Sequential binding of SeqA protein to nascent DNA segments at replication forks in synchronized cultures of Escherichia coli

Abstract

SummaryTo demonstrate that sequestration A (SeqA) protein binds preferentially to hemimethylated GATC sequences at replication forks and forms clusters in Escherichia coli growing cells, we analysed, by the chromatin immunoprecipitation (ChIP) assay using anti-SeqA antibody, a synchronized culture of a temperature-sensitive dnaC mutant strain in which only one round of chromosomal DNA replication was synchronously initiated. After synchronized initiation of chromosome replication, the replication origin oriC was first detected by the ChIP assay, and other six chromosomal regions having multiple GATC sequences were sequentially detected according to bidirectional replication of the chromosome. In contrast, DNA regions lacking the GATC sequence were not detected by the ChIP assay. These results indicate that SeqA binds hemimethylated nascent DNA segments according to the proceeding of replication forks in the chromosome, and SeqA releases from the DNA segments when fully methylated. Immunofluorescence microscopy reveals that a single SeqA focus containing paired replication apparatuses appears at the middle of the cell immediately after initiation of chromosome replication and the focus is subsequently separated into two foci that migrate to 1/4 and 3/4 cellular positions, when replication forks proceed bidirectionally an approximately one-fourth distance from the replication origin towards the terminus. This supports the translocating replication apparatuses model.

The GATC-Binding Protein SeqA Is Required for Bile Resistance and Virulence inSalmonella entericaSerovar Typhimurium

“…The cell division abnormalities observed in SeqA Ϫ mutants of S. enterica closely resemble those previously described in their E. coli counterparts, which have been the subject of more numerous and deeper studies (1,4,10,25,27,39,46,48). Other analogies between E. coli and Salmonella SeqA Ϫ mutants include SOS induction, requirement for the SOS regulon for viability, envelope instability, and sensitivity to bile salts and other envelope-damaging agents.…”

“…Wild-type ATCC 14028 and its isogenic SeqA Ϫ derivative, SV4752, were grown in LB medium, and RNA extraction for microarray analysis was carried out in exponential-and stationary-phase cultures. Use of these two conditions was justified by the fact that SeqA binds to methylated DNA, found in both exponential-and stationary-phase cells, and hemimethylated DNA, present in dividing cells only (48). The experiments were based on the rationale that higher RNA content in a SeqA Ϫ background would indicate that SeqA represses gene expression in the wild type.…”

“…Proper chromosome segregation also requires SeqA (1). Furthermore, SeqA trails the DNA replication fork and may contribute to nucleoid organization in newly replicated DNA (4,24,25,48). Aside from its roles in chromosome replication and nucleoid segregation, SeqA is known to regulate the transcription of certain genes.…”

DNA compaction in the early part of the SOS response is dependent on RecN and RecA

“…or fluorescently tagged SeqA protein (Brendler et al, 2000;Fossum et al, 2007;Hiraga et al, 1998;Molina & Skarstad, 2004;Onogi et al, 1999;Yamazoe et al, 2005). Formation of such SeqA foci depends on ongoing replication (Hiraga et al, 2000).…”

Functional Taxonomy of Bacterial Hyperstructures

“…But what explains it? One possibility is that the continuing production of new methylated and hemimethylated GATC sequences titrates away SeqA and so weakens the giant sequestration hyperstructure; in the case of E. coli synchronized for chromosome replication, there is evidence that the SeqA foci change in composition as replication proceeds (288). Another, complementary, possibility is related to the existence of a specific segregation machine that might help pull apart both origins and sequestration hyperstructure.…”