Identification of a novel type of spacer element required for imprinting in fission yeast

Asymmetrical segregation of differentiated sister chromatids is thought to be important for cellular differentiation in higher eukaryotes. Similarly, in fission yeast, cellular differentiation involves the asymmetrical segregation of a chromosomal imprint. This imprint has been shown to consist of two ribonucleotides that are incorporated into the DNA during laggingstrand synthesis in response to a replication pause, but the underlying mechanism remains unknown. Here we present key novel discoveries important for unravelling this process. Our data show that cis-acting sequences within the mat1 cassette mediate pausing of replication forks at the proximity of the imprinting site, and the results suggest that this pause dictates specific priming at the position of imprinting in a sequence-independent manner. Also, we identify a novel type of cis-acting spacer region important for the imprinting process that affects where subsequent primers are put down after the replication fork is released from the pause. Thus, our data suggest that the imprint is formed by ligation of a not-fullyprocessed Okazaki fragment to the subsequent fragment. The presented work addresses how differentiated sister chromatids are established during DNA replication through the involvement of replication barriers

A NON-LINEAR OPTIMISATION APPROACH TO IN-PIT HAUL ROAD DESIGN

Truck haul costs, as one of the predominant operational costs for mining and quarrying operations, are known to be heavily dependent on the design parameters of haul roads. Furthermore, in-pit haul road design parameters determine the pit limits and therefore, the potential feasibility of the mining operation. Thus, when in search of an optimal solution in terms of in-pit haul roads, one should primarily consider the location of the in-pit haul road, its design features and the deriving operational costs regarding extraction and haul costs. A suitable objective function in this case may be the undiscounted profit for the ultimate pit design. However, for each considered scenario, truck and excavator operational costs can be calculated using simulation techniques for better accuracy. Furthermore, finding an optimal solution requires the execution of a reliable and efficient algorithm, depending on the shape of the objective function. Hence, a non-linear optimisation approach was proposed in this paper for solving the in-pit haul road optimisation problem, based on a simulation of the materials allocation, which was used for calculating the objective function. Design parameters were assumed to be predetermined, while the only variable used for finding an optimal solution was the location of the in-pit haul road inside the pit contour. In addition, two 1-D algorithms were compared for finding the optimal solution (Search with accelerated step size and 1-D Simplex method). Furthermore, two regression models are proposed (Multiple Linear Regression – MLR and Non-Linear Regression - NLR), which could identify the more feasible region for the in-pit haul road location and reduce the number of iterations required for convergence

A diffusion model for the coordination of DNA replication in Schizosaccharomyces pombe

The locations of proteins and epigenetic marks on the chromosomal DNA sequence are believed to demarcate the eukaryotic genome into distinct structural and functional domains that contribute to gene regulation and genome organization. However, how these proteins and epigenetic marks are organized in three dimensions remains unknown. Recent advances in proximity-ligation methodologies and high resolution microscopy have begun to expand our understanding of these spatial relationships. Here we use polymer models to examine the spatial organization of epigenetic marks, euchromatin and heterochromatin, and origins of replication within the Schizosaccharomyces pombe genome. These models incorporate data from microscopy and proximity-ligation experiments that inform on the positions of certain elements and contacts within and between chromosomes. Our results show a striking degree of compartmentalization of epigenetic and genomic features and lead to the proposal of a diffusion based mechanism, centred on the spindle pole body, for the coordination of DNA replication in S. pombe

Single-strand nicks induce homologous recombination with less toxicity than double-strand breaks using an AAV vector template

Gene targeting by homologous recombination (HR) can be induced by double-strand breaks (DSBs), however these breaks can be toxic and potentially mutagenic. We investigated the I-AniI homing endonuclease engineered to produce only nicks, and found that nicks induce HR with both plasmid and adeno-associated virus (AAV) vector templates. The rates of nick-induced HR were lower than with DSBs (24-fold lower for plasmid transfection and 4- to 6-fold lower for AAV vector infection), but they still represented a significant increase over background (240- and 30-fold, respectively). We observed severe toxicity with the I-AniI ‘cleavase’, but no evidence of toxicity with the I-AniI ‘nickase.’ Additionally, the frequency of nickase-induced mutations at the I-AniI site was at least 150-fold lower than that induced by the cleavase. These results, and the observation that the surrounding sequence context of a target site affects nick-induced HR but not DSB-induced HR, strongly argue that nicks induce HR through a different mechanism than DSBs, allowing for gene correction without the toxicity and mutagenic activity of DSBs

СОВРЕМЕННЫЕ ПРИНЦИПЫ В ХИРУРГИИ ТРАВМ И ЗАБОЛЕВАНИЙ ПОЗВОНОЧНИКА

ABSTRACT. This article describes the current trends concerning diagnostics and treatment of spinal trauma as well as tumors and diseases of vertebra column and spinal cord. The assessment of computer tomography and magnetic resonance imaging capabilities to reveal various pathologies of vertebra column and spinal cord is given. The clinical cases of usage the minimally invasive, endoscopic and transcutaneous technologies for treatment of various spinal pathologies are presented. РЕЗЮМЕ. В статье описан современный подход к диагностике и лечению травм, заболеваний и опухолей позвоночника. Дана оценка возможностей компьютерной и магнитно-резонансной томографии в выявлении патологии позвоночника и спинного мозга. Приведены клинические примеры использования минимально инвазивных, эндоскопических, транскутанных технологий при различной патологии позвоночного столба и спинного мозга.

Characterization of SpPol4, a unique X-family DNA polymerase in Schizosaccharomyces pombe

As predicted by the amino acid sequence, the purified protein coded by Schizosaccharomyces pombe SPAC2F7.06c is a DNA polymerase (SpPol4) whose biochemical properties resemble those of other X family (PolX) members. Thus, this new PolX is template-dependent, polymerizes in a distributive manner, lacks a detectable 3′→5′ proofreading activity and its preferred substrates are small gaps with a 5′-phosphate group. Similarly to Polμ, SpPol4 can incorporate a ribonucleotide (rNTP) into a primer DNA. However, it is not responsible for the 1–2 rNTPs proposed to be present at the mating-type locus and those necessary for mating-type switching. Unlike Polμ, SpPol4 lacks terminal deoxynucleotidyltransferase activity and realigns the primer terminus to alternative template bases only under certain sequence contexts and, therefore, it is less error-prone than Polμ. Nonetheless, the biochemical properties of this gap-filling DNA polymerase are suitable for a possible role of SpPol4 in non-homologous end-joining. Unexpectedly based on sequence analysis, SpPol4 has deoxyribose phosphate lyase activity like Polβ and Polλ, and unlike Polμ, suggesting also a role of this enzyme in base excision repair. Therefore, SpPol4 is a unique enzyme whose enzymatic properties are hybrid of those described for mammalian Polβ, Polλ and Polμ

Checkpoint-Dependent and -Independent Roles of Swi3 in Replication Fork Recovery and Sister Chromatid Cohesion in Fission Yeast

Multiple genome maintenance processes are coordinated at the replication fork to preserve genomic integrity. How eukaryotic cells accomplish such a coordination is unknown. Swi1 and Swi3 form the replication fork protection complex and are involved in various processes including stabilization of replication forks, activation of the Cds1 checkpoint kinase and establishment of sister chromatid cohesion in fission yeast. However, the mechanisms by which the Swi1–Swi3 complex achieves and coordinates these tasks are not well understood. Here, we describe the identification of separation-of-function mutants of Swi3, aimed at dissecting the molecular pathways that require Swi1–Swi3. Unlike swi3 deletion mutants, the separation-of-function mutants were not sensitive to agents that stall replication forks. However, they were highly sensitive to camptothecin that induces replication fork breakage. In addition, these mutants were defective in replication fork regeneration and sister chromatid cohesion. Interestingly, unlike swi3-deleted cell, the separation-of-functions mutants were proficient in the activation of the replication checkpoint, but their fork regeneration defects were more severe than those of checkpoint mutants including cds1Δ, chk1Δ and rad3Δ. These results suggest that, while Swi3 mediates full activation of the replication checkpoint in response to stalled replication forks, Swi3 activates a checkpoint-independent pathway to facilitate recovery of collapsed replication forks and the establishment of sister chromatid cohesion. Thus, our separation-of-function alleles provide new insight into understanding the multiple roles of Swi1-Swi3 in fork protection during DNA replication, and into understanding how replication forks are maintained in response to different genotoxic agents

Chromosome Duplication in <i>Saccharomyces cerevisiae</i>

The accurate and complete replication of genomic DNA is essential for all life. In eukaryotic cells, the assembly of the multi-enzyme replisomes that perform replication is divided into stages that occur at distinct phases of the cell cycle. Replicative DNA helicases are loaded around origins of DNA replication exclusively during G 1 phase. The loaded helicases are then activated during S phase and associate with the replicative DNA polymerases and other accessory proteins. The function of the resulting replisomes is monitored by checkpoint proteins that protect arrested replisomes and inhibit new initiation when replication is inhibited. The replisome also coordinates nucleosome disassembly, assembly, and the establishment of sister chromatid cohesion. Finally, when two replisomes converge they are disassembled. Studies in Saccharomyces cerevisiae have led the way in our understanding of these processes. Here, we review our increasingly molecular understanding of these events and their regulation. Keywords: DNA replication; cell cycle; chromatin; chromosome duplication; genome stability; YeastBookNational Institutes of Health (U.S.) (Grant GM-052339

A SIMULATION OF THE MATERIALS FLOW IN A QUARRYING OPERATION

ABSTRACT. Determining the exact number of different types of mining equipment on an annual base or for the life of a mine is one of the most crucial elements in a mining project. In most cases, such estimates are based on analytical models which utilise certain empirical coefficients as correction factors, as they account for the individual conditions under which the deposit is exploited. However, their exact values to some extent is unknown and their values used for calculations can be based on subjectivity, leading to a biased estimate of the productivity of different mining equipment. The reason for such bias can originate from the use of data from similar mining operations. Therefore, for this study, a simulation approach was used to more accurately estimate the productivity of different mining equipment for a quarrying operation. Following this approach, certain design decisions can be validated or invalidated in a more robust way. In addition, results from the simulation model are compared to one another, which leads to the adoption of a hybrid solution between two of the considered scenarios