title: Replication fork inhibition in seqA mutants of Escherichia coli triggers replication fork breakage.
abstract: SeqA protein negatively regulates replication initiation in Escherichia coli and is also proposed to organize maturation and segregation of the newly replicated DNA. The seqA mutants suffer from chromosomal fragmentation; since this fragmentation is attributed to defective segregation or nucleoid compaction, two-ended breaks are expected. Instead, we show that, in SeqA's absence, chromosomes mostly suffer one-ended DNA breaks, indicating disintegration of replication forks. We further show that replication forks are unexpectedly slow in seqA mutants. Quantitative kinetics of origin and terminus replication from aligned chromosomes not only confirm origin overinitiation in seqA mutants, but also reveal terminus under-replication, indicating inhibition of replication forks. Pre-/post-labelling studies of the chromosomal fragmentation in seqA mutants suggest events involving single forks, rather than pairs of forks from consecutive rounds rear-ending into each other. We suggest that, in the absence of SeqA, the sister-chromatid cohesion 'safety spacer' is destabilized and completely disappears if the replication fork is inhibited, leading to the segregation fork running into the inhibited replication fork and snapping the latter at single-stranded DNA regions.
authors.affiliation: Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
authors.forename: Ella
authors.initials: E
authors.lastname: Rotman
authors.forename: Sharik R
authors.initials: SR
authors.lastname: Khan
authors.forename: Elena
authors.initials: E
authors.lastname: Kouzminova
authors.forename: Andrei
authors.initials: A
authors.lastname: Kuzminov
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D002876:Chromosomes, Bacterial; D053960:DNA Breaks; D004260:DNA Repair; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D008957:Models, Genetic; D009154:Mutation; D018741:Replication Origin
chemical_list: D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C089746:SeqA protein, E coli