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Sequential binding of SeqA protein to nascent DNA segments at replication forks in synchronized cultures of Escherichia coli
Author(s) -
Yamazoe Mitsuyoshi,
Adachi Shun,
Kanaya Shigehiko,
Ohsumi Katsufumi,
Hiraga Sota
Publication year - 2005
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/j.1365-2958.2004.04389.x
Subject(s) - seqa protein domain , biology , dna replication , control of chromosome duplication , dna , origin recognition complex , genetics , chromosome , microbiology and biotechnology , origin of replication , chromosome segregation , pre replication complex , eukaryotic dna replication , gene
Summary To 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.