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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

SeqA binding is reduced in rRNA operons. The figure shows ChIP-chip data for SeqA and RNA polymerase binding close to the replication origin and the rrnC rRNA operon. The data were generated from synchronized cultures of E. coli where chromosome replication had been reinitiated in synchronicity for a period of 6 min. The ChIP-chip data sets have been aligned with a graph showing the locations of and frequencies at which GATC sites occur in the underlying DNA sequence.
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f6: SeqA binding is reduced in rRNA operons. The figure shows ChIP-chip data for SeqA and RNA polymerase binding close to the replication origin and the rrnC rRNA operon. The data were generated from synchronized cultures of E. coli where chromosome replication had been reinitiated in synchronicity for a period of 6 min. The ChIP-chip data sets have been aligned with a graph showing the locations of and frequencies at which GATC sites occur in the underlying DNA sequence.

Mentions: Our initial attempts to detect effects of SeqA on transcription, using reverse transcription (RT)-PCR and RNA extracted from CMT940 cells at different time points, was uninformative (data not shown). For example, we saw no changes in mukF and pyrD mRNA levels between time points B and C, despite SeqA binding at these loci and altering substantially (Fig. 5). We reasoned that a better strategy would be to compare genome-wide patterns of SeqA and RNA polymerase binding in unsynchronized cultures (time point A) using ChIP-chip (30). Thus, problems due to low levels of many transcripts, RNA instability, and cell cycle-dependent effects on gene transcription were avoided. Our data show that regions with a strong SeqA binding signal (for example, the locations listed in Table 1) tend to give a low RNA polymerase binding signal, while locations with a strong RNA polymerase binding signal (for example, the rRNA operons) are not bound by SeqA. Some examples of binding profiles are shown in Fig. S4 in the supplemental material, which shows an overall negative correlation between the binding of SeqA and RNA polymerase. The comparison was then repeated for a culture sampled shortly after synchronized initiation of replication (time point C). Figure 6 shows a detailed view of the SeqA and RNA polymerase binding data across the region encompassing both oriC and the rrnC rRNA operon at time point C. The inverse correlation between the binding of SeqA and RNA polymerase is most apparent in the rrnC operon, which has a lower GATC content than the surrounding DNA.


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)

SeqA binding is reduced in rRNA operons. The figure shows ChIP-chip data for SeqA and RNA polymerase binding close to the replication origin and the rrnC rRNA operon. The data were generated from synchronized cultures of E. coli where chromosome replication had been reinitiated in synchronicity for a period of 6 min. The ChIP-chip data sets have been aligned with a graph showing the locations of and frequencies at which GATC sites occur in the underlying DNA sequence.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: SeqA binding is reduced in rRNA operons. The figure shows ChIP-chip data for SeqA and RNA polymerase binding close to the replication origin and the rrnC rRNA operon. The data were generated from synchronized cultures of E. coli where chromosome replication had been reinitiated in synchronicity for a period of 6 min. The ChIP-chip data sets have been aligned with a graph showing the locations of and frequencies at which GATC sites occur in the underlying DNA sequence.
Mentions: Our initial attempts to detect effects of SeqA on transcription, using reverse transcription (RT)-PCR and RNA extracted from CMT940 cells at different time points, was uninformative (data not shown). For example, we saw no changes in mukF and pyrD mRNA levels between time points B and C, despite SeqA binding at these loci and altering substantially (Fig. 5). We reasoned that a better strategy would be to compare genome-wide patterns of SeqA and RNA polymerase binding in unsynchronized cultures (time point A) using ChIP-chip (30). Thus, problems due to low levels of many transcripts, RNA instability, and cell cycle-dependent effects on gene transcription were avoided. Our data show that regions with a strong SeqA binding signal (for example, the locations listed in Table 1) tend to give a low RNA polymerase binding signal, while locations with a strong RNA polymerase binding signal (for example, the rRNA operons) are not bound by SeqA. Some examples of binding profiles are shown in Fig. S4 in the supplemental material, which shows an overall negative correlation between the binding of SeqA and RNA polymerase. The comparison was then repeated for a culture sampled shortly after synchronized initiation of replication (time point C). Figure 6 shows a detailed view of the SeqA and RNA polymerase binding data across the region encompassing both oriC and the rrnC rRNA operon at time point C. The inverse correlation between the binding of SeqA and RNA polymerase is most apparent in the rrnC operon, which has a lower GATC content than the surrounding DNA.

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