Tetrameric UvrD helicase is located at theE. colireplisome due to frequent replication blocks

Abstract

DNA replication in all organisms must overcome nucleoprotein blocks to complete genome duplication. Accessory replicative helicases in Escherichia coli, Rep and UvrD, help replication machinery overcome blocks by removing incoming nucleoprotein complexes or aiding the re-initiation of replication. Mechanistic details of Rep function have emerged from recent live cell studies, however, the activities of UvrD in vivo remain unclear. Here, by integrating biochemical analysis and super-resolved single-molecule fluorescence microscopy, we discovered that UvrD self-associates into a tetramer and, unlike Rep, is not recruited to a specific replisome protein despite being found at approximately 80% of replication forks. By deleting rep and DNA repair factors mutS and uvrA, perturbing transcription by mutating RNA polymerase, and antibiotic inhibition; we show that the presence of UvrD at the fork is dependent on its activity. This is likely mediated by the very high frequency of replication blocks due to DNA bound proteins, including RNA polymerase, and DNA damage. UvrD is recruited to sites of nucleoprotein blocks via distinctly different mechanisms to Rep and therefore plays a more important and complementary role than previously realised in ensuring successful DNA replication.

Correlating fluorescence microscopy, optical and magnetic tweezers to study single chiral biopolymers such as DNA

“…And, with trivial adaption, the technology can be implemented to study other filamentous chiral biopolymers; RNA is an obvious candidate, but also modular proteins such as silk show evidence for interesting torsional properties 65 which have yet to be explored at the single-molecule level in regards to their response to twist. It may also be valuable to use COMBI-Tweez to explore specific mechanistic questions relating to emergent features of DNA topology, including the dependence on force and ionic strength on DNA buckling, to further probe the source of very rapid fluctuations we observe in DNA supercoiling using bfp detection, of putative long-range rapid plectoneme hopping mobility reported previously 22 , as well as investigating the role of DNA topology in crucial cell processes involving interaction with DNA binding proteins, such as DNA repair mechanisms 66 . The collation of single-molecule tools in COMBI-Tweez around a single optical microscope also presents valuable future opportunities to integrate even more single-molecule biophysics tools, many of which are developed around optical microscopy [67][68][69] .…”

“…Similarly, our observation of an increase in the lower frequency power spectral components of buckling DNA merits further investigation to study whether this effect is influenced by sequence, salt and plectoneme formation. Also, there may be value in using this instrumentation to investigate long-range rapid plectoneme hopping mobility reported previously 22 , as well as probing the role of DNA topology in crucial cell processes involving interaction with DNA binding proteins, such as DNA repair mechanisms 66 . The collation of single-molecule tools in COMBI-Tweez around a single optical microscope also presents valuable future opportunities to integrate even more single-molecule biophysics tools, many of which are developed around optical microscopy [67][68][69] .…”

“…Multiple cell processes involve chiral biopolymers experiencing pN scale forces 1 and torques of tens of pN.nm 2 , exemplified by molecular machines on DNA 3 where torsion is a critical physical factor 4 . Although lacking torsional information, optical tweezers (OT) combined 5 with fluorescence microscopy of dye-labelled DNA was used to image DNA extension in response to fluid drag 6 , and though missing topological details from fluorescence visualisation, magnetic tweezers (MT) experiments have demonstrated how DNA molecules respond to force combined with supercoiling 7 . Developments in OT and MT have enabled molecular study of DNA over-stretching 8 , protein binding 9 , dependence of mechanical properties to ionicity 10 , and DNA-protein bridge formation 11 .…”

iPAR: a new reporter for eukaryotic cytoplasmic protein aggregation

“…Using these reagents with newly designed image analysis techniques, we were able to quantify the induced protein aggregation following hyperosmotic and elevated temperature cell stresses, and also to assess the capacity for mother cells to retain protein aggregates during the process of asymmetric cell division, during which other cellular organelles such as the nucleus and lytic vacuole are inherited in budding daughter cells. We further visualise iPAR in vivo using Slimfield microscopy, a rapid fluorescence imaging modality which can detect single fluorescent dye molecules including fluorescent proteins in the cytoplasm of a range of organisms including single bacteria (9,(60)(61)(62)(63)(64)(65)(66)(67), yeast (68)(69)(70), algae (71,72) and mammalian (73, 74) cells, as well as animal (75) and plant (76) tissue, with below millisecond sampling capability (77). Analysis of iPAR aggregate Slimfield tracks indicate that aggregates are mobile in the vacuolar and nuclear compartments and possess between a few tens and a few hundred iPAR molecules per aggregate whose mean value increases upon extracellular hyperosmotic stress.…”