scholarly journals Multiple interfaces between a serine recombinase and an enhancer control site-specific DNA inversion

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Meghan M McLean ◽  
Yong Chang ◽  
Gautam Dhar ◽  
John K Heiss ◽  
Reid C Johnson
Keyword(s):  
Catalytic Domain ◽  
Control Site ◽  
Dna Looping ◽  
Enhancer Element ◽  
Dna Inversion ◽  
Serine Recombinases ◽  
Multiple Interfaces ◽  
Nucleoprotein Complexes ◽  
Beta Hairpin ◽  
Basic Region

Serine recombinases are often tightly controlled by elaborate, topologically-defined, nucleoprotein complexes. Hin is a member of the DNA invertase subclass of serine recombinases that are regulated by a remote recombinational enhancer element containing two binding sites for the protein Fis. Two Hin dimers bound to specific recombination sites associate with the Fis-bound enhancer by DNA looping where they are remodeled into a synaptic tetramer competent for DNA chemistry and exchange. Here we show that the flexible beta-hairpin arms of the Fis dimers contact the DNA binding domain of one subunit of each Hin dimer. These contacts sandwich the Hin dimers to promote remodeling into the tetramer. A basic region on the Hin catalytic domain then contacts enhancer DNA to complete assembly of the active Hin tetramer. Our results reveal how the enhancer generates the recombination complex that specifies DNA inversion and regulates DNA exchange by the subunit rotation mechanism.

scholarly journals The emerging diversity of transpososome architectures

2012 ◽  
Vol 45 (4) ◽  
pp. 493-521 ◽  
Author(s):  
Fred Dyda ◽  
Michael Chandler ◽  
Alison Burgess Hickman
Keyword(s):  
Chemical Reactions ◽  
Conformational Changes ◽  
Catalytic Domain ◽  
Functional Requirements ◽  
Dna Breaks ◽  
Target Dna ◽  
Nucleoprotein Complexes ◽  
Dna Strand ◽  
Transposition Reaction ◽  
Subsequent Integration

AbstractDNA transposases are enzymes that catalyze the movement of discrete pieces of DNA from one location in the genome to another. Transposition occurs through a series of controlled DNA strand cleavage and subsequent integration reactions that are carried out by nucleoprotein complexes known as transpososomes. Transpososomes are dynamic assemblies which must undergo conformational changes that control DNA breaks and ensure that, once started, the transposition reaction goes to completion. They provide a precise architecture within which the chemical reactions involved in transposon movement occur, but adopt different conformational states as transposition progresses. Their components also vary as they must, at some stage, include target DNA and sometimes even host-encoded proteins. A very limited number of transpososome states have been crystallographically captured, and here we provide an overview of the various structures determined to date. These structures include examples of DNA transposases that catalyze transposition by a cut-and-paste mechanism using an RNaseH-like nuclease catalytic domain, those that transpose using only single-stranded DNA substrates and targets, and the retroviral integrases that carry out an integration reaction very similar to DNA transposition. Given that there are a number of common functional requirements for transposition, it is remarkable how these are satisfied by complex assemblies that are so architecturally different.

scholarly journals Calmodulin-binding and Autoinhibitory Domains ofAcanthamoebaMyosin I Heavy Chain Kinase, a p21-activated Kinase (PAK)

2001 ◽  
Vol 276 (50) ◽  
pp. 47468-47473 ◽  
Author(s):  
Hanna Brzeska ◽  
Rachel Young ◽  
Cristina Tan ◽  
Joanna Szczepanowska ◽  
Edward D. Korn
Keyword(s):  
Heavy Chain ◽  
Kinase Activity ◽  
Insect Cells ◽  
Catalytic Domain ◽  
Full Length ◽  
Calmodulin Binding ◽  
Myosin I ◽  
P21 Activated Kinase ◽  
Basic Region ◽  
Stimulation Of

The sequence homology betweenAcanthamoebamyosin I heavy chain kinase (MIHCK) and other p21-activated kinases (PAKs) is relatively low, including only the catalytic domain and a short PAK N-terminal motif (PAN), and even these regions are not highly homologous. In this paper, we report the expression in insect cells of full-length, fully regulatedAcanthamoebaMIHCK and further characterize the regulation of this PAK by Rac, calmodulin, and autoinhibition. We map the autoinhibitory region of MIHCK to its PAN region and show that the PAN region inhibits autophosphorylation and kinase activity of unphosphorylated full-length MIHCK and its expressed catalytic domain but has very little effect on either when they are phosphorylated. These properties are similar to those reported for mammalian PAK1. Unlike PAK1, MIHCK is activated by Rac only in the presence of phospholipid. However, peptides containing the PAN region of MIHCK bind Rac in the absence of lipid, and Rac binding reverses the inhibition of the MIHCK catalytic domain by PAN peptides. Our data suggest that a region N-terminal to PAN is required for optimal binding of Rac. Also unlike mammalian PAK, phospholipid stimulation ofAcanthamoebaMIHCK andDictyosteliumMIHCK) (which is also a PAK) is inhibited by Ca2+-calmodulin. In contrast toDictyosteliumMIHCK, however, Ca2+-calmodulin also inhibits Rac-induced activity ofAcanthamoebaMIHCK. The basic region N-terminal to PAN is essential for calmodulin binding.

Snapshot blotting: The transfer of nucleic acids and nucleoprotein complexes from electrophoresis gels to EM grids

1992 ◽  
Vol 50 (1) ◽  
pp. 762-763
Author(s):  
Stephen D. Jett
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Nucleic Acid Binding ◽  
Mobility Shift ◽  
Carbon Coated ◽  
Nucleoprotein Complexes ◽  
Mobility Shift Assay ◽  
Protein Nucleic Acid ◽  
Gel Mobility Shift ◽  
Nucleic Acid Interactions

The electrophoresis gel mobility shift assay is a popular method for the study of protein-nucleic acid interactions. The binding of proteins to DNA is characterized by a reduction in the electrophoretic mobility of the nucleic acid. Binding affinity, stoichiometry, and kinetics can be obtained from such assays; however, it is often desirable to image the various species in the gel bands using TEM. Present methods for isolation of nucleoproteins from gel bands are inefficient and often destroy the native structure of the complexes. We have developed a technique, called “snapshot blotting,” by which nucleic acids and nucleoprotein complexes in electrophoresis gels can be electrophoretically transferred directly onto carbon-coated grids for TEM imaging.

Mechanism for Activation of the EGF Receptor Catalytic Domain by the Juxtamembrane Segment

2009 ◽  
Vol 138 (3) ◽  
pp. 604-604
Author(s):  
Natalia Jura ◽  
Nicholas F. Endres ◽  
Kate Engel ◽  
Sebastian Deindl ◽  
Rahul Das ◽  
...  
Keyword(s):  
Egf Receptor ◽  
Catalytic Domain

Mechanism for Activation of the EGF Receptor Catalytic Domain by the Juxtamembrane Segment

2009 ◽  
Vol 138 (33) ◽  
pp. 604-604
Author(s):  
Natalia Jura ◽  
Nicholas F. Endres ◽  
Kate Engel ◽  
Sebastian Deindl ◽  
Rahul Das ◽  
...  
Keyword(s):  
Egf Receptor ◽  
Catalytic Domain
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