Dynamic Escherichia coli SeqA complexes organize the newly replicated DNA at a considerable distance from the replisome.
The Escherichia coli SeqA protein binds to newly replicated, hemimethylated DNA behind replication forks and forms structures consisting of several hundred SeqA molecules bound to about 100 kb of DNA. It has been suggested that SeqA structures either direct the new sister DNA molecules away from each other or constitute a spacer that keeps the sisters together. We have developed an image analysis script that automatically measures the distance between neighboring foci in cells. Using this tool as well as direct stochastic optical reconstruction microscopy (dSTORM) we find that in cells with fluorescently tagged SeqA and replisome the sister SeqA structures were situated close together (less than about 30 nm apart) and relatively far from the replisome (on average 200-300 nm). The results support the idea that newly replicated sister molecules are kept together behind the fork and suggest the existence of a stretch of DNA between the replisome and SeqA which enjoys added stabilization. This could be important in facilitating DNA transactions such as recombination, mismatch repair and topoisomerase activity. In slowly growing cells without ongoing replication forks the SeqA protein was found to reside at the fully methylated origins prior to initiation of replication.
Department of Cell Biology, Institute for Cancer Research, Oslo University Hospital, Radiumhospitalet, 0310 Oslo, Norway.
Emily
E
Helgesen
Department of Cell Biology, Institute for Cancer Research, Oslo University Hospital, Radiumhospitalet, 0310 Oslo, Norway School of Pharmacy, Faculty of Mathematics and Natural Sciences, University of Oslo, 0316 Oslo, Norway.
Solveig
S
Fossum-Raunehaug
Department of Cell Biology, Institute for Cancer Research, Oslo University Hospital, Radiumhospitalet, 0310 Oslo, Norway.
Frank
F
Sætre
Department of Biochemistry, Institute for Cancer Research, Oslo University Hospital, Radiumhospitalet, 0310 Oslo, Norway.
Kay Oliver
KO
Schink
Department of Cell Biology, Institute for Cancer Research, Oslo University Hospital, Radiumhospitalet, 0310 Oslo, Norway School of Pharmacy, Faculty of Mathematics and Natural Sciences, University of Oslo, 0316 Oslo, Norway kirsten.skarstad@labmed.uio.no.
Kirsten
K
Skarstad
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D002453:Cell Cycle / Q000235:genetics; D002455:Cell Division / Q000235:genetics; D002876:Chromosomes, Bacterial / Q000235:genetics; D004261:DNA Replication / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D005434:Flow Cytometry; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D012093:Replicon / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; C476430:SSB protein, E coli; C089746:SeqA protein, E coli