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Molecular Dissection of the Essential Features of the Origin of Replication of the Second Vibrio cholerae Chromosome.

Gerding MA, Chao MC, Davis BM, Waldor MK - MBio (2015)

Bottom Line: RctB displayed a reduced affinity for most of the low-efficacy origins tested, although its characteristic cooperative binding was generally maintained.Together, our results reveal the remarkable evolutionary honing of oriCII and provide new insight into the complex interplay between RctB and oriCII.Finally, our findings suggest that the OriSeq approach is likely to be widely applicable for defining critical bases in cis-acting sequences.

View Article: PubMed Central - PubMed

Affiliation: Program in Biological and Biomedical Sciences, Graduate School of Arts and Sciences, Harvard Medical School, Boston, Massachusetts, USA Division of Infectious Diseases, Brigham and Women's Hospital, Boston, Massachusetts, USA.

No MeSH data available.


Related in: MedlinePlus

Individual 12-mer changes modestly reduce RctB binding affinity Representative EMSAs of RctB wt (A-F) or ΔC159 (G-L) with pseudo-origins depicted above gels. The concentrations of RctB ranged from 0.000006125-409.6 nM in a 4-fold dilution series. Red arrows below the gels indicate RctB wt and ΔC159 concentrations where near maximal binding is seen with the methylated 12×6 probe in Fig. 3B and 3C respectively.
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fig6: Individual 12-mer changes modestly reduce RctB binding affinity Representative EMSAs of RctB wt (A-F) or ΔC159 (G-L) with pseudo-origins depicted above gels. The concentrations of RctB ranged from 0.000006125-409.6 nM in a 4-fold dilution series. Red arrows below the gels indicate RctB wt and ΔC159 concentrations where near maximal binding is seen with the methylated 12×6 probe in Fig. 3B and 3C respectively.

Mentions: In the transformation assay, methylation site mutations within single 12-mers in the array reduced but did not abolish the capacity of the origin constructs to support replication, suggesting that no individual 12-mer makes an essential contribution to the functionality of the array. Consistent with these results, WT RctB and RctB ΔC159 bound to the corresponding probes (12-1, 12-2, 12-3, 12-4, 12-5, 12-6) with affinities similar to or slightly lower than those they had for the WT array (Table 1; Fig. 3B and C and 6; see Fig. S5 in the supplemental material). These binding experiments did not provide a clear explanation of why some mutations had a larger effect upon RctB ΔC159-based replication; no marked variation was observed in the truncated protein’s pattern of binding to the set of probes, and there was no precise correspondence between binding affinity and replication capacity (Table 1; Fig. 3C, 4, and 6G to L; see Fig. S5C in the supplemental material). However, an apparent correspondence between replication capacity and RctB binding was observed for the constructs with mutations in multiple 12-mers (12-135, 12-246, and 12-12456). Both WT RctB and RctB ΔC159 bound to these probes with markedly reduced affinity, suggesting that contiguous methylated 12-mers are important for high-affinity RctB binding to the origin (Table 1; see Fig. S5B and D and S6 in the supplemental material). WT RctB’s cooperative binding to these probes was slightly reduced as well, as shown by the appearance of weak intermediate species in the gels (see Fig. S6B to D, green arrows).


Molecular Dissection of the Essential Features of the Origin of Replication of the Second Vibrio cholerae Chromosome.

Gerding MA, Chao MC, Davis BM, Waldor MK - MBio (2015)

Individual 12-mer changes modestly reduce RctB binding affinity Representative EMSAs of RctB wt (A-F) or ΔC159 (G-L) with pseudo-origins depicted above gels. The concentrations of RctB ranged from 0.000006125-409.6 nM in a 4-fold dilution series. Red arrows below the gels indicate RctB wt and ΔC159 concentrations where near maximal binding is seen with the methylated 12×6 probe in Fig. 3B and 3C respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4551981&req=5

fig6: Individual 12-mer changes modestly reduce RctB binding affinity Representative EMSAs of RctB wt (A-F) or ΔC159 (G-L) with pseudo-origins depicted above gels. The concentrations of RctB ranged from 0.000006125-409.6 nM in a 4-fold dilution series. Red arrows below the gels indicate RctB wt and ΔC159 concentrations where near maximal binding is seen with the methylated 12×6 probe in Fig. 3B and 3C respectively.
Mentions: In the transformation assay, methylation site mutations within single 12-mers in the array reduced but did not abolish the capacity of the origin constructs to support replication, suggesting that no individual 12-mer makes an essential contribution to the functionality of the array. Consistent with these results, WT RctB and RctB ΔC159 bound to the corresponding probes (12-1, 12-2, 12-3, 12-4, 12-5, 12-6) with affinities similar to or slightly lower than those they had for the WT array (Table 1; Fig. 3B and C and 6; see Fig. S5 in the supplemental material). These binding experiments did not provide a clear explanation of why some mutations had a larger effect upon RctB ΔC159-based replication; no marked variation was observed in the truncated protein’s pattern of binding to the set of probes, and there was no precise correspondence between binding affinity and replication capacity (Table 1; Fig. 3C, 4, and 6G to L; see Fig. S5C in the supplemental material). However, an apparent correspondence between replication capacity and RctB binding was observed for the constructs with mutations in multiple 12-mers (12-135, 12-246, and 12-12456). Both WT RctB and RctB ΔC159 bound to these probes with markedly reduced affinity, suggesting that contiguous methylated 12-mers are important for high-affinity RctB binding to the origin (Table 1; see Fig. S5B and D and S6 in the supplemental material). WT RctB’s cooperative binding to these probes was slightly reduced as well, as shown by the appearance of weak intermediate species in the gels (see Fig. S6B to D, green arrows).

Bottom Line: RctB displayed a reduced affinity for most of the low-efficacy origins tested, although its characteristic cooperative binding was generally maintained.Together, our results reveal the remarkable evolutionary honing of oriCII and provide new insight into the complex interplay between RctB and oriCII.Finally, our findings suggest that the OriSeq approach is likely to be widely applicable for defining critical bases in cis-acting sequences.

View Article: PubMed Central - PubMed

Affiliation: Program in Biological and Biomedical Sciences, Graduate School of Arts and Sciences, Harvard Medical School, Boston, Massachusetts, USA Division of Infectious Diseases, Brigham and Women's Hospital, Boston, Massachusetts, USA.

No MeSH data available.


Related in: MedlinePlus