Semiconservative Research Articles

Aortic Graft Infections – A Nationwide Study

Introduction: Aortic graft and endograft infections (AGI) are rare, but a dreaded complication after aortic surgery. The frequency is difficult to assess due to varying definitions but has been reported to range from 0.3-4%. Only recently a proposal for a unanimous definition of AGI (the MAGIC-criteria) was published. As of now, three major surgical techniques are used to treat AGI; 1) a semiconservative (SC) approach with infection drainage and preservation of the vascular prosthesis, 2) resection of the infected vascular prosthesis with extra-anatomic bypass (EAB), or 3) in-situ repair (ISR) with a vein-, aortic allo-, or antibiotic soaked graft. The literature lacks comparative studies and mainly consists of small single center retrospective studies with a high degree of selection and publication bias. The aim of this study was to assess the outcome and risk factors of surgical treatment of AGI in a large nationwide cohort. Methods: Patients with an AGI-related surgery in a specific European country1 between 1995 and 2016 were identified using the nationwide registry for vascular surgeries1. The MAGIC-criteria were used as definition of an AGI. All vascular centers in the country were invited to participate in the study. A predetermined protocol for data extraction was designed and used for a retrospective case record review. Survival was compared between the three surgical groups at 30-days, 1-year and 5-years (Kaplan-Meier analysis). Predictors for long-term mortality were assessed with Cox-regression. Results: A total of 122 patients with a surgically managed AGI were identified with an additional estimate of 40-50 cases still under review2. The case mix consisted of 25 (20.5%) SC, 58 (37.5%) EAB and 39 (32.0%) ISR. A graft enteric fistula (GEF) was present in 61(50.0%). Within the ISR-group there were 21 vein-grafts, eight Silver-Dacron grafts, seven antibiotic-soaked grafts and three standard PTFE or Dacron grafts. Patients undergoing SC had a higher frequency of coronary heart disease and congestive heart failure (p< 0.05), there were no other differences in base-line comorbidities, age, frequency of emergency surgery or GEF between the groups. The median follow-up among survivors was 5.3 years. The estimated survival was; 30 day: SC 80.8% vs EAB 82.5% vs ISR 83.8% (p = ns), 1 year: SC 46.2% vs EAB 69.1% vs ISR 70.3% (p = ns), 5 years: SC 26.9% vs EAB 41.9% vs ISR 49.2% (p = ns). Survival after SC was significantly worse than EAB and ISR (pooled survival curve analysis EAB + ISR vs SC, p = 0.043). In a Cox-regression analysis including age, surgical technique, presence of GEF and clinical shock at presentation as covariables, only age had a significant correlation with overall survival (HR 1.08, p< 0.001). Conclusion: In a large non-selected nationwide AGI-cohort, mortality after surgical repair remains high. In contrast with some previous studies we could not identify any clear survival benefit using ISR vs EAB. Advanced age correlated with worse outcome. A semi-conservative approach was associated with a worse long-term outcome, at least partially explained by a more advanced comorbidity burden within this cohort. [Kaplain-Meier estimated overall survival comparing the three surgical groups.] Disclosure: Nothing to disclose

Fork Cleavage-Religation Cycle and Active Transcription Mediate Replication Restart after Fork Stalling at Co-transcriptional R-Loops.

Formation of co-transcriptional R-loops underlies replication fork stalling upon head-on transcription-replication encounters. Here, we demonstrate that RAD51-dependent replication fork reversal induced by R-loops is followed by the restart of semiconservative DNA replication mediated by RECQ1 and RECQ5 helicases, MUS81/EME1 endonuclease, RAD52 strand-annealing factor, the DNA ligase IV (LIG4)/XRCC4 complex, and the non-catalytic subunit of DNA polymerase δ, POLD3. RECQ5 disrupts RAD51 filaments assembled on stalled forks after RECQ1-mediated reverse branch migration, preventing a new round of fork reversal and facilitating fork cleavage by MUS81/EME1. MUS81-dependent DNA breaks accumulate in cells lacking RAD52 or LIG4 upon induction of R-loop formation, suggesting that RAD52 acts in concert with LIG4/XRCC4 to catalyze fork religation, thereby mediating replication restart. The resumption of DNA synthesis after R-loop-associated fork stalling also requires active transcription, the restoration of which depends onMUS81, RAD52, LIG4, and the transcription elongation factor ELL. These findings provide mechanistic insights into transcription-replication conflict resolution.

Origins of DNA replication.

In all kingdoms of life, DNA is used to encode hereditary information. Propagation of the genetic material between generations requires timely and accurate duplication of DNA by semiconservative replication prior to cell division to ensure each daughter cell receives the full complement of chromosomes. DNA synthesis of daughter strands starts at discrete sites, termed replication origins, and proceeds in a bidirectional manner until all genomic DNA is replicated. Despite the fundamental nature of these events, organisms have evolved surprisingly divergent strategies that control replication onset. Here, we discuss commonalities and differences in replication origin organization and recognition in the three domains of life.

RNA insertion in DNA as the imprint moiety: the fission yeast paradigm.

This review elaborates on the findings of a new report which possibly resolves the biochemical nature of a novel type of DNA imprint as ribonucleotide and the mechanism of its formation during cell differentiation in fission yeast. The process of mating-type switching in fission yeast, Schizosaccharomyces pombe, displays characteristics of a typical mammalian stem cell lineage, wherein a cell divides to produce an identical cell and a differentiated cell after every two cell divisions. This developmental asymmetry has been ascribed to play a role in generation of a DNA strand-specific imprint at the mat1 locus during lagging strand synthesis and its segregation to one of the two daughter cells by the process of asymmetric, semi-conservative DNA replication. The nature of this imprint and mechanisms of its generation have been a subject of research and debate. A recent report by Singh et al. (Nucleic Acids Res 47:3422-3433. https://doi.org/10.1093/nar/gkz092 , 2019) provides compelling evidence in support of a ribonucleotide as the imprint moiety within the mat1 DNA and demonstrates the role of Mcm10/Cdc23, an important, evolutionarily conserved component of DNA replication machinery in eukaryotes, in installing the imprint through a non-canonical primase activity and interaction with DNA Polα and Swi1. The high degree of conservation of DNA replication machinery, especially the presence of the T7 gene 4 helicase/primase domain in the mammalian orthologs of Mcm10 suggests that similar mechanisms of DNA imprinting may play a role during cell differentiation in metazoans.

Breaking Bad: Uncoupling of Modularity in Centriole Biogenesis and the Generation of Excess Centrioles in Cancer.

Centrosomes are tiny yet complex cytoplasmic structures that perform a variety of roles related to their ability to act as microtubule-organizing centers. Like the genome, centrosomes are single copy structures that undergo a precise semi-conservative replication once each cell cycle. Precise replication of the centrosome is essential for genome integrity, because the duplicated centrosomes will serve as the poles of a bipolar mitotic spindle, and any number of centrosomes other than two will lead to an aberrant spindle that mis-segregates chromosomes. Indeed, excess centrosomes are observed in a variety of human tumors where they generate abnormal spindles in situ that are thought to participate in tumorigenesis by driving genomic instability. At the heart of the centrosome is a pair of centrioles, and at the heart of centrosome duplication is the replication of this centriole pair. Centriole replication proceeds through a complex macromolecular assembly process. However, while centrosomes may contain as many as 500 proteins, only a handful of proteins have been shown to be essential for centriole replication. Our observations suggest that centriole replication is a modular, bottom-up process that we envision akin to building a house; the proper site of assembly is identified, a foundation is assembled at that site, and subsequent modules are added on top of the foundation. Here, we discuss the data underlying our view of modularity in the centriole assembly process, and suggest that non-essential centriole assembly factors take on greater importance in cancer cells due to their function in coordination between centriole modules, using the Monopolar spindles 1 protein kinase and its substrate Centrin 2 to illustrate our model.

Differential Incremental Equilibrium Geometry - Spatial Folding of Protein Particles, Genome Expression and Bidirectional Semiconservative Replication of Ring Chromosomes

The research direction of this paper is to study the interdisciplinary subjects of life science, mathematics and computer science at the molecular level from the life science Molecular Cell Biology. On the basis of mathematical primitive innovation Differential Incremental Balanced Geometry, the cell modification of normal chromosome mitosis was established at the molecular level, and the normal cell tissue spatial morphology with initial boundary was established. DNA is used to unravel double helix and separate double strands to solve the protein skeleton structure of bi-directional Semi-Reserved replication of cyclic chromosomes in life sciences at the molecular level. Therefore, it establishes and reveals the duplication fork and bidirectional duplication of molecular cell biology model, the internal structure and regularity of cyclic chromosomes bound by cyclic DNA double helix and many proteins. New mathematics is integrated into the micro-activities of cell modification in life sciences. The topological geometric image of the solitary wavelet with supersymmetric structure is constructed, which reflects the correct abstract model of cell modification and provides dynamic structure for DNA gene sequencing, etc. It also provides a mature mathematical basis for reliable predictability of gene editing.

Treatment of intertrochanteric fracture femur by Ilizarov fixator in elderly patients

Objective To assess the effectiveness of fixation of intertrochanteric fracture femur in elderly high-risk patients using Ilizarov fixator. Background Internal fixation of intertrochanteric fractures is a routine procedure, but in patients with anesthetic risks, external fixation can be considered a semiconservative method to reduce operative time and complications. In this study, the functional and radiological results of management of intertrochanteric fracture femur in elderly patients using Ilizarov fixator is evaluated. Patients and methods A total of 20 elderly high-risk patients with intertrochanteric fracture were treated by Ilizarov fixator. All patients had uncontrolled concomitant medical disorders such as hypertension, diabetes mellitus, and chronic hepatic or renal failure. The age range was 50–85 years. Results The patients were followed up for a mean of 7.5 months with a range from 6 to 9 months. Excellent and good (satisfactory) results were found in 16 patients and fair and poor (unsatisfactory) results were found in four patients. The time of radiological union and fixator removal ranged from 10 to 16 weeks. Pin-tract infection occurred in all patients. Varus malalignment occurred in 20% of patients. Shortening ranged from 0 to 3°. There were two cases of penetration of the femoral head. Conclusion It was found that external fixation of intertrochanteric fractures in elderly high-risk patients is a reliable, effective, and safe treatment option. It offers minimal operative risk, no blood loss, early mobilization, short hospital stay, and low morbidity and mortality.

DNA Replication

Nearly every cell in a person’s body has the same DNA. Most DNA is located in the cell nucleus, but a small amount of DNA can also be found in the mitochondria. DNA was thought to be a simple molecule, consisting of nucleotides strung together like beads on a string.By the late 1940s biochemists knew that DNA was a very long polymer made up of millions of nucleotides. DNA is made up of two strands and each strand of the original DNA molecule serves as a template for the production of the complementary strand, a process referred to as semi conservative replication. Cellular proofreading and error-checking mechanisms ensure near perfect fidelity for DNA replication. DNA usually exists as a double-stranded structure, with both strands coiled together to form the characteristic double- helix. Each single strand of DNA is a chain of four types of nucleotides. Nucleotides in DNA contain a deoxyribose sugar, a phosphate, and a nucleobase. The four types of nucleotide correspond to the four-nucleobase adenine; cytosine, guanine, and thymine, commonly abbreviated as A, C, G and T. This review was assumed to have the historical background of DNA replicationand major DNA replication steps and its function.

Perennial Prescience.

Perennial Prescience.

Single-Stranded Condensation Stochastically Blocks G-Quadruplex Assembly in Human Telomeric RNA.

TERRA is an RNA molecule transcribed from human subtelomeric regions toward chromosome ends potentially involved in regulation of heterochromatin stability, semiconservative replication, and telomerase inhibition, among others. TERRA contains tandem repeats of the sequence GGGUUA, with a strong tendency to fold into a four-stranded arrangement known as a parallel G-quadruplex. Here, we demonstrate by using single-molecule force spectroscopy that this potential is limited by the inherent capacity of RNA to self-associate randomly and further condense into entropically more favorable structures. We stretched RNA constructions with more than four and less than eight hexanucleotide repeats, thus unable to form several G-quadruplexes in tandem, flanked by non-G-rich overhangs of random sequence by optical tweezers on a one by one basis. We found that condensed RNA stochastically blocks G-quadruplex folding pathways with a near 20% probability, a behavior that is not found in DNA analogous molecules.

Ficellomycin: an aziridine alkaloid antibiotic with potential therapeutic capacity.

Ficellomycin is an aziridine antibiotic produced by Streptomyces ficellus, which displays high in vitro activity against Gram-positive bacteria including multidrug resistant strains of Staphylococcus aureus. Compared to currently available antibiotics, ficellomycin exhibits a unique mechanism of action-it impairs the semiconservative DNA replication by inducing the formation of deficient 34S DNA fragments, which lack the ability to integrate into larger DNA pieces and eventually the complete bacterial chromosome. Until recently, some important progress has been made in research on ficellomycin synthesis and biosynthesis, opening the perspective to develop a new generation of antibiotics with better clinical performance than the currently used ones. In this review, we will cover the discovery and biological activity of ficellomycin, its biosynthesis, mode of action, and related synthetic analogs. The role of ficellomycin and its analogs as an important source of drug prototypes will be discussed together with future research prospects.

Persistence and Tolerance of DNA Damage Induced by Chronic UVB Irradiation of the Human Genome

Exposure to solar UVB radiation leads to the formation of the highly mutagenic cyclobutane pyrimidine dimers (CPDs), the DNA damage responsible for mutations found in skin cancer. The frequency of CPD formation and the repair rate of those lesions are two important parameters to determine the probability of UVR-induced mutations. Previous work has shown that chronic irradiation with sublethal doses of UVB radiation (chronic low-dose UVB radiation) leads to the accumulation of residual CPD that persists over time. We have thus investigated the persistence, localization, and consequences on genome stability of those chronic low-dose UVB radiation-induced residual CPDs. We show that chronic low-dose UVB radiation-induced residual CPDs persist on DNA and are diluted via semiconservative replication. They are overrepresented in the heterochromatin and at the TT dipyrimidine sites, and they catalyze the incidence of sister chromatin exchange. Our results shed some light on the impact of chronic UVB radiation exposure on DNA, with a focus on residual CPDs, their distribution, and consequences.

Identification and characterization of the ficellomycin biosynthesis gene cluster from Streptomyces ficellus.

Ficellomycin is a peptide-like antibiotic which exhibits potent in vitro activity against Penicillium oxalicum and Staphylococcus aureus, even against strains resistant to most clinically used antibiotics. The gene cluster responsible for ficellomycin biosynthesis was cloned from Streptomyces ficellus and sequenced. It was found to contain 26 ORFs and is located within 30kb of contiguous DNA. Targeted disruption of the encoding genes revealed that most were involved in the functional section of ficellomycin biosynthesis, such as peptide assembly, regulation, resistance, and biosynthesis of the precursor of ficellomycin 2-[4-guanidyl-1-azabicyclo[3.1.0]hexan-2-yl] glycine (2-GAHG). Within the 2-GAHG synthesis pathway, a sulfate adenylyltransferase appears to be involved in the synthesis of the pharmaceutically important 1-azabicyclo[3.1.0]hexane ring moiety, which has been reported to cause DNA cross-linking or impairment of semiconservative DNA replication.

The Inherent Asymmetry of DNA Replication.

Semiconservative DNA replication has provided an elegant solution to the fundamental problem of how life is able to proliferate in a way that allows cells, organisms, and populations to survive and replicate many times over. Somewhat lost, however, in our admiration for this mechanism is an appreciation for the asymmetries that occur in the process of DNA replication. As we discuss in this review, these asymmetries arise as a consequence of the structure of the DNA molecule and the enzymatic mechanism of DNA synthesis. Increasing evidence suggests that asymmetries in DNA replication are able to play a central role in the processes of adaptation and evolution by shaping the mutagenic landscape of cells. Additionally, in eukaryotes, recent work has demonstrated that the inherent asymmetries in DNA replication may play an important role in the process of chromatin replication. As chromatin plays an essential role in defining cell identity, asymmetries generated during the process of DNA replication may play critical roles in cell fate decisions related to patterning and development.

DNA Replication Origins and Fork Progression at Mammalian Telomeres.

Telomeres are essential chromosomal regions that prevent critical shortening of linear chromosomes and genomic instability in eukaryotic cells. The bulk of telomeric DNA is replicated by semi-conservative DNA replication in the same way as the rest of the genome. However, recent findings revealed that replication of telomeric repeats is a potential cause of chromosomal instability, because DNA replication through telomeres is challenged by the repetitive telomeric sequences and specific structures that hamper the replication fork. In this review, we summarize current understanding of the mechanisms by which telomeres are faithfully and safely replicated in mammalian cells. Various telomere-associated proteins ensure efficient telomere replication at different steps, such as licensing of replication origins, passage of replication forks, proper fork restart after replication stress, and dissolution of post-replicative structures. In particular, shelterin proteins have central roles in the control of telomere replication. Through physical interactions, accessory proteins are recruited to maintain telomere integrity during DNA replication. Dormant replication origins and/or homology-directed repair may rescue inappropriate fork stalling or collapse that can cause defects in telomere structure and functions.

Fission yeast Stn1 is crucial for semi-conservative replication at telomeres and subtelomeres.

The CST complex is a phylogenetically conserved protein complex consisting of CTC1/Cdc13, Stn1 and Ten1 that protects telomeres on linear chromosomes. Deletion of the fission yeast homologs stn1 and ten1 results in complete telomere loss; however, the precise function of Stn1 is still largely unknown. Here, we have isolated a high-temperature sensitive stn1 allele (termed stn1-1). stn1-1 cells abruptly lost telomeric sequence almost completely at the restrictive temperature. The loss of chromosomal DNA happened without gradual telomere shortening, and extended to 30 kb from the ends of chromosomes. We found transient and modest single-stranded G-strand exposure, but did not find any evidence of checkpoint activation in stn1-1 at the restrictive temperature. When we probed neutral-neutral 2D gels for subtelomere regions, we found no Y-arc-shaped replication intermediates in cycling cells. We conclude that the loss of telomere and subtelomere DNAs in stn1-1 cells at the restrictive temperature is caused by very frequent replication fork collapses specifically in subtelomere regions. Furthermore, we identified two independent suppressor mutants of the high-temperature sensitivity of stn1-1: a multi-copy form of pmt3 and a deletion of rif1. Collectively, we propose that fission yeast Stn1 primarily safeguards the semi-conservative DNA replication at telomeres and subtelomeres.

From HDACi to KDACi: we need to revisit non-epigenetic pathways affected by inhibiting lysine deacetylases in therapy.

Various human maladies during aging are associated with substantial transcriptomic deregulation. Such widespread alterations of gene expression are thought to be mediated, at least partially, by changes in histone modifications. Large amount of data suggest that changes in histone acetylation levels are associated with severe diseases as well as aging [1]. Drugs that target and reverse the deregulated histone acetylation sites have therefore attracted considerable attention in recent years as an efficient approach for epigenetic therapy [1], [2]. Indeed, previous studies suggested that administrating histone deacetylase inhibitors could serve as a therapeutic avenue in age‐associated maladies by transcriptional reprogramming [ …

Plant microbiomes and sustainable agriculture: Deciphering the plant microbiome and its role in nutrient supply and plant immunity has great potential to reduce the use of fertilizers and biocides in agriculture.

Bacteria had been around long before multi‐cellular eukaryotes evolved and quickly began to colonize these new life forms as another suitable habitat and potential source of nutrients. Nearly all higher organisms across taxa now have bacterial communities living on and within them—from mutually beneficial symbioses to antagonistic parasitic conflicts. They have co‐evolved with their microbiota and come to depend on it for a range of functions in immunity, metabolism and nutrient absorption. In humans, the microbiota, especially bacteria in the gut, play important roles in all areas, by breaking down indigestible polysaccharides, providing vitamins, mediating nutrient absorption in the gut and helping train the immune system. While research on the human microbiome has stolen the limelight, plant scientists have also been making progress towards elucidating the composition and function of plant microbiomes. Their work has great potential for the future of humanity, as it could help to establish more sustainable agriculture with less dependence on fertilizers, pesticides and herbicides, while increasing yield and nutrient content. Research on the plant microbiome actually predates the animal and human counterparts because microorganisms play an obvious and crucial role in fixing nutrients for the plant, especially nitrogen and phosphorus. Given their vital importance for protein and DNA synthesis and energy production, many plants have evolved close symbiotic relationships with soil bacteria and fungi that supply them with both elements in convenient forms with minimum energy expenditure. Indeed, the close relationships with two specific families of microorganisms set apart the plant microbiome from its animal counterpart: rhizobia nitrogen‐fixing bacteria and mycorrhizal fungi, which greatly enhance the ability of roots to extract various nutrients from the soil. Neither group can survive without the host plant that in return supplies oxygen and products of photosynthesis, mainly proteins and carbohydrates. ### A three‐way relationship The relationship with mycorrhizal fungi seems to be …

Shelterin

Schmutz and de Lange discuss the shelterin complex, which binds to mammalian telomeres and allows for the natural ends of chromosomes to be distinguished from sites of DNA damage.

Alternative Okazaki Fragment Ligation Pathway by DNA Ligase III.

Higher eukaryotes have three types of DNA ligases: DNA ligase 1 (Lig1), DNA ligase 3 (Lig3) and DNA ligase 4 (Lig4). While Lig1 and Lig4 are present in all eukaryotes from yeast to human, Lig3 appears sporadically in evolution and is uniformly present only in vertebrates. In the classical, textbook view, Lig1 catalyzes Okazaki-fragment ligation at the DNA replication fork and the ligation steps of long-patch base-excision repair (BER), homologous recombination repair (HRR) and nucleotide excision repair (NER). Lig4 is responsible for DNA ligation at DNA double strand breaks (DSBs) by the classical, DNA-PKcs-dependent pathway of non-homologous end joining (C-NHEJ). Lig3 is implicated in a short-patch base excision repair (BER) pathway, in single strand break repair in the nucleus, and in all ligation requirements of the DNA metabolism in mitochondria. In this scenario, Lig1 and Lig4 feature as the major DNA ligases serving the most essential ligation needs of the cell, while Lig3 serves in the cell nucleus only minor repair roles. Notably, recent systematic studies in the chicken B cell line, DT40, involving constitutive and conditional knockouts of all three DNA ligases individually, as well as of combinations thereof, demonstrate that the current view must be revised. Results demonstrate that Lig1 deficient cells proliferate efficiently. Even Lig1/Lig4 double knockout cells show long-term viability and proliferate actively, demonstrating that, at least in DT40, Lig3 can perform all ligation reactions of the cellular DNA metabolism as sole DNA ligase. Indeed, in the absence of Lig1, Lig3 can efficiently support semi-conservative DNA replication via an alternative Okazaki-fragment ligation pathway. In addition, Lig3 can back up NHEJ in the absence of Lig4, and can support NER and HRR in the absence of Lig1. Supporting observations are available in less elaborate genetic models in mouse cells. Collectively, these observations raise Lig3 from a niche-ligase to a universal DNA ligase, which can potentially substitute or backup the repair and replication functions of all other DNA ligases in the cell nucleus. Thus, the old model of functionally dedicated DNA ligases is now replaced by one in which only Lig4 remains dedicated to C-NHEJ, with Lig1 and Lig3 showing an astounding functional flexibility and interchangeability for practically all nuclear ligation functions. The underlying mechanisms of Lig3 versus Lig1 utilization in DNA repair and replication are expected to be partly different and remain to be elucidated.