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Dynamic distribution of seqa protein across the chromosome of escherichia coli K-12.

Sánchez-Romero MA, Busby SJ, Dyer NP, Ott S, Millard AD, Grainger DC - MBio (2010)

Bottom Line: Less SeqA is found in highly transcribed regions, as well as in the ter macrodomain.Using synchronized cultures, we show that SeqA distribution differs with the cell cycle.SeqA remains bound to some targets after replication has ceased, and these targets locate to genes encoding factors involved in nucleotide metabolism, chromosome replication, and methyl transfer.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, the University of Birmingham, Edgbaston, Birmingham, United Kingdom.

ABSTRACT
The bacterial SeqA protein binds to hemi-methylated GATC sequences that arise in newly synthesized DNA upon passage of the replication machinery. In Escherichia coli K-12, the single replication origin oriC is a well-characterized target for SeqA, which binds to multiple hemi-methylated GATC sequences immediately after replication has initiated. This sequesters oriC, thereby preventing reinitiation of replication. However, the genome-wide DNA binding properties of SeqA are unknown, and hence, here, we describe a study of the binding of SeqA across the entire Escherichia coli K-12 chromosome, using chromatin immunoprecipitation in combination with DNA microarrays. Our data show that SeqA binding correlates with the frequency and spacing of GATC sequences across the entire genome. Less SeqA is found in highly transcribed regions, as well as in the ter macrodomain. Using synchronized cultures, we show that SeqA distribution differs with the cell cycle. SeqA remains bound to some targets after replication has ceased, and these targets locate to genes encoding factors involved in nucleotide metabolism, chromosome replication, and methyl transfer.

No MeSH data available.


Related in: MedlinePlus

Relationship between SeqA binding and the occurrence of GATC motifs in vivo. The SeqA binding values are Cy5/Cy3 ratios generated from ChIP experiments analyzed using a DNA microarray. DNA microarray probes were grouped according to the number of GATC motifs present in the probe, and the average Cy5/Cy3 ratio was calculated for each group of probes. Data presented were generated from unsynchronized cultures of E. coli (time point A), cultures where chromosome replication had been blocked for 1 h (time point B), and cultures where chromosome replication had been reinitiated in synchronicity for a period of 6 min (time point C).
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f3: Relationship between SeqA binding and the occurrence of GATC motifs in vivo. The SeqA binding values are Cy5/Cy3 ratios generated from ChIP experiments analyzed using a DNA microarray. DNA microarray probes were grouped according to the number of GATC motifs present in the probe, and the average Cy5/Cy3 ratio was calculated for each group of probes. Data presented were generated from unsynchronized cultures of E. coli (time point A), cultures where chromosome replication had been blocked for 1 h (time point B), and cultures where chromosome replication had been reinitiated in synchronicity for a period of 6 min (time point C).

Mentions: Figure 3 shows a comparison of the SeqA binding signal (i.e., the Cy5/Cy3 ratio) generated for each probe on the DNA microarray with the number of GATC sites present in each probe. Thus, probes with the same number of GATC motifs were grouped and the average SeqA binding signal was calculated for each group of probes for each time point. For the sample from the unsynchronized culture (time point A) there is a clear correlation between the SeqA binding signals and probe GATC content. In contrast, for the samples from synchronized cultures (time points B and C), there is little correlation. This is expected because it is only in the unsynchronized cells that each location with a GATC has an equal chance of being hemi-methylated. At time points B and C, many GATC motifs will be fully methylated and hence have a greatly reduced affinity for SeqA.


Dynamic distribution of seqa protein across the chromosome of escherichia coli K-12.

Sánchez-Romero MA, Busby SJ, Dyer NP, Ott S, Millard AD, Grainger DC - MBio (2010)

Relationship between SeqA binding and the occurrence of GATC motifs in vivo. The SeqA binding values are Cy5/Cy3 ratios generated from ChIP experiments analyzed using a DNA microarray. DNA microarray probes were grouped according to the number of GATC motifs present in the probe, and the average Cy5/Cy3 ratio was calculated for each group of probes. Data presented were generated from unsynchronized cultures of E. coli (time point A), cultures where chromosome replication had been blocked for 1 h (time point B), and cultures where chromosome replication had been reinitiated in synchronicity for a period of 6 min (time point C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2912659&req=5

f3: Relationship between SeqA binding and the occurrence of GATC motifs in vivo. The SeqA binding values are Cy5/Cy3 ratios generated from ChIP experiments analyzed using a DNA microarray. DNA microarray probes were grouped according to the number of GATC motifs present in the probe, and the average Cy5/Cy3 ratio was calculated for each group of probes. Data presented were generated from unsynchronized cultures of E. coli (time point A), cultures where chromosome replication had been blocked for 1 h (time point B), and cultures where chromosome replication had been reinitiated in synchronicity for a period of 6 min (time point C).
Mentions: Figure 3 shows a comparison of the SeqA binding signal (i.e., the Cy5/Cy3 ratio) generated for each probe on the DNA microarray with the number of GATC sites present in each probe. Thus, probes with the same number of GATC motifs were grouped and the average SeqA binding signal was calculated for each group of probes for each time point. For the sample from the unsynchronized culture (time point A) there is a clear correlation between the SeqA binding signals and probe GATC content. In contrast, for the samples from synchronized cultures (time points B and C), there is little correlation. This is expected because it is only in the unsynchronized cells that each location with a GATC has an equal chance of being hemi-methylated. At time points B and C, many GATC motifs will be fully methylated and hence have a greatly reduced affinity for SeqA.

Bottom Line: Less SeqA is found in highly transcribed regions, as well as in the ter macrodomain.Using synchronized cultures, we show that SeqA distribution differs with the cell cycle.SeqA remains bound to some targets after replication has ceased, and these targets locate to genes encoding factors involved in nucleotide metabolism, chromosome replication, and methyl transfer.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, the University of Birmingham, Edgbaston, Birmingham, United Kingdom.

ABSTRACT
The bacterial SeqA protein binds to hemi-methylated GATC sequences that arise in newly synthesized DNA upon passage of the replication machinery. In Escherichia coli K-12, the single replication origin oriC is a well-characterized target for SeqA, which binds to multiple hemi-methylated GATC sequences immediately after replication has initiated. This sequesters oriC, thereby preventing reinitiation of replication. However, the genome-wide DNA binding properties of SeqA are unknown, and hence, here, we describe a study of the binding of SeqA across the entire Escherichia coli K-12 chromosome, using chromatin immunoprecipitation in combination with DNA microarrays. Our data show that SeqA binding correlates with the frequency and spacing of GATC sequences across the entire genome. Less SeqA is found in highly transcribed regions, as well as in the ter macrodomain. Using synchronized cultures, we show that SeqA distribution differs with the cell cycle. SeqA remains bound to some targets after replication has ceased, and these targets locate to genes encoding factors involved in nucleotide metabolism, chromosome replication, and methyl transfer.

No MeSH data available.


Related in: MedlinePlus