  DNA-interacting proteins have roles multiple processes, many operating as
molecular machines which undergo dynamic metastable transitions to bring about
their biological function. To fully understand this molecular heterogeneity,
DNA and the proteins that bind to it must ideally be interrogated at a single
molecule level in their native in vivo environments, in a time-resolved manner
fast to sample the molecular transitions across the free energy landscape.
Progress has been made over the past decade in utilising cutting-edge tools of
the physical sciences to address challenging biological questions concerning
the function and modes of action of several different proteins which bind to
DNA. These physiologically relevant assays are technically challenging, but can
be complemented by powerful and often more tractable in vitro experiments which
confer advantages of the chemical environment with enhanced detection
single-to-noise of molecular signatures and transition events. Here, we discuss
a range of techniques we have developed to monitor DNA-protein interactions in
vivo, in vitro and in silico. These include bespoke single-molecule
fluorescence microscopy techniques to elucidate the architecture and dynamics
of the bacterial replisome and the structural maintenance of bacterial
chromosomes, as well as new computational tools to extract single-molecule
molecular signatures from live cells to monitor stoichiometry, spatial
localization and mobility in living cells. We also discuss recent developments
from our lab made in vitro, complementing these in vivo studies, which combine
optical and magnetic tweezers to manipulate and image single molecules of DNA,
with and without bound protein, in a new superresolution fluorescence
microscope.
