Elucidating Structural and Cellular Mechanisms of Bacterial Single-stranded Dna-binding Proteins (ssb) PDF Download
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Author: Katarzyna Maria Dubiel
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Book Description
Single-stranded (ss) DNA-binding proteins (SSBs) perform critical functions in genome maintenance by protecting ssDNA intermediates and recruiting numerous genome maintenance proteins to their sites of action. Escherichia coli SSB (EcSSB) consists of an N-terminal DNA binding/tetramerization domain and C-terminal intrinsically disordered linker (IDL) capped by an SSB-Ct motif that mediates protein interactions. EcSSB tetramers bind ssDNA using multiple modes differing in the number of bases bound per tetramer and the magnitude of the binding cooperativity. In this work, I describe the role of SSB intra-tetramer interfaces in cooperativity and present functional SSB fusion proteins to probe SSBs in vivo functions. The mechanisms underlying cooperative binding by SSBs were largely unknown. I solved a crystal structure of Bacillus subtilis SsbA bound to ssDNA that resolves SsbA tetramers joined together by a ssDNA "bridge." This structure identifies a novel interface, termed the "bridge interface," that links adjacent SSB tetramers. I investigated the role of these interfaces in SSB cooperativity and DNA binding. EcSSB variants with altered bridge interface residues bind ssDNA with reduced cooperativity and with an altered distribution of DNA binding modes. I propose a model in which the bridge interface contributes to cooperative ssDNA binding and SSB function, however, destabilization of the bridge interface is tolerated in vivo. Tools to probe SSB function in cells have been limited with all current SSB fluorescent fusions requiring a gene duplication of wild type ssb for viability. I have generated functional SSB fusion proteins in which fluorescent proteins are inserted with EcSSB's IDL. SSB-IDL fusions maintain wild type DNA- and protein-binding activities in vitro while a direct C-terminal fluorescent fusion of SSB (analogous to those in previous studies) was non-function for protein interactions. E. coli strains with chromosomally incorporated SSB-IDL fusions are viable and without requiring a second ssb gene. Further, these strains have wild type growth rates and fitness. SSB-IDL fusions form SSB foci in cells with similar frequencies as proteins that colocalize with the replisome. Cells expressing SSB-IDL fusions are sensitized to certain DNA damaging agents. My work highlights the utility of SSB-IDL fusions for biochemical and cellular studies of genome maintenance reactions.
Author: Katarzyna Maria Dubiel
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Book Description
Single-stranded (ss) DNA-binding proteins (SSBs) perform critical functions in genome maintenance by protecting ssDNA intermediates and recruiting numerous genome maintenance proteins to their sites of action. Escherichia coli SSB (EcSSB) consists of an N-terminal DNA binding/tetramerization domain and C-terminal intrinsically disordered linker (IDL) capped by an SSB-Ct motif that mediates protein interactions. EcSSB tetramers bind ssDNA using multiple modes differing in the number of bases bound per tetramer and the magnitude of the binding cooperativity. In this work, I describe the role of SSB intra-tetramer interfaces in cooperativity and present functional SSB fusion proteins to probe SSBs in vivo functions. The mechanisms underlying cooperative binding by SSBs were largely unknown. I solved a crystal structure of Bacillus subtilis SsbA bound to ssDNA that resolves SsbA tetramers joined together by a ssDNA "bridge." This structure identifies a novel interface, termed the "bridge interface," that links adjacent SSB tetramers. I investigated the role of these interfaces in SSB cooperativity and DNA binding. EcSSB variants with altered bridge interface residues bind ssDNA with reduced cooperativity and with an altered distribution of DNA binding modes. I propose a model in which the bridge interface contributes to cooperative ssDNA binding and SSB function, however, destabilization of the bridge interface is tolerated in vivo. Tools to probe SSB function in cells have been limited with all current SSB fluorescent fusions requiring a gene duplication of wild type ssb for viability. I have generated functional SSB fusion proteins in which fluorescent proteins are inserted with EcSSB's IDL. SSB-IDL fusions maintain wild type DNA- and protein-binding activities in vitro while a direct C-terminal fluorescent fusion of SSB (analogous to those in previous studies) was non-function for protein interactions. E. coli strains with chromosomally incorporated SSB-IDL fusions are viable and without requiring a second ssb gene. Further, these strains have wild type growth rates and fitness. SSB-IDL fusions form SSB foci in cells with similar frequencies as proteins that colocalize with the replisome. Cells expressing SSB-IDL fusions are sensitized to certain DNA damaging agents. My work highlights the utility of SSB-IDL fusions for biochemical and cellular studies of genome maintenance reactions.
Author: James L. Keck
Publisher: Humana Press
ISBN: 9781493962518
Category : Science
Languages : en
Pages : 272
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Book Description
This Methods in Molecular Biology book offers techniques for examining fundamental properties of SSBs and for exploiting the biochemical functions of SSBs as in vitro and in vivo reagents. Includes materials lists, protocols, troubleshooting tips and more."
Author:
Publisher:
ISBN: 9780815332183
Category : Cells
Languages : en
Pages : 0
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Book Description
Author: Fumio Hanaoka
Publisher: Springer
ISBN: 443155873X
Category : Science
Languages : en
Pages : 548
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Book Description
This book is a comprehensive review of the detailed molecular mechanisms of and functional crosstalk among the replication, recombination, and repair of DNA (collectively called the "3Rs") and the related processes, with special consciousness of their biological and clinical consequences. The 3Rs are fundamental molecular mechanisms for organisms to maintain and sometimes intentionally alter genetic information. DNA replication, recombination, and repair, individually, have been important subjects of molecular biology since its emergence, but we have recently become aware that the 3Rs are actually much more intimately related to one another than we used to realize. Furthermore, the 3R research fields have been growing even more interdisciplinary, with better understanding of molecular mechanisms underlying other important processes, such as chromosome structures and functions, cell cycle and checkpoints, transcriptional and epigenetic regulation, and so on. This book comprises 7 parts and 21 chapters: Part 1 (Chapters 1–3), DNA Replication; Part 2 (Chapters 4–6), DNA Recombination; Part 3 (Chapters 7–9), DNA Repair; Part 4 (Chapters 10–13), Genome Instability and Mutagenesis; Part 5 (Chapters 14–15), Chromosome Dynamics and Functions; Part 6 (Chapters 16–18), Cell Cycle and Checkpoints; Part 7 (Chapters 19–21), Interplay with Transcription and Epigenetic Regulation. This volume should attract the great interest of graduate students, postdoctoral fellows, and senior scientists in broad research fields of basic molecular biology, not only the core 3Rs, but also the various related fields (chromosome, cell cycle, transcription, epigenetics, and similar areas). Additionally, researchers in neurological sciences, developmental biology, immunology, evolutionary biology, and many other fields will find this book valuable.
Author: Andrei Kuzminov
Publisher: Landes Bioscience
ISBN:
Category : Medical
Languages : en
Pages : 234
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Book Description
Author: Mark R. Kelley
Publisher: Academic Press
ISBN: 0128035994
Category : Science
Languages : en
Pages : 466
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Book Description
DNA Repair and Cancer Therapy: Molecular Targets and Clinical Applications, Second Edition provides a comprehensive and timely reference that focuses on the translational and clinical use of DNA repair as a target area for the development of diagnostic biomarkers and the enhancement of cancer treatment. Experts on DNA repair proteins from all areas of cancer biology research take readers from bench research to new therapeutic approaches. This book provides a detailed discussion of combination therapies, in other words, how the inhibition of repair pathways can be coupled with chemotherapy, radiation, or DNA damaging drugs. Newer areas in this edition include the role of DNA repair in chemotherapy induced peripheral neuropathy, radiation DNA damage, Fanconi anemia cross-link repair, translesion DNA polymerases, BRCA1-BRCA2 pathway for HR and synthetic lethality, and mechanisms of resistance to clinical PARP inhibitors. - Provides a comprehensive overview of the basic and translational research in DNA repair as a cancer therapeutic target - Includes timely updates from the earlier edition, including Fanconi Anemia cross-link repair, translesion DNA polymerases, chemotherapy induced peripheral neuropathy, and many other new areas within DNA repair and cancer therapy - Saves academic, medical, and pharma researchers time by allowing them to quickly access the very latest details on DNA repair and cancer therapy - Assists researchers and research clinicians in understanding the importance of the breakthroughs that are contributing to advances in disease-specific research
Author: Robert K. Delong
Publisher: Academic Press
ISBN: 0128010126
Category : Science
Languages : en
Pages : 123
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Book Description
Introductory Experiments on Biomolecules and their Interactions provides a novel approach to teaching biomolecules in the lab. While featuring the requisite fundamentals, it also captures the author's experience in industry, thus providing unique, up-to-date experiments which take the learning experience one-step further. The text parallels lectures using a standard biochemistry undergraduate text. Unlike most current lab manuals available in the market which simply emphasize an introduction of techniques, this lab manual provides students with opportunities to demonstrate and prove the knowledge and theories they learn from class. - Features quantitative analysis of RNA degradation by RNase - Contains problem sets, calculations, and references for each lab fully immersing students in the learning process - Includes instruction on how to maintain a lab notebook and write a formal lab report - Provides hands-on engagement with the four major types of biomolecules and "real-life and better applied examples of molecular interactions
Author: Byung Hong Kim
Publisher: Cambridge University Press
ISBN: 1107171733
Category : Medical
Languages : en
Pages : 509
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Book Description
Extensive and up-to-date review of key metabolic processes in bacteria and archaea and how metabolism is regulated under various conditions.
Author: Loutfy H. Madkour
Publisher: Academic Press
ISBN: 0128197781
Category : Science
Languages : en
Pages : 652
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Book Description
Nucleic Acids as Gene Anticancer Drug Delivery Therapy highlights the most recent developments in cancer treatment using nucleic acids, nanoparticles and polymer nanoparticles for genomic nanocarriers as drug delivery, including promising opportunities for targeted and combination therapy. The development of a wide spectrum of nanoscale technologies is beginning to change the scientific landscape in terms of disease diagnosis, treatment, and prevention. This book presents the use of nanotechnology for medical applications, focusing on its use for anticancer drug delivery. Various intelligent drug delivery systems such as inorganic nanoparticles and polymer-based drug delivery are discussed. The use of smart drug delivery systems seems to be a promising approach for developing intelligent therapeutic systems for cancer immunotherapies and is discussed in detail along with nucleic acid-targeted drug delivery combination therapy for cancer. Nucleic Acids as Gene Anticancer Drug Delivery Therapy will be a useful reference for pharmaceutical scientists, pharmacologiests, and those involved in nanotechnology and cancer research. - Discusses intelligent drug delivery systems such as inorganic nanoparticles and polymer-based drug delivery - Contains a comprehensive comparison of various delivery systems, listing their advantages and limitations - Presents combination therapy as a new hope for enhancing current gene-based treatment efficacy
Author: Chang-Hui Shen
Publisher: Elsevier
ISBN: 0323986099
Category : Science
Languages : en
Pages : 590
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Book Description
Diagnostic Molecular Biology, Second Edition describes the fundamentals of molecular biology in a clear, concise manner with each technique explained within its conceptual framework and current applications of clinical laboratory techniques comprehensively covered. This targeted approach covers the principles of molecular biology, including basic knowledge of nucleic acids, proteins and chromosomes; the basic techniques and instrumentations commonly used in the field of molecular biology, including detailed procedures and explanations; and the applications of the principles and techniques currently employed in the clinical laboratory. Topics such as whole exome sequencing, whole genome sequencing, RNA-seq, and ChIP-seq round out the discussion. Fully updated, this new edition adds recent advances in the detection of respiratory virus infections in humans, like influenza, RSV, hAdV, hRV but also corona. This book expands the discussion on NGS application and its role in future precision medicine. - Provides explanations on how techniques are used to diagnosis at the molecular level - Explains how to use information technology to communicate and assess results in the lab - Enhances our understanding of fundamental molecular biology and places techniques in context - Places protocols into context with practical applications - Includes extra chapters on respiratory viruses (Corona)
