Journal articles

The formation of G-quadruplexes in G-rich regions of DNA is believed to affect DNA transcription and replication. However, it is currently unclear how this formation occurs in the presence of a complementary strand. We have used atomic force microscopy (AFM) to image stable RNA/DNA hybrid loops generated by transcription of the plasmid pPH600, which contains a 604-bp fragment of the murine immunoglobulin Sgamma3 switch region. We show that the non-RNA-containing portion folds into G-quadruplexes, consistent with computational predictions.

We have previously described a high conductance calcium-activated 'maxi K' channel in primary cultures of human eccrine sweat gland cells both from normal subjects and those with cystic fibrosis. In further studies we have now identified a potassium-selective channel of much lower conductance which shows outward-rectification and which is present in sweat glands isolated from cystic fibrosis subjects.

Atomic force microscopy (AFM) allows the study of single protein-DNA interactions such as those observed with the Type I Restriction-Modification systems. The mechanisms employed by these systems are complicated and understanding them has proved problematic. It has been known for years that these enzymes translocate DNA during the restriction reaction, but more recent AFM work suggested that the archetypal EcoKI protein went through an additional dimerization stage before the onset of translocation. The results presented here extend earlier findings confirming the dimerization.

Recordings have been made of a potassium-selective ion channel in primary cultures of cells derived from explants of human eccrine sweat glands obtained from normal subjects and from subjects suffering from cystic fibrosis. There appears to be no functional difference between potassium channels derived from normal subjects and those from cystic fibrosis subjects. The channel falls into the group generally known as "Maxi-K" channels, and has a slope conductance, with symmetrical solutions in bath and pipette containing 140 mM K+, of approximately 230 pS.

There has been confusion about the subunit stoichiometry of the degenerin family of ion channels. Recently, a crystal structure of acid-sensing ion channel (ASIC) 1a revealed that it assembles as a trimer. Here, we used atomic force microscopy (AFM) to image unprocessed ASIC1a bound to mica. We detected a mixture of subunit monomers, dimers and trimers. In some cases, triple-subunit clusters were clearly visible, confirming the trimeric structure of the channel, and indicating that the trimer sometimes disaggregated after adhesion to the mica surface.

Synaptotagmin I (syt), an integral protein of the synaptic vesicle membrane, is believed to act as a Ca2+ sensor for neuronal exocytosis. Syt's cytoplasmic domain consists largely of two C2 domains, C2A and C2B. In response to Ca2+ binding, the C2 domains interact with membranes, becoming partially embedded in the lipid bilayer. We have imaged syt C2AB in association with lipid bilayers under fluid, using AFM. As expected, binding of C2AB to bilayers required both an anionic phospholipid [phosphatidylserine (PS)] and Ca2+.

The GABA(A) receptor is a chloride-selective ligand-gated ion channel of the Cys-loop superfamily. The receptor consists of five subunits arranged pseudosymmetrically around a central pore. The predominant form of the receptor in the brain contains alpha(1)-, beta(2)-, and gamma(2)-subunits in the arrangement alphabetaalphagammabeta, counter-clockwise around the pore.

Fast neurotransmission in the nervous system is mediated by ionotropic receptors, all of which contain several subunits surrounding an integral ion channel. There are three major families of ionotropic receptors: the 'Cys-loop' receptors (including the nicotinic receptor for acetylcholine, the 5-HT(3) receptor, the GABA(A) receptor and the glycine receptor), the glutamate receptors (including the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, kainate and N-methyl-D: -aspartic acid receptors) and the P2X receptors for adenosine triphosphate.

EcoP15I is a type III restriction enzyme that requires two recognition sites in a defined orientation separated by up to 3.5 kbp to efficiently cleave DNA. The mechanism through which site-bound EcoP15I enzymes communicate between the two sites is unclear. Here, we use atomic force microscopy to study EcoP15I-DNA pre-cleavage complexes. From the number and size distribution of loops formed, we conclude that the loops observed do not result from translocation, but are instead formed by a contact between site-bound EcoP15I and a nonspecific region of DNA.

Many DNA-modifying enzymes act in a manner that requires communication between two noncontiguous DNA sites. These sites can be brought into contact either by a diffusion-mediated chance interaction between enzymes bound at the two sites, or by active translocation of the intervening DNA by a site-bound enzyme. EcoP15I, a type III restriction enzyme, needs to interact with two recognition sites separated by up to 3,500 bp before it can cleave DNA.