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Replication fork inhibition in seqA mutants of E scherichia coli triggers replication fork breakage
Author(s) -
Rotman Ella,
Khan Sharik R.,
Kouzminova Elena,
Kuzminov Andrei
Publication year - 2014
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/mmi.12638
Subject(s) - seqa protein domain , biology , mutant , genetics , microbiology and biotechnology , dna replication , dna , control of chromosome duplication , gene
Summary SeqA protein negatively regulates replication initiation in E scherichia 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.