of genes carrying an expanded repeat
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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