21 research outputs found
Single-molecule experiments in biological physics: methods and applications
I review single-molecule experiments (SME) in biological physics. Recent
technological developments have provided the tools to design and build
scientific instruments of high enough sensitivity and precision to manipulate
and visualize individual molecules and measure microscopic forces. Using SME it
is possible to: manipulate molecules one at a time and measure distributions
describing molecular properties; characterize the kinetics of biomolecular
reactions and; detect molecular intermediates. SME provide the additional
information about thermodynamics and kinetics of biomolecular processes. This
complements information obtained in traditional bulk assays. In SME it is also
possible to measure small energies and detect large Brownian deviations in
biomolecular reactions, thereby offering new methods and systems to scrutinize
the basic foundations of statistical mechanics. This review is written at a
very introductory level emphasizing the importance of SME to scientists
interested in knowing the common playground of ideas and the interdisciplinary
topics accessible by these techniques. The review discusses SME from an
experimental perspective, first exposing the most common experimental
methodologies and later presenting various molecular systems where such
techniques have been applied. I briefly discuss experimental techniques such as
atomic-force microscopy (AFM), laser optical tweezers (LOT), magnetic tweezers
(MT), biomembrane force probe (BFP) and single-molecule fluorescence (SMF). I
then present several applications of SME to the study of nucleic acids (DNA,
RNA and DNA condensation), proteins (protein-protein interactions, protein
folding and molecular motors). Finally, I discuss applications of SME to the
study of the nonequilibrium thermodynamics of small systems and the
experimental verification of fluctuation theorems. I conclude with a discussion
of open questions and future perspectives.Comment: Latex, 60 pages, 12 figures, Topical Review for J. Phys. C (Cond.
Matt
A specific PP2A regulatory subunit, B56Ī³, mediates DNA damage-induced dephosphorylation of p53 at Thr55
Protein phosphatase 2A (PP2A) has been implicated to exert its tumor suppressive function via a small subset of regulatory subunits. In this study, we reported that the specific B regulatory subunits of PP2A B56Ī³1 and B56Ī³3 mediate dephosphorylation of p53 at Thr55. Ablation of the B56Ī³ protein by RNAi, which abolishes the Thr55 dephosphorylation in response to DNA damage, reduces p53 stabilization, Bax expression and cell apoptosis. To investigate the molecular mechanisms, we have shown that the endogenous B56Ī³ protein level and association with p53 increase after DNA damage. Finally, we demonstrate that Thr55 dephosphorylation is required for B56Ī³3-mediated inhibition of cell proliferation and cell transformation. These results suggest a molecular mechanism for B56Ī³-mediated tumor suppression and provide a potential route for regulation of B56Ī³-specific PP2A complex function
Structural basis for translocation by AddAB helicaseānuclease and its arrest at Ļ sites
In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is dependent upon the recombination hotspot sequence Ļ (Chi)1, 2 and is catalysed by either an AddAB- or RecBCD-type helicaseānuclease (reviewed in refs 3, 4). These enzyme complexes unwind and digest the DNA duplex from the broken end until they encounter a Ļ sequence5, whereupon they produce a 3ā² single-stranded DNA tail onto which they initiate loading of the RecA protein6. Consequently, regulation of the AddAB/RecBCD complex by Ļ is a key control point in DNA repair and other processes involving genetic recombination. Here we report crystal structures of Bacillus subtilis AddAB in complex with different Ļ-containing DNA substrates either with or without a non-hydrolysable ATP analogue. Comparison of these structures suggests a mechanism for DNA translocation and unwinding, suggests how the enzyme binds specifically to Ļ sequences, and explains how Ļ recognition leads to the arrest of AddAB (and RecBCD) translocation that is observed in single-molecule experiments7, 8, 9