Structural Studies of the Single-strand Binding Protein of Escherichia Coli PDF Download
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Author: Jennifer Marie Thorn
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Author: Jennifer Marie Thorn
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Author: Duo Lu
Publisher:
ISBN:
Category :
Languages : en
Pages : 149
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ISBN:
Category :
Languages : en
Pages : 0
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Author: Nico Tjandra
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Category :
Languages : en
Pages : 0
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Author: Marcos T. Oliveira
Publisher: Humana
ISBN: 9781071612897
Category : Science
Languages : en
Pages : 376
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Book Description
This volume provides a comprehensive set of protocols that can be used by any research lab to investigate diverse functional and structural properties of Single Stranded DNA Binding Proteins (SSBs) from eubacterial, archaeal, eukaryotic, mitochondrial and viral systems. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Single Stranded DNA Binding Proteins aims to be a useful practical guide to researchers to help further their study in this field.
Author: Katarzyna Maria Dubiel
Publisher:
ISBN:
Category :
Languages : en
Pages : 243
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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:
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ISBN: 9780815332183
Category : Cells
Languages : en
Pages : 0
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Author: Frederick W. Perrino
Publisher:
ISBN:
Category : DNA.
Languages : en
Pages : 366
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Author:
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Category :
Languages : en
Pages : 264
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In bacterial cells, most if not all replication forks encounter some form of DNA damage or roadblock that stall or inactivate the fork during normal cell growth. Numerous pathways exist for repairing and reactivating replication forks and these pathways are crucial for maintaining genome stability and cell viability. Bacterial "Maintenance of Genome Stability Protein A" (MgsA) and related eukaryotic enzymes are implicated in cellular responses to stalled DNA replication processes. MgsA enzymes are members of the clamp loader clade of AAA+ proteins but their structures and biochemical properties are poorly characterized. We describe the first complete crystal structure of Escherichia coli MgsA that reveals a highly intertwined homotetrameric arrangement for the protein that distinguishes it from other clamp-loader clade AAA+ proteins. An extended oligomerization domain relative to the clamp loader proteins accounts for the unique oligomeric state. The structure represents the inactive conformation of MgsA due to displacement of the arginine finger residues from the neighboring active sites. Thus, a conformational rearrangement is required to engage the arginine finger and activate MgsA ATPase activity. Association with double stranded DNA ends appears to be the trigger that induces the conformational rearrangement and activates ATP hydrolysis. We also describe a potential switch residue, Arginine92, that appears to coordinate DNA binding and ATP hydrolysis within MgsA. MgsA physically interacts with the single-stranded DNA binding protein (SSB). The interaction requires SSB's highly conserved C terminus (SSB Ct) and we define a likely SSB Ct binding site on MgsA. This interaction adds another member to the growing list of SSB interacting proteins and we propose that the interaction is critical for proper MgsA localization to the replisome. Collectively, this thesis presents a structural and biochemical characterization of Escherichia coli MgsA and provides insights into the mechanisms of MgsA-family proteins.
Author: Arnold Revzin
Publisher: CRC Press
ISBN: 9780849361777
Category : Medical
Languages : en
Pages : 278
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Book Description
This important publication addresses the interactions of proteins with nonspecific binding sites on DNA as they play critical roles in fundamental cellular processes such as transcription, DNA replication, and recombination. The book presents current reviews of the biochemistry of representative nonspecific DNA-protein systems, and of their physiological functions. It includes chapters on the techniques used to characterize the complexes, on their thermodynamic properties, and on the role of nonspecific binding as gene regulatory proteins search for specific target sites on the chromosome. Systems considered include the effects of nonspecific binding in regulation of the lactose operon of Escherichia coli, the T4 bacteriophage gene 32 protein, the E. coli single strand binding (SSB) protein and recA protein, eukaryotic SSB's and histone-DNA complexes. The book presents those proteins displaying multiple modes of DNA binding as participants in more than one cellular process. This monograph combines rigorous descriptions of new findings for these important systems with provocative interpretations of the biological significance of the results. It is of great value to researchers ranging from graduate students to senior scientists in the areas of biochemistry, microbiology and molecular/cell biology.
