Significance of Knotted Structures for Function of Proteins and Nucleic Acids
Saturday Abstracts
Escherichia coli Live Cell Super-resolution Analysis of Topoisomerase IV Action
Pawel Zawadzki
1,2
, David Sherratt
1
.
1
Oxford University, Oxford, United Kingdom,
2
Oxford University, Oxford, United Kingdom.
~440, 000 links between duplex strands need to be removed every E. coli cell generation. Type II
topoisomerses perform the majority of this task. Gyrase relaxes (+)ve supercoils in front of
replication fork, while TopoIV plays an essential function in decatenation of newly replicated
DNA and can additionally relax both (+)ve and (-)ve supercoils. We counted and followed the in
vivo behaviour of single molecules of the two TopoIV subunits, ParC and ParE by using live-cell
super-resolution PALM imaging. Approximately 40% of both subunits are present as an
immobile population that forms discrete foci suggesting that action of Topo IV is precisely
localised in slowly growing cells. Analysis of colocalisation with DNA regions and protein
complexes showed that TopoIV is enriched at ori, where it co-localizes with MukBEF, and at ter
in cell approaching replication termination and division. The localisation and diffusional profiles
of both subunits were independent of replication. Removal of functional MukBEF, or
interference of the interaction between MukBEF and TopoIV led to the loss of most of the
immobile TopoIV molecules associated with ori (and MukBEF), thereby demonstrating that the
immobile TopoIV molecules are physically associated with MukBEF molecules within foci.
Additionally we show that the TopoIV molecules associated with MukBEF foci are catalytically
active, by showing that after covalent attachment to DNA during inhibition of catalysis with
norfloxacin, that their association with MukBEF foci is retained. Taken together our results show
where, when and how TopoIV performs its function in living cell.
Fine-tuning the Activity of DNA Bridging Proteins
Remus Dame
.
Leiden University, Leiden, Netherlands.
Loop formation is key to the global organization of genomes in organisms from all three
domains of life. Genomes are organized dynamically and their re-modelling is implied in
translating external signals into specific gene products. The bacterial chromatin proteins H-NS
and the archaeal chromatin protein Alba are capable of forming bridges between DNA
segments
in vitro
and thus candidates for genomic loop formation
in vivo
. H-NS is a global
regulator of transcription and a similar role has been suggested for Alba. We have investigated
how the DNA bridging activity of these proteins is fine-tuned by physico-chemical conditions
and interaction with other proteins (1,2). We demonstrate that for both proteins there is a delicate
balance between two binding modes (stiffening and bridging), which can be shifted when
conditions are changed or interaction partners are present. Our observations yield models for
fine-tuning of genome organization and for the translation of changes in genome organization
into transcriptional activity.
1) Laurens
et al
., Nature comm. (2012)
2) Van der Valk
et al.
Submitted.
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