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Neisseria conserved hypothetical protein DMP12 is a DNA mimic that binds to histone-like HU protein.

Wang HC, Wu ML, Ko TP, Wang AH - Nucleic Acids Res. (2013)

Bottom Line: Our gel filtration and analytical ultracentrifugation results showed that the DMP12 monomer interacts with the dimeric form of the bacterial histone-like protein HU.Functionally, HU proteins participate in bacterial nucleoid formation, as well as recombination, gene regulation and DNA replication.The interaction between DMP12 and HU protein might, therefore, play important roles in these DNA-related mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan.

ABSTRACT
DNA mimic proteins are unique factors that control the DNA-binding activity of target proteins by directly occupying their DNA-binding sites. To date, only a few DNA mimic proteins have been reported and their functions analyzed. Here, we present evidence that the Neisseria conserved hypothetical protein DMP12 should be added to this list. Our gel filtration and analytical ultracentrifugation results showed that the DMP12 monomer interacts with the dimeric form of the bacterial histone-like protein HU. Subsequent structural analysis of DMP12 showed that the shape and electrostatic surface of the DMP12 monomer are similar to those of the straight portion of the bent HU-bound DNA and complementary to those of HU protein dimer. DMP12 also protects HU protein from limited digestion by trypsin and enhances the growth rate Escherichia coli. Functionally, HU proteins participate in bacterial nucleoid formation, as well as recombination, gene regulation and DNA replication. The interaction between DMP12 and HU protein might, therefore, play important roles in these DNA-related mechanisms.

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Related in: MedlinePlus

DMP12 partially prevents the DNA binding of Neisseria HU protein. A preliminary EMSA showed that the addition of 4 µM Neisseria HU protein produced a marked band shift of the 2.5 nM plasmid DNA substrate (Supplementary Figure S5), and this concentration was, therefore, used in this competitive EMSA assay. The results show that the DNA mimic DMP12 partially reduces the DNA shift of Neisseria HU in a dosage-dependent manner.
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gkt201-F4: DMP12 partially prevents the DNA binding of Neisseria HU protein. A preliminary EMSA showed that the addition of 4 µM Neisseria HU protein produced a marked band shift of the 2.5 nM plasmid DNA substrate (Supplementary Figure S5), and this concentration was, therefore, used in this competitive EMSA assay. The results show that the DNA mimic DMP12 partially reduces the DNA shift of Neisseria HU in a dosage-dependent manner.

Mentions: ITC was used to investigate the binding affinity of Niesseria HU and DMP12 (Figure 3). The calorimetric titration of Neisseria HU dimer into a solution of DMP12 showed that the proteins bind with a Kd of 2.81 ± 0.43 µM (Table 3). This is ∼4-fold lower than the 0.72 ± 0.06 µM for HU dimer binding to the 8mer dsDNA. A competitive electrophoresis mobility shift assay (EMSA) further showed that HU-plasmid DNA complexes were partially disrupted by the addition of DMP12 (Figure 4). The dosage-dependent decrease in the shifted bands when DMP12 was pre-incubated with Neisseria HU protein suggests that DMP12 prevented the binding of HU protein to the plasmid DNA. Also of note is that a high concentration of DMP12 did not completely return the plasmid DNA to its original unbound form. A similar finding has been previously reported for the HU-bound DNA mimic protein HI1450 (22). In the present case, the relatively weak affinity of DMP12 to Neisseria HU suggests that it may not fully block the HU protein–plasmid binding. These results suggest that although DMP12 and DNA evidently share the same binding sites on HU protein, DMP12 is more likely to act as a regulator than a competitive inhibitor.Figure 3.


Neisseria conserved hypothetical protein DMP12 is a DNA mimic that binds to histone-like HU protein.

Wang HC, Wu ML, Ko TP, Wang AH - Nucleic Acids Res. (2013)

DMP12 partially prevents the DNA binding of Neisseria HU protein. A preliminary EMSA showed that the addition of 4 µM Neisseria HU protein produced a marked band shift of the 2.5 nM plasmid DNA substrate (Supplementary Figure S5), and this concentration was, therefore, used in this competitive EMSA assay. The results show that the DNA mimic DMP12 partially reduces the DNA shift of Neisseria HU in a dosage-dependent manner.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643605&req=5

gkt201-F4: DMP12 partially prevents the DNA binding of Neisseria HU protein. A preliminary EMSA showed that the addition of 4 µM Neisseria HU protein produced a marked band shift of the 2.5 nM plasmid DNA substrate (Supplementary Figure S5), and this concentration was, therefore, used in this competitive EMSA assay. The results show that the DNA mimic DMP12 partially reduces the DNA shift of Neisseria HU in a dosage-dependent manner.
Mentions: ITC was used to investigate the binding affinity of Niesseria HU and DMP12 (Figure 3). The calorimetric titration of Neisseria HU dimer into a solution of DMP12 showed that the proteins bind with a Kd of 2.81 ± 0.43 µM (Table 3). This is ∼4-fold lower than the 0.72 ± 0.06 µM for HU dimer binding to the 8mer dsDNA. A competitive electrophoresis mobility shift assay (EMSA) further showed that HU-plasmid DNA complexes were partially disrupted by the addition of DMP12 (Figure 4). The dosage-dependent decrease in the shifted bands when DMP12 was pre-incubated with Neisseria HU protein suggests that DMP12 prevented the binding of HU protein to the plasmid DNA. Also of note is that a high concentration of DMP12 did not completely return the plasmid DNA to its original unbound form. A similar finding has been previously reported for the HU-bound DNA mimic protein HI1450 (22). In the present case, the relatively weak affinity of DMP12 to Neisseria HU suggests that it may not fully block the HU protein–plasmid binding. These results suggest that although DMP12 and DNA evidently share the same binding sites on HU protein, DMP12 is more likely to act as a regulator than a competitive inhibitor.Figure 3.

Bottom Line: Our gel filtration and analytical ultracentrifugation results showed that the DMP12 monomer interacts with the dimeric form of the bacterial histone-like protein HU.Functionally, HU proteins participate in bacterial nucleoid formation, as well as recombination, gene regulation and DNA replication.The interaction between DMP12 and HU protein might, therefore, play important roles in these DNA-related mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan.

ABSTRACT
DNA mimic proteins are unique factors that control the DNA-binding activity of target proteins by directly occupying their DNA-binding sites. To date, only a few DNA mimic proteins have been reported and their functions analyzed. Here, we present evidence that the Neisseria conserved hypothetical protein DMP12 should be added to this list. Our gel filtration and analytical ultracentrifugation results showed that the DMP12 monomer interacts with the dimeric form of the bacterial histone-like protein HU. Subsequent structural analysis of DMP12 showed that the shape and electrostatic surface of the DMP12 monomer are similar to those of the straight portion of the bent HU-bound DNA and complementary to those of HU protein dimer. DMP12 also protects HU protein from limited digestion by trypsin and enhances the growth rate Escherichia coli. Functionally, HU proteins participate in bacterial nucleoid formation, as well as recombination, gene regulation and DNA replication. The interaction between DMP12 and HU protein might, therefore, play important roles in these DNA-related mechanisms.

Show MeSH
Related in: MedlinePlus