DNA Replication
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Rolling Circle Mutagenesis of GST-mCherry to Understand Mutation, Gene Expression, and Regulation †
- Authors: Jessica Cole, Amanda Ferguson, Verónica A. Segarra, Susan Walsh*
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Citation: Cole J, Ferguson A, Segarra V, Walsh S. 2017. Rolling circle mutagenesis of gst-mcherry to understand mutation, gene expression, and regulation † . 18(1): doi:10.1128/jmbe.v18i1.1201
- DOI 10.1128/jmbe.v18i1.1201
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Undergraduates are often familiar with textbook examples of human mutations that affect coding regions and the subsequent disorders, but they may struggle with understanding the implications of mutations in the regulatory regions of genes. We have designed a laboratory sequence that will allow students to explore the effect random mutagenesis can have on protein function, expression, and ultimately phenotype. Students design and perform a safe and time-efficient random mutagenesis experiment using error-prone rolling circular amplification of a plasmid expressing the inducible fusion protein glutathione S-transferase (GST)-mCherry. Mutagenized and wild-type control plasmid DNA, respectively, are then purified and transformed into bacteria to assess phenotypic changes. While bacteria transformed with the wild type control should be pink, some bacterial colonies transformed with mutagenized plasmids will exhibit a different color. Students attempt to identify their mutations by isolating plasmid from these mutant colonies, sequencing, and comparing their mutant sequence to the wild-type sequence. Additionally, students evaluate the potential effects of mutations on protein production by inducing GST-mCherry expression in cultures, generating cell lysates, and analyzing them using SDS-PAGE. Students who have a phenotypic difference but do not obtain a coding region mutation will be able to think critically about plasmid structure and regulation outside of the gene sequence. Students who do not obtain bacterial transformants have the chance to contemplate how mutation of antibiotic resistance genes or replication origins may have contributed to their results. Overall, this series of laboratories exposes students to basic genetic techniques and helps them conceptualize mutation beyond coding regions.
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Laboratory Activity to Promote Student Understanding of UV Mutagenesis and DNA Repair †
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Citation: Kouassi J, Waldron I, Tripepi M, Pohlschroder M. 2017. Laboratory activity to promote student understanding of uv mutagenesis and dna repair † . 18(1): doi:10.1128/jmbe.v18i1.1202
- DOI 10.1128/jmbe.v18i1.1202
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Changes in DNA molecules are common, due to the effects of UV light and other external and internal mutagens. Cells have a variety of repair mechanisms which serve to maintain the accuracy of the genetic code. This activity includes a low-cost, safe and technically feasible experiment, which allows students to observe the effects of UV mutagenesis and DNA photorepair in the halophilc archaeon, Haloferax volcanii. An optional extension links this activity to topics of immediate concern to students – how exposure to UVC light contributes to skin cancer risk and the protective effects of sunscreen. Students design and carry out an experiment to test whether SPF 15 sunscreen increases the lethal exposure time for H. volcanii by a factor of 15. Throughout the activity, discussion questions engage students in actively thinking about the biological phenomena and experimental procedures and analysis. This activity is designed for students in college or university genetics, microbiology, or introductory biology courses as well as in high school honors biology courses. Teachers report that this activity was valuable in helping students understand mutagenesis and photorepair and in developing student skills in designing and analyzing experiments.
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Small Things Considered
- Author: Merry Youle
- Publication Date : May 2016
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Author: Merry YouleAbstract:
Bacterial DNA can be damaged by UV radiation, toxins, or by DNA replication error, but bacteria have sophisticated mechanisms to detect and repair such damage. One widely conserved system is the SOS response, which activates a net-work of genes to repair the damage. A key agent in recovery is the recombinase RecA. When regions of single-stranded DNA (ssDNA) result from damage, they must be protected from attack by cellu-lar nucleases. Such regions eventually become coated by RecA monomers that polymerize into long filaments wrap-ping around the DNA. The filament form of RecA(RecA*) drives increased expression of the more than 43 genes that comprise the SOS regulon through its interaction with the repressor LexA.
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Herpes Simplex Virus Turns on DNA Synthesis in Nearby Uninfected Cells
- Author: David C. Holzman
- Publication Date : November 2015
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Author: David C. HolzmanAbstract:
Through some unexpected and still-unidentified form of molecular texting, the herpes simplex virus (HSV) induces a hormone-like stimulation of cellular DNA replication in nearby, uninfected cells, according to Peter O'Hare of St. Mary's Medical School, Imperial College in London, United Kingdom, and his collaborators. Details appeared 26 August 2015 in the Journal of Virology (doi:10.1128/JVI.01950–15/).
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Changes in Chromosome Structure Regulate DNA Replication Initiation
- Publication Date : October 2015
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Bacterial chromosomes change organization and structure during the cell cycle, for reasons as yet unclear. Now David Magnan and David Bates of Baylor College of Medicine, Houston, show in Escherichia coli that programmed changes in chromosome structure may regulate initiation of DNA replication. Negative supercoiling, which favors duplex melting, is required to initiate replication. Based on their previous observations, they posited that structural differences before and after initiation of replication changed chromosome supercoiling. They found that artificially tethering the chromosome to the cell membrane decreased negative supercoiling, and blocked replication initiation without affecting other DNA metabolic processes. “This finding may reveal new targets for antibiotics, and may explain why existing antibiotics that affect DNA supercoiling are so effective,” says Bates. “More significantly, this research may lead to better understanding of bacterial cell cycle control, which is a black box, as well as eukaryotic replication initiation, which is also sensitive to changes in chromosome structure.”
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Prop Demonstrations in Biology Lectures Facilitate Student Learning and Performance †
- Authors: Farshad Tamari*, Kevin M. Bonney, Kristin Polizzotto
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Citation: Tamari F, Bonney K, Polizzotto K. 2015. Prop demonstrations in biology lectures facilitate student learning and performance † . 16(1):6-12 doi:10.1128/jmbe.v16i1.756
- DOI 10.1128/jmbe.v16i1.756
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Abstract:
Science students can benefit from visual aids. In biology lectures, visual aids are usually limited to tables, figures, and PowerPoint presentations. In this IRB-approved study, we examined the effectiveness of the use of five prop demonstrations, three of which are at the intersection of biology and chemistry, in three community college biology courses. We hypothesized that students’ performance on test questions is enhanced by the use of prop demonstrations. Consistent with our hypothesis, we showed that students learn more effectively and perform better on questions that relate to demonstrations than on questions related to lessons that do not have a demonstration component.
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Using Lecture Demonstrations to Visualize Biological Concepts
- Authors: Kristin Polizzotto, Farshad Tamari*
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Citation: Polizzotto K, Tamari F. 2015. Using lecture demonstrations to visualize biological concepts. 16(1):79-81 doi:10.1128/jmbe.v16i1.840
- DOI 10.1128/jmbe.v16i1.840
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Teaching complex biology concepts successfully can be challenging. The use of visual demonstrations has been shown to aid in this process, so we describe here two lecture demonstrations that instructors can use in introductory biology and genetics classes, and that students can easily remember and repeat on their own. The demonstrations do not require expensive equipment and use either props, which the instructor can borrow from the students, or participation of volunteer students. These demonstrations facilitate learning by allowing the student to more actively engage in the process, as we have formally documented in a separate study. They are described in detail here with the hope that other instructors and their students will also find them helpful. Three other props demonstrations for DNA replication, protein structure and linkage can be found using the url: https://sites.google.com/site/tamarif26. The effectiveness of the five props demonstrations was assessed. The results from an IRB approved study, which complements the descriptions provided here, also appear in this issue.
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Case Study Teaching Method Improves Student Performance and Perceptions of Learning Gains †
- Author: Kevin M. Bonney
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Citation: Bonney K. 2015. Case study teaching method improves student performance and perceptions of learning gains † . 16(1):21-28 doi:10.1128/jmbe.v16i1.846
- DOI 10.1128/jmbe.v16i1.846
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Following years of widespread use in business and medical education, the case study teaching method is becoming an increasingly common teaching strategy in science education. However, the current body of research provides limited evidence that the use of published case studies effectively promotes the fulfillment of specific learning objectives integral to many biology courses. This study tested the hypothesis that case studies are more effective than classroom discussions and textbook reading at promoting learning of key biological concepts, development of written and oral communication skills, and comprehension of the relevance of biological concepts to everyday life. This study also tested the hypothesis that case studies produced by the instructor of a course are more effective at promoting learning than those produced by unaffiliated instructors. Additionally, performance on quantitative learning assessments and student perceptions of learning gains were analyzed to determine whether reported perceptions of learning gains accurately reflect academic performance. The results reported here suggest that case studies, regardless of the source, are significantly more effective than other methods of content delivery at increasing performance on examination questions related to chemical bonds, osmosis and diffusion, mitosis and meiosis, and DNA structure and replication. This finding was positively correlated to increased student perceptions of learning gains associated with oral and written communication skills and the ability to recognize connections between biological concepts and other aspects of life. Based on these findings, case studies should be considered as a preferred method for teaching about a variety of concepts in science courses.
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From Pipe Cleaners and Pony Beads to Apps and 3D Glasses: Teaching Protein Structure †
- Author: Pamela A. Marshall
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Citation: Marshall P. 2014. From pipe cleaners and pony beads to apps and 3d glasses: teaching protein structure † . 15(2):304-306 doi:10.1128/jmbe.v15i2.714
- DOI 10.1128/jmbe.v15i2.714
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Abstract:
Students often self-identify as visual learners and prefer to engage with a topic in an active, hands-on way. Indeed, much research has shown that students who actively engage with the material and are engrossed in the topics retain concepts better than students who are passive receivers of information. However, much of learning life science concepts is still driven by books and static pictures. One concept students have a hard time grasping is how a linear chain of amino acids folds to becomes a 3D protein structure. Adding three dimensional activities to the topic of protein structure and function should allow for a deeper understanding of the primary, secondary, tertiary, and quaternary structure of proteins and how proteins function in a cell. Here, I review protein folding activities and describe using Apps and 3D visualization to enhance student understanding of protein structure.
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Making Heads or Tails: Planarian Stem Cells in the Classroom †
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Citation: Srougi M, Thomas-Swanik J, Chan J, Marchant J, Carson S. 2014. Making heads or tails: planarian stem cells in the classroom † . 15(1):18-25 doi:10.1128/jmbe.v15i1.692
- DOI 10.1128/jmbe.v15i1.692
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Stem cells hold great promise in the treatment of diseases ranging from cancer to dementia. However, as rapidly as the field of stem cell biology has emerged, heated political debate has followed, scrutinizing the ethical implications of stem cell use. It is therefore imperative to promote scientific literacy by educating students about stem cell biology. Yet, there is a definite lack of material to engage students in this subject at the basic science level. Therefore, we have developed and implemented a hands-on introductory laboratory module that introduces students to stem cell biology and can be easily incorporated into existing curricula. Students learn about stem cell biology using an in vivo planarian model system in which they down-regulate two genes important in stem cell differentiation using RNA interference and then observe the regenerative phenotype. The module was piloted at the high school, community college, and university levels. Here, we report that introductory biology students enrolled at a community college were able to demonstrate gains in learning after completion of a one-hour lecture and four 45-minute laboratory sessions over the course of three weeks. These gains in learning outcomes were objectively evaluated both before and after its execution using a student quiz and experimental results. Furthermore, students’ self-assessments revealed increases in perceived knowledge as well as a general interest in stem cells. Therefore, these data suggest that this module is a simple, useful way to engage and to teach students about stem cell biology.
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Review of: iBioSeminars and iBioMagazine
- Author: Pamela A. Marshall
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Citation: Marshall P. 2013. Review of: ibioseminars and ibiomagazine. 14(2):285-286 doi:10.1128/jmbe.v14i2.657
- DOI 10.1128/jmbe.v14i2.657
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Review of: iBioSeminars (http://ibioseminars.org/) and iBio-Magazine (http://ibiomagazine.org/).
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Homologous Recombination—Enzymes and Pathways
- Authors: Bénédicte Michel, and David Leach
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Citation: Michel B, Leach D. 2012. Homologous Recombination—Enzymes and Pathways, EcoSal Plus 2012; doi:10.1128/ecosalplus.7.2.7
- DOI 10.1128/ecosalplus.7.2.7
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Homologous recombination is an ubiquitous process that shapes genomes and repairs DNA damage. The reaction is classically divided into three phases: presynaptic, synaptic, and postsynaptic. In Escherichia coli, the presynaptic phase involves either RecBCD or RecFOR proteins, which act on DNA double-stranded ends and DNA single-stranded gaps, respectively; the central synaptic steps are catalyzed by the ubiquitous DNA-binding protein RecA; and the postsynaptic phase involves either RuvABC or RecG proteins, which catalyze branch-migration and, in the case of RuvABC, the cleavage of Holliday junctions. Here, we review the biochemical properties of these molecular machines and analyze how, in light of these properties, the phenotypes of null mutants allow us to define their biological function(s). The consequences of point mutations on the biochemical properties of recombination enzymes and on cell phenotypes help refine the molecular mechanisms of action and the biological roles of recombination proteins. Given the high level of conservation of key proteins like RecA and the conservation of the principles of action of all recombination proteins, the deep knowledge acquired during decades of studies of homologous recombination in bacteria is the foundation of our present understanding of the processes that govern genome stability and evolution in all living organisms.
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The Use of Open-Ended Problem-Based Learning Scenarios in an Interdisciplinary Biotechnology Class: Evaluation of a Problem-Based Learning Course Across Three Years †
- Authors: Todd R. Steck*, Warren DiBiase, Chuang Wang, Anatoli Boukhtiarov
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Citation: Steck T, DiBiase W, Wang C, Boukhtiarov A. 2012. The use of open-ended problem-based learning scenarios in an interdisciplinary biotechnology class: evaluation of a problem-based learning course across three years † . 13(1):2-10 doi:10.1128/jmbe.v13i1.389
- DOI 10.1128/jmbe.v13i1.389
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Use of open-ended Problem-Based Learning (PBL) in biology classrooms has been limited by the difficulty in designing problem scenarios such that the content learned in a course can be predicted and controlled, the lack of familiarity of this method of instruction by faculty, and the difficulty in assessment. Here we present the results of a study in which we developed a team-based interdisciplinary course that combined the fields of biology and civil engineering across three years. We used PBL scenarios as the only learning tool, wrote the problem scenarios, and developed the means to assess these courses and the results of that assessment. Our data indicates that PBL changed students’ perception of their learning in content knowledge and promoted a change in students’ learning styles. Although no statistically significant improvement in problem-solving skills and critical thinking skills was observed, students reported substantial changes in their problem-based learning strategies and critical thinking skills.
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Barriers to the Formation of Inversion Rearrangements in Salmonella
- Author: Lynn Miesel
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Source: The Lure of Bacterial Genetics , pp 233-243
Publication Date :
January 2011
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Abstract:
This review illustrates the logic style that Roth taught in investigation of chromosome rearrangements and highlights the interesting findings. A few explanations were proposed for the rarity of inversions: inverse-order repeats occur infrequently in the chromosomes of these organisms; inversion rearrangements may cause deleterious effects that cause a selective disadvantage; and mechanistic constraints may prevent the formation of the rearrangements. Inversions were frequently recovered among recombinants when the repeated sequences flanked certain chromosome segments, termed “permissive". The author focuses on the Roth lab’s approach to studying the barriers to inversion of nonpermissive chromosome segments in S. Typhimurium and will correlate findings with outcomes of investigations in E. coli. Alternatively, barriers to the recombination events required for inversion may block formation of the rearrangements. The crosses yielded inversion rearrangements at frequencies expected of two-fragment transduction events. The role of DNA replication is considered in the homologous recombination events that form inversion rearrangements. Investigation of inversion formation by homologous recombination in E. coli found that replication pausing at inverted ter sites is not the only explanation for nonpermissive and restrictive intervals. It remains to be determined if other barriers limit inversion in Salmonella. The findings summarized here highlight topics to investigate regarding the mechanism of inversion formation and barriers to this process for nonpermissive chromosomal segments. The role of DNA replication in inversion formation could be examined by testing the outcome of placing blocked ter sites within permissive arcs of the chromosome.
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Initiation of DNA Replication
- Authors: Alan C. Leonard, and Julia E. Grimwade
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Citation: Leonard A, Grimwade J. 2010. Initiation of DNA Replication, EcoSal Plus 2010; doi:10.1128/ecosalplus.4.4.1
- DOI 10.1128/ecosalplus.4.4.1
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In recent years it has become clear that complex regulatory circuits control the initiation step of DNA replication by directing the assembly of a multicomponent molecular machine (the orisome) that separates DNA strands and loads replicative helicase at oriC, the unique chromosomal origin of replication. This chapter discusses recent efforts to understand the regulated protein-DNA interactions that are responsible for properly timed initiation of chromosome replication. It reviews information about newly identified nucleotide sequence features within Escherichia coli oriC and the new structural and biochemical attributes of the bacterial initiator protein DnaA. It also discusses the coordinated mechanisms that prevent improperly timed DNA replication. Identification of the genes that encoded the initiators came from studies on temperature-sensitive, conditional-lethal mutants of E. coli, in which two DNA replication-defective phenotypes, "immediate stop" mutants and "delayed stop" mutants, were identified. The kinetics of the delayed stop mutants suggested that the defective gene products were required specifically for the initiation step of DNA synthesis, and subsequently, two genes, dnaA and dnaC, were identified. The DnaA protein is the bacterial initiator, and in E. coli, the DnaC protein is required to load replicative helicase. Regulation of DnaA accessibility to oriC, the ordered assembly and disassembly of a multi-DnaA complex at oriC, and the means by which DnaA unwinds oriC remain important questions to be answered and the chapter discusses the current state of knowledge on these topics.
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DNA Helicases
- Author: Piero R. Bianco
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Citation: Bianco P. 2010. DNA Helicases, EcoSal Plus 2010; doi:10.1128/ecosalplus.4.4.8
- DOI 10.1128/ecosalplus.4.4.8
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DNA and RNA helicases are organized into six superfamilies of enzymes on the basis of sequence alignments, biochemical data, and available crystal structures. DNA helicases, members of which are found in each of the superfamilies, are an essential group of motor proteins that unwind DNA duplexes into their component single strands in a process that is coupled to the hydrolysis of nucleoside 5'-triphosphates. The purpose of this DNA unwinding is to provide nascent, single-stranded DNA (ssDNA) for the processes of DNA repair, replication, and recombination. Not surprisingly, DNA helicases share common biochemical properties that include the binding of single- and double-stranded DNA, nucleoside 5'-triphosphate binding and hydrolysis, and nucleoside 5'-triphosphate hydrolysis-coupled, polar unwinding of duplex DNA. These enzymes participate in every aspect of DNA metabolism due to the requirement for transient separation of small regions of the duplex genome into its component strands so that replication, recombination, and repair can occur. In Escherichia coli, there are currently twelve DNA helicases that perform a variety of tasks ranging from simple strand separation at the replication fork to more sophisticated processes in DNA repair and genetic recombination. In this chapter, the superfamily classification, role(s) in DNA metabolism, effects of mutations, biochemical analysis, oligomeric nature, and interacting partner proteins of each of the twelve DNA helicases are discussed.
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Role of Cyclic Di-GMP in Caulobacter crescentus Development and Cell Cycle Control
- Authors: Sören Abel, Urs Jenal
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Source: The Second Messenger Cyclic Di-GMP , pp 120-136
Publication Date :
January 2010
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Abstract:
This chapter summarizes the current knowledge of cyclic di-GMP (c-di-GMP)-mediated control in Caulobacter crescentus. Several members of this family dynamically position to distinct polar sites during the C. crescentus cell cycle, where they contribute to the temporal and spatial regulation of pole morphogenesis and cell cycle progression. The finding that C. crescentus pole development is regulated by c-di-GMP raised several important questions. Recent in vitro and in vivo studies provided convincing evidence that DivK acts as an allosteric regulator of PleC kinase activity. The primary function of the complex regulatory mechanism responsible for cell cycle-dependent PleD phosphorylation is to limit PleD diguanylate cyclase (DGC) activity to the sessile stalked (ST) cell and exclude it from the motile swarmer (SW) cell. The wide range of different cellular processes and molecular targets that are regulated by c-di-GMP reflects its remarkable versatility as a signaling device. Recently, cell cycle control and regulated proteolysis have been added to this growing list of cellular functions controlled by c-di-GMP. The C. crescentus cell cycle is controlled by a cascade of four master regulators that are activated sequentially and in a hierarchical manner. The molecular and cellular mechanisms that underlie the characteristic behavior of Caulobacter cells and its regulation by c-di-GMP might thus be of general relevance for the understanding of processes involved in the motile-sessile transition in many other bacteria.
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Replisome Dynamics during Chromosome Duplication
- Authors: Isabel Kurth, and Mike O’Donnell
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Citation: Kurth I, O’Donnell M. 2009. Replisome Dynamics during Chromosome Duplication, EcoSal Plus 2009; doi:10.1128/ecosalplus.4.4.2
- DOI 10.1128/ecosalplus.4.4.2
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This review describes the components of the Escherichia coli replisome and the dynamic process in which they function and interact under normal conditions. It also briefly describes the behavior of the replisome during situations in which normal replication fork movement is disturbed, such as when the replication fork collides with sites of DNA damage. E. coli DNA Pol III was isolated first from a polA mutant E. coli strain that lacked the relatively abundant DNA Pol I activity. Further biochemical studies, and the use of double mutant strains, revealed Pol III to be the replicative DNA polymerase essential to cell viability. In a replisome, DnaG primase must interact with DnaB for activity, and this constraint ensures that new RNA primers localize to the replication fork. The leading strand polymerase continually synthesizes DNA in the direction of the replication fork, whereas the lagging-strand polymerase synthesizes short, discontinuous Okazaki fragments in the opposite direction. Discontinuous lagging-strand synthesis requires that the polymerase rapidly dissociate from each new completed Okazaki fragment in order to begin the extension of a new RNA primer. Lesion bypass can be thought of as a two-step reaction that starts with the incorporation of a nucleotide opposite the lesion, followed by the extension of the resulting distorted primer terminus. A remarkable property of E. coli, and many other eubacterial organisms, is the speed at which it propagates. Rapid cell division requires the presence of an extremely efficient replication machinery for the rapid and faithful duplication of the genome.
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DNA Topoisomerases
- Authors: Katherine Evans-Roberts, and Anthony Maxwell
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Citation: Evans-Roberts K, Maxwell A. 2009. DNA Topoisomerases, EcoSal Plus 2009; doi:10.1128/ecosalplus.4.4.9
- DOI 10.1128/ecosalplus.4.4.9
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DNA topoisomerases are enzymes that control the topological state of DNA in all cells; they have central roles in DNA replication and transcription. They are classified into two types, I and II, depending on whether they catalyze reactions involving the breakage of one or both strands of DNA. Structural and mechanistic distinctions have led to further classifications: IA, IB, IC, IIA, and IIB. The essence of the topoisomerase reaction is the ability of the enzymes to stabilize transient breaks in DNA, via the formation of tyrosyl-phosphate covalent intermediates. The essential nature of topoisomerases and their ability to stabilize DNA breaks has led to them being key targets for antibacterial and anticancer agents. This chapter reviews the basic features of topoisomerases focussing mainly on the prokaryotic enzymes. We highlight recent structural advances that have given new insight into topoisomerase mechanisms and into the molecular basis of the action of topoisomerase-specific drugs.
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DNA Replication
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Source: Molecular Biology and Biotechnology: A Guide for Teachers, Third Edition , pp 179-186
Publication Date :
January 2008
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DNA replication is a topic usually presented in 9th-grade biology. The essential fact of DNA replication is that the base-pairing rules make it very easy to generate two identical new helices from one helix. The first part is appropriate for young students; more advanced students will perform both parts of the lesson. The first activity described in this chapter is a simple (and necessarily inaccurate) paper simulation of DNA replication. The second activity is a student reading about DNA polymerase, the central DNA replication enzyme. The background information in the introduction that follows contains far more detail about DNA replication. The two aspects of DNA synthesis that your advanced students need to know are that synthesis is unidirectional and that it absolutely requires a primer. Not surprisingly, the characteristics of DNA polymerase determine the overall features of DNA replication inside the cell and in the test tube. The feature of DNA replication means that DNA synthesis is unidirectional, from 5' to 3'. Uni-directionality presents a problem for chromosome replication that is discussed in this chapter. Chromosomal DNA replication is usually initiated at specific sites along the DNA called replication origins. Instead, several different strategies circumvent the problem created by the specificity of DNA replication enzymes.
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