Journal: Nucleic Acids Research

Article Title: Replication fork movement and methylation govern SeqA binding to the Escherichia coli chromosome

doi: 10.1093/nar/gks187

Figure Lengend Snippet: Strains used in this study

Article Snippet: Hybridization was for about 36 h at 55°C to custom E. coli microarrays from Oxford Gene Technology.

Techniques:

Journal: Nucleic Acids Research

Article Title: Replication fork movement and methylation govern SeqA binding to the Escherichia coli chromosome

doi: 10.1093/nar/gks187

Figure Lengend Snippet: SeqA binding in synchronized E. coli dnaC2 cells at 15, 16 and 17 min after initiation. The experiment and data processing was as described . Relative values for the three time points were aligned at the oriC region based on the finding that SeqA binding there is relatively stable. Only the SeqA bound part of the chromosome is shown with oriC in the middle and the distance to oriC as indicated (for whole genome plot of 15 min time point see ). Values for the different time points are colored according to legend. The blue scale bar indicates the number of base-pairs replicated per minute in the used system according to .

Article Snippet: Hybridization was for about 36 h at 55°C to custom E. coli microarrays from Oxford Gene Technology.

Techniques: Binding Assay

Journal: Nucleic Acids Research

Article Title: Replication fork movement and methylation govern SeqA binding to the Escherichia coli chromosome

doi: 10.1093/nar/gks187

Figure Lengend Snippet: Whole genome SeqA binding in synchronized E. coli dnaC2 cells. Cells were grown exponentially in AB medium supplemented with glucose and CAA at 30°C and shifted to 39°C for 70 min. Synchronous initiation of replication was induced by a downshift to 30°C. Parts of the culture (60 ml) were removed and subjected to cross-linking at the indicated time points after downshift. ChIP-Chip was performed with a SeqA specific antibody and enrichment values are relative to values of an unsynchronized wt culture (LB) with a moving window of 60 kbp and a step size of 6 kb . Data are plotted as a function of their chromosomal position with oriC in the middle and the distance to oriC indicated. The first pair of SeqA peaks/replication forks and the second pair after reinitiation are indicated by white and grey arrows, respectively.

Article Snippet: Hybridization was for about 36 h at 55°C to custom E. coli microarrays from Oxford Gene Technology.

Techniques: Binding Assay, ChIP-chip

Journal: Nucleic Acids Research

Article Title: Replication fork movement and methylation govern SeqA binding to the Escherichia coli chromosome

doi: 10.1093/nar/gks187

Figure Lengend Snippet: Methylation of synchronized E. coli dnaC2 cells at 15 min after initiation. Cell growth was as described . 15 min after synchronous initiation of replication cells were collected and chromosomal DNA isolated. For details on the methylation analysis, see ‘Results’ and ‘Materials and Methods’ sections. Values shown are average enrichment factors for a moving window of 60 kbp, step size of 6 kb. Red, methylation analysis. Grey, SeqA binding data from at similar time point.

Article Snippet: Hybridization was for about 36 h at 55°C to custom E. coli microarrays from Oxford Gene Technology.

Techniques: Methylation, Isolation, Binding Assay

Journal: Nucleic Acids Research

Article Title: Replication fork movement and methylation govern SeqA binding to the Escherichia coli chromosome

doi: 10.1093/nar/gks187

Figure Lengend Snippet: Chromosome-wide SeqA binding varies according to the replication pattern. ( A) Top panel : Whole-genome plot of the SeqA binding in unsynchronized, slow-growing E. coli MG1655 cells (AB-acetate) with no overlapping replication (red) and unsynchronized, fast-growing E. coli MG1655 cells (LB-glucose) with overlapping replication (blue). The sum of SeqA ChIP signals in windows of 60 kbp are shown (step size 1 kbp; only values >1 were included). Red/blue second circle from inside: mean centred line graph with ratios of AB-acetate (red) and LB-glucose (blue). Inner circle : mean centred line graph of number of GATC sites in windows of 5 kbp (step size 1 kbp). Outer lines indicate the region replicated by the new (black) and old (grey) forks during the origin sequestration time found to be 1/3 of the cell cycle . Outer green circle (see below for explanation): regions replicated by new and old forks at different stages of the cell cycle presented with the same colour code as in the bottom panel. Bottom panel : Schematic of the replication pattern of the cells grown in LB-glucose medium. The width of the rectangle represents one generation with the different greens indicating the different events in the life of the average cell: the period from cell birth to termination (0–14 min), from termination to initiation (14–18 min) and from initiation to cell division (18–22 min). Examples of cells (rods in shades of green) are shown above each of the three intervals with chromosomes (black lines) and origins (black dots). Horizontal lines represent the C period (black) and D period (white line; the total duration of which spans more than three generations) and should be read from top to bottom (see ‘Materials and Methods’ section for calculation details). Initiation of chromosome replication occurs in 18 min old cells leading to 16 copies of oriC (dark green). After cell division at an age of 22 min the cells contain eight oriC copies (light green). The chromosomes are then replicated with four old and eight new forks (light green cell). Old forks finish replication at 14 min cell age. ( B ) Examples of SeqA binding to chromosomal sites in AB-acetate (red) and LB-glucose medium (blue). Values are mean values of two replicates. Chromosomal positions of sites are marked by roman numbers in A.

Article Snippet: Hybridization was for about 36 h at 55°C to custom E. coli microarrays from Oxford Gene Technology.

Techniques: Binding Assay

Journal: Nucleic Acids Research

Article Title: Replication fork movement and methylation govern SeqA binding to the Escherichia coli chromosome

doi: 10.1093/nar/gks187

Figure Lengend Snippet: SeqA binding to the GATCs cluster at different insertion sites. E. coli strains carrying cluster insertions at indicated sites were grown in LB batch culture. SeqA ChIP enrichment was quantified by qPCR with primers specific for the GATC cluster or oriC . Respective ratios of three experiments are shown with the standard deviation. The distance of the cluster insertion to oriC are indicated in Mbps.

Article Snippet: Hybridization was for about 36 h at 55°C to custom E. coli microarrays from Oxford Gene Technology.

Techniques: Binding Assay, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Replication fork movement and methylation govern SeqA binding to the Escherichia coli chromosome

doi: 10.1093/nar/gks187

Figure Lengend Snippet: Insertion of a GATC cluster affects SeqA binding to neighbouring sites. E. coli strains with a cluster inserted at indicated regions (arrow) of the chromosome were grown in LB-glucose batch culture. SeqA ChIP-Chip was performed in duplicate for each strain. ChIP signals are plotted for a region of 250 probes up and down stream of the insertion (∼60 kbp). Red, values for the strain with the insertion. Blue, average of the respective three other strains. Line plots below the diagrams show a rank based data analysis. Every black line marks one data point out of the 500 most enriched in the respective strain compared to the three others. Colour code as above.

Article Snippet: Hybridization was for about 36 h at 55°C to custom E. coli microarrays from Oxford Gene Technology.

Techniques: Binding Assay, ChIP-chip