Analysis of Bacteriophage [lambda] Site-specific Recombination Intermediates PDF Download

Author: Geoffrey Cassell
Publisher:
ISBN:
Category : Bacteriophage lambda
Languages : en
Pages : 320

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Author: Lea Jessop
Publisher:
ISBN:
Category : Bacteriophage lambda
Languages : en
Pages : 330

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Book Description
Site-specific recombination occurs in many cells types, providing a variety of functions ranging from controlling gene expression to integrating and excising viral and bacteriophage genomes into and out of host chromosomes. We have focused on the 4 site-specific recombination pathways mediated by the bacteriophage Lambda Int protein. In order to better understand the determinants of directionality and efficiency in these recombination pathways, we have developed two assays to determine the architecture of the synaptic intermediates. Crystal structures of these large nucleoprotein complexes are unavailable, consequently we have taken a biochemical approach to address 4 specific questions: (1) What is the multimeric state of Int during recombination? (2) Where do the 4 cysteines of Int map relative to multimerization interfaces? (3) What effect, if any, does tethering through these interfaces have on recombination? (4) Which Int binding sites are bridged by a single Int monomer within the recombination intermediates? Using protein-protein crosslinking we have shown that Int forms a tetramer during recombination conditions, independent of the recombination pathway. There are at least two multimerization interfaces within the tetramer; C25 is located at one of these interfaces while C197 and C262 are located at the second. C217 is also present at or near a multimerization interface, but no cysteine from the adjacent monomer is present to allow tethering by sulfhydryl reactive compounds. Modification of C217 affects excision differently than bent-L recombination. Specifically, modification at C217 is not tolerated during excision but is allowed for bent-L recombination, suggesting differences in the architecture of intermediates between these two pathways. We have also developed an assay to identify intra- and intermolecular bridges formed by Int within the nucleoprotein complexes. In the straight-L recombination pathway, Int is delivered to the point of top strand cleavage, the B core, from the P2 arm site of the partner attL site. During excision, the monomer of Int which cleaves the top strand of attL, the B core, is delivered from the P2 arm site of attR. Thus for two pathways, Int is delivered to the same core site from two different arm sites, which may influence the contacts made between Int and the core sites. We suggest that differences in the way in which Int contacts the core sites may control directionality and efficiency of the Int-mediated recombination pathways.

Author: Troy Michael Bankhead
Publisher:
ISBN:
Category : Bacteriophage lambda
Languages : en
Pages : 348

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Author: Jeffrey A. Engler
Publisher:
ISBN:
Category : Bacteriophage lambda
Languages : en
Pages : 492

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Author: Zhao Wu
Publisher:
ISBN:
Category :
Languages : en
Pages : 318

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Author: Dane Hazelbaker
Publisher:
ISBN: 9780549675563
Category : Genetic recombination
Languages : en
Pages : 288

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Book Description
The site-specific recombinase Integrase encoded by bacteriophage lambda promotes integration and excision of the viral chromosome into and out of its E. coli host chromosome through a Holliday junction recombination intermediate. This intermediate contains an Integrase tetramer bound via its catalytic carboxy-terminal domains to the four "core-type" sites of the Holliday junction DNA and via its amino-terminal domains to distal "arm-type" sites. The two classes of Integrase binding sites are brought into close proximity by an ensemble of accessory proteins that bind and bend the intervening DNA. In this dissertation, aspects of Integrase higher-order structure and function are examined; the C-terminal tail of Integrase and the arm-type Integrase DNA binding sites. The C-terminal tail of Int comprises a context-sensitive regulatory element that links catalytic function with protein multimerization and that also coordinates Integrase activity within the multimeric recombinogenic complex. The experiments reported here show that the beta5-strand of Integrase is not simply a placeholder for the C-terminal tail but rather exerts its own allosteric effects on Integrase function in response to the incoming tail. In the study of Int arm-type sites, we have used a Biotin Interference Assay that probes the requirement for major groove protein binding at specified DNA loci, in conjunction with DNA protection, gel mobility shift, and genetic experiments to test several predictions of the models derived from the X-ray crystal structures of minimized and symmetrized surrogates of recombination intermediates lacking the accessory proteins and their cognate DNA targets. Our data do not support the predictions of "non-canonical" DNA targets for the N-domain of Integrase. We suggest that the difference in the asymmetric interaction profiles of the N-domains and arm-type sites in integrative versus excisive recombinogenic complexes reflects the regulation of recombination while the asymmetry of these patterns within each reaction contributes to directionality.

Author: Alfred Day Hershey
Publisher:
ISBN:
Category : Science
Languages : en
Pages : 810

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Author: Kevin Vaughn Kepple
Publisher:
ISBN:
Category : Genetic recombination
Languages : en
Pages : 390

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Book Description
Bacteriophage lambda uses the recombination protein Integrase (Int) to incorporate and remove its genome from the chromosomal DNA of E. coli . Intermediates of the recombination reaction are difficult to study because of the high efficiency of the reaction and lack of high-energy cofactor requirements. Previously, peptide libraries were screened in order to isolate inhibitors of the recombination reaction. These peptides function by targeting specific recombination intermediates, but also inhibit the growth of bacterial cells. The exact mechanism by which the peptides function both in vitro and in vivo is not known with certainty. In this work, I am interested in how peptide inhibitors interact with their target structure and if this can be applied to explain their antibiotic activity. The specific questions I set out to answer include: what is the site of interaction between the peptide inhibitors and recombination intermediates? What role does substrate conformation play during recombination and inhibition of recombination? What types of biochemical interactions are important for peptide binding? Are potential in vivo protein targets inhibited by the peptides? If inhibition of these potential targets is observed, what is the mechanism of inhibition? I show that peptide inhibitors bind to the center of the protein-bound Holliday junction complex, in agreement with crystal structure data. The data indicate that contacts are mediated by interactions that mimic base stacking. In addition, peptides bind Holliday junctions in the square-planar conformation even in the absence of Int. This opens up the possibility that inhibitors of lambda recombination that trap Holliday junctions may also inhibit many other proteins that process DNA junctions. Indeed, the peptides prevent unwinding of branched DNA substrates by the RecG helicase of E. coli and interfere with the resolution of Holliday junction substrates by the RuvABC complex. The results indicate that inhibition of lambda Integrase, the RecG helicase, and the RuvABC complex all occur by a similar mechanism and imply that there are a number of potential targets for these peptides in vivo. Finally, the results emphasize the importance of Holliday junction conformation for peptide activity and as a determinant for the directionality of catalysis by lambda Int.

Author: K Low
Publisher: Elsevier
ISBN: 0323147275
Category : Science
Languages : en
Pages : 517

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Book Description
The Recombination of Genetic Material aims to introduce the elementary properties of recombinational phenomena. Genetic recombination is a favorite research topic in biology due to its significance. In fact, a simple recombination event can have a profound effect and sometimes can mean the difference between the survival and the demise of an organism. Examples of this are provided in this book. This work also describes numerous recombination systems, mechanisms of the major types of recombination, and the macroscopic products of this biological process. Molecular analyses of recombination enzymes and substrates that have been identified or implicated are also shown. This book will be valuable as a reference material to those interested in this field of study.

Author: Andrés Aguilera
Publisher: Springer Science & Business Media
ISBN: 3540710213
Category : Science
Languages : en
Pages : 536

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Book Description
This work offers a fascinating insight into a crucial genetic process. Recombination is, quite simply, one of the most important topics in contemporary biology. This book is a totally comprehensive treatment of the subject, summarizing all existing views on the topic and at the same time putting them into context. It provides in-depth and up-to-date analysis of the chapter topics, and has been written by international experts in the field.