SeqA Chip-chip analysis of E. coli MG1655 and derivatives

ABSTRACT: Chromosomes are composed of enormously long DNA molecules which must be distributed correctly as the cells grow and divide. In Escherichia coli the new DNA behind the replication forks is specifically bound by the SeqA protein. SeqA binds to GATC sequences which are methylated on the A of the old strand but not on the new strand. Binding lasts for a period of time until Dam methyltransferase methylates the new strand. It is therefore believed that a region of hemi-methylated DNA covered by SeqA follows the replication fork. We show that this is indeed the case by using global ChIP on Chip analysis and a newly developed method for methylation analysis. A comparison of rapid and slow growth conditions showed that in cells with multiple replication forks per chromosome, the old forks bind little SeqA. Analysis of strains with strong SeqA binding sites at different chromosomal loci supported this finding. The results indicate that a re-organization of the chromosome occurs at a timepoint when new forks have travelled about 20% and old forks about 75% of the way to the terminus. This timepoint coincides with the end of origin sequestration. It is so far not known what brings about the end of origin sequestration. Here we suggest that a reorganization event occurs resulting in both origin desequestration and loss of old replication forks from the SeqA structures. SeqA ChIP-Chip analysis of unsynchronized E. coli MG1655 and in cells synchronized regarding initiation of DNA replication; SeqA ChIP-Chip of Dam overproducing strain and SeqA4 mutant; SeqA ChIP-Chip of strains with chromosomal insertions of strong SeqA binding site. Methylation analysis of synchronized E. coli MG1655dnaC2 cells 0 and 15 min after initiation.

ORGANISM(S): Escherichia coli str. K-12 substr. MG1655

SUBMITTER: Torsten Waldminghaus 

PROVIDER: E-GEOD-28280 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress