Study on the Molecular Interaction Between Escherichia Coli Single-stranded DNA Binding Protein and Its Partner Protein in DNA Replication Restart PDF Download
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Languages : en
Pages : 77
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Languages : en
Pages : 77
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Languages : en
Pages : 71
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ISBN: 9780815332183
Category : Cells
Languages : en
Pages : 0
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Author: Andrei Kuzminov
Publisher: Landes Bioscience
ISBN:
Category : Medical
Languages : en
Pages : 234
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Author: Aidan Mair McKenzie
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Languages : en
Pages : 0
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Before cells can divide, they must first duplicate their genome through DNA replication. DNA replication is facilitated by the coordinated activities of multiprotein complexes (replisomes). While the speed and abilities of replisomes are mystifying, they are frequently met with challenges to their progression. During active replication, Escherichia coli replisomes are estimated to physically dissociate from the genome at least once per cell cycle after colliding with DNA damage or tightly associated proteins. To sustain cellular viability, DNA replication needs to be reinitiated at these abandoned replication forks through the process of DNA replication restart. Here, I present a genetic, biochemical, and structural investigation of DNA replication restart in E. coli to better define its cellular role and mechanistic details. In Chapter 1, I outline the original discovery of replication restart proteins (RRPs) and the evidence used to propose a three-pathway model for DNA replication restart in E. coli (consisting of the PriA/PriB, PriA/PriC, and PriC/Rep pathways). To facilitate replication restart, each pathway recognizes an abandoned replication fork, remodels the fork, recruits additional RRPs, and ultimately reloads the replicative helicase (DnaB) onto the DNA with the help of its helicase loader (DnaC). Chapter 2 describes a genetic study that systematically defined the roles of specific replication restart pathways in a variety of cellular environments. My results helped integrate DNA replication restart within other cellular processes by revealing a tight link between the repair of double-strand breaks and the PriA/PriB restart pathway. In Chapter 3, I exploited a streamlined DnaB reloading system afforded by PriC to structurally define its manner of binding to the replicative helicase/loader (DnaB6/DnaC6) complex. The cryoEM structure identified dramatic conformational changes required to satisfy the binding interface between PriC and DnaB, and subsequent biochemical investigation identified a hydrophobic packing interface as important for sustained binding. A summary of the findings in Chapters 2 and 3, as well as future directions, are provided in Chapter 4. This work probed mechanistic details of DNA replication restart in E. coli by revealing the cellular processes (and missteps) that necessitate replication restart and helped define the biochemical mechanism of PriC-mediated replicative helicase loading.
Author: Erwin J. G. Peterman
Publisher: Humana Press
ISBN: 9781617792816
Category : Science
Languages : en
Pages : 317
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Life scientists believe that life is driven, directed, and shaped by biomolecules working on their own or in concert. It is only in the last few decades that technological breakthroughs in sensitive fluorescence microscopy and single-molecule manipulation techniques have made it possible to observe and manipulate single biomolecules and measure their individual properties. The methodologies presented in Single Molecule Techniques: Methods and Protocols are being applied more and more to the study of biologically relevant molecules, such as DNA, DNA-binding proteins, and motor proteins, and are becoming commonplace in molecular biophysics, biochemistry, and molecular and cell biology. The aim of Single Molecule Techniques: Methods and Protocols is to provide a broad overview of single-molecule approaches applied to biomolecules on the basis of clear and concise protocols, including a solid introduction to the most widely used single-molecule techniques, such as optical tweezers, single-molecule fluorescence tools, atomic force microscopy, magnetic tweezers, and tethered particle motion. Written in the highly successful Methods in Molecular BiologyTM series format, chapters contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and accessible, Single Molecule Techniques: Methods and Protocols serves as an ideal guide to scientists of all backgrounds and provides a broad and thorough overview of the exciting and still-emerging field of single-molecule biology.
Author: Shiro Iuchi
Publisher: Springer Science & Business Media
ISBN: 0387274219
Category : Science
Languages : en
Pages : 291
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In the early 1980s, a few scientists started working on a Xenopus transcription factor, TFIIIA. They soon discovered a novel domain associated with zinc, and named this domain "zinc finger. " Th e number of proteins with similar zinc fingers grew quickly and these proteins are now called C2H2, Cys2His2 or classical zinc finger proteins. To date, about 24,000 C2H2 zinc finger proteins have been recognized. Approximately 700 human genes, or more than 2% of the genome, have been estimated to encode C2H2 finger proteins. From the beginning these proteins were thought to be numerous, but no one could have predicted such a huge number. Perhaps thousands of scientists are now working on C2H2 zinc finger proteins fi-om variou s viewpoints. This field is a good example of how a new science begins with the insight of a few scientists and how it develops by efforts of numerous independent scientists, in contrast to a policy-driven scientific project, such as the Human Genome Project, with goals clearly set at its inception and with work performed by a huge collaboration throughout the world. As more zinc finger proteins were discovered, several subfamilies, such as C2C2, CCHC, CCCH, LIM, RING, TAZ, and FYVE emerged, increasing our understanding of zinc fingers. The knowledge was overwhelming. Moreover, scientists began defining the term "zinc finger" differently and using various names for identical zinc fingers. These complications may explain why no single comprehensive resource of zinc finger proteins was available before this publication.
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Languages : en
Pages :
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Exonuclease I (ExoI) from Escherichia coli is a monomeric enzyme that processively degrades single stranded DNA in the 3' to 5' direction and has been implicated in DNA recombination and repair. It functions in numerous genome maintenance pathways, with particularly well defined roles in methyl-directed mismatch repair (MMR). The Escherichia coli MMR pathway can be reconstituted in vitro with the activities of eight proteins (8). MutS, MutL and MutH are involved in initiation of repair including mismatch recognition and generation of a nick at a nearby GATC sequence (53, 54, 55, 56). The hemimethylated state of GATC sequences immediately following replication serves as a signal to direct repair to the nascent strand of the DNA duplex (57, 58). DNA helicase II and one of several exonucleases (Exonucleas I, Exonuclease VII and RecJ) are required to excise the error-containing DNA strand beginning at the nicked GATC site (34, 35). Restoration of the correct DNA sequence by repair synthesis involves DNA polymerase III holoenzyme and SSB, and the final nick is sealed by DNA ligase (34). To identify interactions with ExoI involved in MMR repair system, we used the yeast two-hybrid system with ExoI as bait. By screening an E.coli genomic library, E. coli DNA helicase II (UvrD) was identified as a potential interacting protein. UvrD has been shown to be required for DNA excision repair, methyl-directed mismatch repair and has some undefined, role in DNA replication and recombination. In this report, in vitro experiments confirm that UvrD and ExoI make a direct physical interaction that may be required for function of the methyl-directed mismatch repair. Werner Syndrome is a rare autosomal recessive disease characterized by a premature aging phenotype, genomic instability and a dramatically increased incidence of cancer and heart disease. Mutations in a single gene encoding a 1,432 amino-acid helicase/exonuclease (hWRN) have been shown to be responsible for the development o
Author: Peter J. McInerney
Publisher:
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Category :
Languages : en
Pages : 312
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Category :
Languages : en
Pages : 440
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Accurate and reliable duplication of the genome is essential for all organisms. In Escherichia coli, the replication machinery assembled at the origin frequently encounters obstacles that can cause it to stall and dissociate from the template, leaving an abandoned replication fork at a region distinct from the origin. To ensure that complete genome duplication occurs and to avoid chromosomal rearrangements and ultimately cell death, cells must be able to reload the replisome in an origin independent manner. DNA replication restart is an essential process in bacteria that enables reloading of the replicative helicase (DnaB in E. coli) at stalled replication forks. In E.coli, the replication restart proteins PriA, PriB, PriC, and DnaT catalyze this activity. The PriC protein is unique among them in that it can mediate in vitro DnaB reloading in the absence of other replication restart proteins, making PriC-mediated DNA replication restart an excellent system for defining the minimal requirements that allow restart to occur. In this work I have characterized an interaction between PriC and the single-stranded DNA-binding protein (SSB). PriC forms a direct complex with SSB mediated by evolutionarily conserved residues from both proteins. Disruption of this interface leads to a significantly impaired ability to mediate DnaB loading in vitro and replication restart in vivo. Binding of PriC to SSB alters SSB/DNA complexes, which is predicted to expose ssDNA as a platform for DnaB loading. Additionally, I show that PriC interacts with the DnaB/DnaC (helicase/helicase loader) complex. I predict that PriC binding to this complex localizes DnaBC to stalled forks so that once ssDNA is exposed from the SSB/DNA complex, DnaB binds (and DnaC dissociates) and resumes DNA replication. Finally, I present the full-length NMR structure of PriC, demonstrating that PriC is a single-domain with 5 alpha-helices arranged in a bundle with an extended loop connecting alpha helices 1 and 2. Residues involved in ssDNA and SSB binding map to distinct sites, indicating the binding sites are adjacent and not overlapping. The data presented in this thesis have led to a model for PriC-mediated DNA replication restart and define minimal requirements for DNA replication restart systems.
