scholarly journals The Amino Terminus of Bacteriophage λ Integrase Is Involved in Protein-Protein Interactions during Recombination

2000 ◽  
Vol 182 (4) ◽  
pp. 1024-1034 ◽  
Author(s):  
Lea Jessop ◽  
Troy Bankhead ◽  
David Wong ◽  
Anca M. Segall
Keyword(s):  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Protein Dna Interactions ◽  
Bacteriophage Lambda Integrase ◽  
Amino Terminal Domain ◽  
Genetic Assay

ABSTRACT Bacteriophage lambda integrase (Int) catalyzes at least four site-specific recombination pathways between pairs of attachment (att) sites. Protein-protein contacts between monomers of Int are presumed to be important for these site-specific recombination events for several reasons: Int binds to the att sites cooperatively, catalytic Int mutants can complement each other for strand cleavage, and crystal structures for two other recombinases in the Int family (Cre from phage P1 and Int from Haemophilus influenzae phage HP1) show extensive protein-protein contacts between monomers. We have begun to investigate interactions between Int monomers by three approaches. First, using a genetic assay, we show that regions of protein-protein interactions occur throughout Int, including in the amino-terminal domain. This domain was previously thought to be important only for high-affinity protein-DNA interactions. Second, we have found that an amino-terminal His tag reduces cooperative binding to DNA. This disruption in cooperativity decreases the stable interaction of Int with core sites, where catalysis occurs. Third, using protein-protein cross-linking to investigate the multimerization of Int during recombination, we show that Int predominantly forms dimers, trimers, and tetramers. Moreover, we show that the cysteine at position 25 is present at or near the interface between monomers that is involved in the formation of dimers and tetramers. Our evidence indicates that the amino-terminal domain of Int is involved in protein-protein interactions that are likely to be important for recombination.

scholarly journals Interactions between Integrase and Excisionase in the Phage Lambda Excisive Nucleoprotein Complex

2002 ◽  
Vol 184 (18) ◽  
pp. 5200-5203 ◽  
Author(s):  
Eun Hee Cho ◽  
Richard I. Gumport ◽  
Jeffrey F. Gardner
Keyword(s):  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Mobility Shift ◽  
Host Chromosome ◽  
Amino Terminal ◽  
Nucleoprotein Complex ◽  
Carboxyl Terminal ◽  
Α Helix ◽  
Gel Mobility Shift ◽  
Amino Terminal Domain

ABSTRACT Bacteriophage lambda site-specific recombination comprises two overall reactions, integration into and excision from the host chromosome. Lambda integrase (Int) carries out both reactions. During excision, excisionase (Xis) helps Int to bind DNA and introduces a bend in the DNA that facilitates formation of the proper excisive nucleoprotein complex. The carboxyl-terminal α-helix of Xis is thought to interact with Int through direct protein-protein interactions. In this study, we used gel mobility shift assays to show that the amino-terminal domain of Int maintained cooperative interactions with Xis. This finding indicates that the amino-terminal arm-type DNA binding domain of Int interacts with Xis.

scholarly journals Amino-Terminal Domain Exchange Redirects Origin-Specific Interactions of Adeno-Associated Virus Rep78 In Vitro

2001 ◽  
Vol 75 (7) ◽  
pp. 3230-3239 ◽  
Author(s):  
Miran Yoon ◽  
Deborah H. Smith ◽  
Peter Ward ◽  
Francisco J. Medrano ◽  
Aneel K. Aggarwal ◽  
...  
Keyword(s):  
Dna Replication ◽  
Catalytic Domain ◽  
Adeno Associated Virus ◽  
Free System ◽  
Site Specific ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Site Specific Integration ◽  
Amino Terminal Domain

ABSTRACT The unique ability of adeno-associated virus type 2 (AAV) to site-specifically integrate its genome into a defined sequence on human chromosome 19 (AAVS1) makes it of particular interest for use in targeted gene delivery. The objective underlying this study is to provide evidence for the feasibility of retargeting site-specific integration into selected loci within the human genome. Current models postulate that AAV DNA integration is initiated through the interactions of the products of a single viral open reading frame,REP, with sequences present in AAVS1 that resemble the minimal origin for AAV DNA replication. Here, we present a cell-free system designed to dissect the Rep functions required to target site-specific integration using functional chimeric Rep proteins derived from AAV Rep78 and Rep1 of the closely related goose parvovirus. We show that amino-terminal domain exchange efficiently redirects the specificity of Rep to the minimal origin of DNA replication. Furthermore, we establish that the amino-terminal 208 amino acids of Rep78/68 constitute a catalytic domain of Rep sufficient to mediate site-specific endonuclease activity.

Protein-protein interactions in a higher-order structure direct lambda site-specific recombination

1987 ◽  
Vol 195 (3) ◽  
pp. 481-493 ◽  
Author(s):  
John F. Thompson ◽  
Lina Moitoso de Vargas ◽  
Sarah E. Skinner ◽  
Arthur Landy
Keyword(s):  
Protein Interactions ◽  
Higher Order ◽  
Order Structure ◽  
Protein Protein Interactions ◽  
Site Specific ◽  
Site Specific Recombination

Molecular mechanisms of androgen receptor-mediated gene regulation: structure-function analysis of the AF-1 domain.

2004 ◽  
Vol 11 (2) ◽  
pp. 281-293 ◽  
Author(s):  
I J McEwan
Keyword(s):  
Androgen Receptor ◽  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Function Analysis ◽  
Transactivation Domain ◽  
Protein Protein Interactions ◽  
Amino Terminal ◽  
Transcription Complexes ◽  
Amino Terminal Domain ◽  
General Transcription Machinery

The androgen receptor is a ligand-activated transcription factor that binds DNA response elements as a homodimer. Binding sites for the receptor have been identified both upstream and downstream of the transcription start site. Once bound to DNA, the receptor contacts chromatin remodelling complexes, coactivator proteins and components of the general transcription machinery in order to regulate target gene expression. The main transactivation domain, termed AF1, is located within the structurally distinct amino-terminal domain. This region is structurally flexible but adopts a more folded conformation in the presence of the binding partner TFIIF, and this in turn enhances subsequent protein-protein interactions. Thus, there is likely to be a dynamic interplay between protein-protein interactions and protein folding, involving AF1, that is proposed to lead to the assembly and/or disassembly of receptor-dependent transcription complexes.

scholarly journals The Amino-terminal Domain of Desmoplakin Binds to Plakoglobin and Clusters Desmosomal Cadherin–Plakoglobin Complexes

1997 ◽  
Vol 139 (3) ◽  
pp. 773-784 ◽  
Author(s):  
Andrew P. Kowalczyk ◽  
Elayne A. Bornslaeger ◽  
Jeffrey E. Borgwardt ◽  
Helena L. Palka ◽  
Avninder S. Dhaliwal ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Interactions ◽  
Critical Role ◽  
Protein S ◽  
Desmosomal Cadherins ◽  
Amino Terminal ◽  
Terminal Domain ◽  
L Cells ◽  
Amino Terminal Domain ◽  
Cell Cell

The desmosome is a highly organized plasma membrane domain that couples intermediate filaments to the plasma membrane at regions of cell–cell adhesion. Desmosomes contain two classes of cadherins, desmogleins, and desmocollins, that bind to the cytoplasmic protein plakoglobin. Desmoplakin is a desmosomal component that plays a critical role in linking intermediate filament networks to the desmosomal plaque, and the amino-terminal domain of desmoplakin targets desmoplakin to the desmosome. However, the desmosomal protein(s) that bind the amino-terminal domain of desmoplakin have not been identified. To determine if the desmosomal cadherins and plakoglobin interact with the amino-terminal domain of desmoplakin, these proteins were co-expressed in L-cell fibroblasts, cells that do not normally express desmosomal components. When expressed in L-cells, the desmosomal cadherins and plakoglobin exhibited a diffuse distribution. However, in the presence of an amino-terminal desmoplakin polypeptide (DP-NTP), the desmosomal cadherins and plakoglobin were observed in punctate clusters that also contained DP-NTP. In addition, plakoglobin and DP-NTP were recruited to cell–cell interfaces in L-cells co-expressing a chimeric cadherin with the E-cadherin extracellular domain and the desmoglein-1 cytoplasmic domain, and these cells formed structures that were ultrastructurally similar to the outer plaque of the desmosome. In transient expression experiments in COS cells, the recruitment of DP-NTP to cell borders by the chimera required co-expression of plakoglobin. Plakoglobin and DP-NTP co-immunoprecipitated when extracted from L-cells, and yeast two hybrid analysis indicated that DP-NTP binds directly to plakoglobin but not Dsg1. These results identify a role for desmoplakin in organizing the desmosomal cadherin–plakoglobin complex and provide new insights into the hierarchy of protein interactions that occur in the desmosomal plaque.

Characterization of a Mode of Specific Binding of Fibrin Monomer through its Amino-Terminal Domain by Macrophages and Macrophage Cell-Lines

1990 ◽  
Vol 63 (02) ◽  
pp. 193-203 ◽  
Author(s):  
John R Shainoff ◽  
Deborah J Stearns ◽  
Patricia M DiBello ◽  
Youko Hishikawa-Itoh
Keyword(s):  
Peritoneal Macrophages ◽  
Affinity Binding ◽  
Macrophage Cell ◽  
High Affinity Binding ◽  
Amino Terminal ◽  
High Affinity ◽  
Terminal Domain ◽  
Weak Binding ◽  
Amino Terminal Domain

SummaryThe studies reported here probe the existence of a receptor-mediated mode of fibrin-binding by macrophages that is associated with the chemical change underlying the fibrinogen-fibrin conversion (the release of fibrinopeptides from the amino-terminal domain) without depending on fibrin-aggregation. The question is pursued by 1) characterization of binding in relation to fibrinopeptide content of both the intact protein and the CNBr-fragment comprising the amino-terminal domain known as the NDSK of the protein, 2) tests of competition for binding sites, and 3) photo-affinity labeling of macrophage surface proteins. The binding of intact monomers of types lacking either fibrinopeptide A alone (α-fibrin) or both fibrinopeptides A and B (αβ-fibrin) by peritoneal macrophages is characterized as proceeding through both a fibrin-specific low density/high affinity (BMAX ≃ 200–800 molecules/cell, KD ≃ 10−12 M) interaction that is not duplicated with fibrinogen, and a non-specific high density/low affinity (BMAX ≥ 105 molecules/cell, KD ≥ 10−6 M) interaction equivalent to the weak binding of fibrinogen. Similar binding characteristics are displayed by monocyte/macrophage cell lines (J774A.1 and U937) as well as peritoneal macrophages towards the NDSK preparations of these proteins, except for a slightly weaker (KD ≃ 10−10 M) high-affinity binding. The high affinity binding of intact monomer is inhibitable by fibrin-NDSK, but not fibrinogen-NDSK. This binding appears principally dependent on release of fibrinopeptide-A, because a species of fibrin (β-fibrin) lacking fibrinopeptide-B alone undergoes only weak binding similar to that of fibrinogen. Synthetic Gly-Pro-Arg and Gly-His-Arg-Pro corresponding to the N-termini of to the α- and the β-chains of fibrin both inhibit the high affinity binding of the fibrin-NDSKs, and the cell-adhesion peptide Arg-Gly-Asp does not. Photoaffinity-labeling experiments indicate that polypeptides with elec-trophoretically estimated masses of 124 and 187 kDa are the principal membrane components associated with specifically bound fibrin-NDSK. The binding could not be up-regulated with either phorbol myristyl acetate, interferon gamma or ADP, but was abolished by EDTA and by lipopolysaccharide. Because of the low BMAX, it is suggested that the high-affinity mode of binding characterized here would be too limited to function by itself in scavenging much fibrin, but may act cooperatively with other, less limited modes of fibrin binding.

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