22 research outputs found
Interaction of APOBEC3A with DNA assessed by atomic force microscopy.
The APOBEC3 family of DNA cytosine deaminases functions to block the spread of endogenous retroelements and retroviruses including HIV-1. Potency varies among family members depending on the type of parasitic substrate. APOBEC3A (A3A) is unique among the human enzymes in that it is expressed predominantly in myeloid lineage cell types, it is strongly induced by innate immune agonists such as type 1 interferon, and it has the capacity to accommodate both normal and 5-methyl cytosine nucleobases. Here we apply atomic force microscopy (AFM) to characterize the interaction between A3A and single- and double-stranded DNA using a hybrid DNA approach in which a single-stranded region is flanked by defined length duplexes. AFM image analyses reveal A3A binding to single-stranded DNA, and that this interaction becomes most evident (ā¼80% complex yield) at high protein-to-DNA ratios (at least 100ā¶1). A3A is predominantly monomeric when bound to single-stranded DNA, and it is also monomeric in solution at concentrations as high as 50 nM. These properties agree well with recent, biochemical, biophysical, and structural studies. However, these characteristics contrast with those of the related enzyme APOBEC3G, which in similar assays can exist as a monomer but tends to form oligomers in a concentration-dependent manner. These AFM data indicate that A3A has intrinsic biophysical differences that distinguish it from APOBEC3G. The potential relationships between these properties and biological functions in innate immunity are discussed
Field-linked States of Ultracold Polar Molecules
We explore the character of a novel set of ``field-linked'' states that were
predicted in [A. V. Avdeenkov and J. L. Bohn, Phys. Rev. Lett. 90, 043006
(2003)]. These states exist at ultralow temperatures in the presence of an
electrostatic field, and their properties are strongly dependent on the field's
strength. We clarify the nature of these quasi-bound states by constructing
their wave functions and determining their approximate quantum numbers. As the
properties of field-linked states are strongly defined by anisotropic dipolar
and Stark interactions, we construct adiabatic surfaces as functions of both
the intermolecular distance and the angle that the intermolecular axis makes
with the electric field. Within an adiabatic approximation we solve the 2-D
Schrodinger equation to find bound states, whose energies correlate well with
resonance features found in fully-converged multichannel scattering
calculations
DNA synapsis through transient tetramerization triggers cleavage by Ecl18kI restriction enzyme
To cut DNA at their target sites, restriction enzymes assemble into different oligomeric structures. The Ecl18kI endonuclease in the crystal is arranged as a tetramer made of two dimers each bound to a DNA copy. However, free in solution Ecl18kI is a dimer. To find out whether the Ecl18kI dimer or tetramer represents the functionally important assembly, we generated mutants aimed at disrupting the putative dimerādimer interface and analysed the functional properties of Ecl18kI and mutant variants. We show by atomic force microscopy that on two-site DNA, Ecl18kI loops out an intervening DNA fragment and forms a tetramer. Using the tethered particle motion technique, we demonstrate that in solution DNA looping is highly dynamic and involves a transient interaction between the two DNA-bound dimers. Furthermore, we show that Ecl18kI cleaves DNA in the synaptic complex much faster than when acting on a single recognition site. Contrary to Ecl18kI, the tetramerization interface mutant R174A binds DNA as a dimer, shows no DNA looping and is virtually inactive. We conclude that Ecl18kI follows the association model for the synaptic complex assembly in which it binds to the target site as a dimer and then associates into a transient tetrameric form to accomplish the cleavage reaction
Models, measurement and inference in epithelial tissue dynamics
The majority of solid tumours arise in epithelia and therefore much research effort has gone into investigating the growth, renewal and regulation of these tissues. Here we review different mathematical and computational approaches that have been used to model epithelia. We compare different models and describe future challenges that need to be overcome in order to fully exploit new data which present, for the first time, the real possibility for detailed model validation and comparison
Results of the volume measurements of A3A complexed with gap-DNA (A) alone (B).
<p>Numbers of complexes analyzed are Nā=ā118 for (A) and Nā=ā148 for (B). The mean volume values (Ā± SD) for monomers (1-mers; 33Ā±16 nm<sup>3</sup>) and dimers (2-mers; 66Ā±26 nm<sup>3</sup>) are indicated with arrows.</p
Gallery of AFM images of A3A complexed with ssDNA (A) or dsDNA (B) regions of the gap-DNA substrate.
<p>Bar size, 30</p
Gallery of AFM images of A3A<sub>E72A</sub> complexed with dsDNA (A) or ssDNA (B) regions of the gap-DNA substrate.
<p>Bar size, 30</p
AFM images of A3A in complex with gap-DNA.
<p>A3A monomer and dimer complexes are labeled 1 and 2, respectively. Bar size, 100</p