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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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Neisseria HU protein is protected from trypsin digestion in the presence of DMP12 or DNA. Because C-terminal–tagged Neisseria HU protein and DMP12 both have similar molecular weight, His–pull-down was used to purify the uncleaved C-terminal–tagged Neisseria HU protein. The uncleaved C-terminal–tagged Neisseria HU proteins were analyzed using a 12% Bis-Tris SDS–PAGE.
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gkt201-F5: Neisseria HU protein is protected from trypsin digestion in the presence of DMP12 or DNA. Because C-terminal–tagged Neisseria HU protein and DMP12 both have similar molecular weight, His–pull-down was used to purify the uncleaved C-terminal–tagged Neisseria HU protein. The uncleaved C-terminal–tagged Neisseria HU proteins were analyzed using a 12% Bis-Tris SDS–PAGE.

Mentions: The DNA-binding β-ribbon arms of bacterial HU protein are disordered in the absence of DNA (23,24). A disordered region is expected to undergo protease digestion ∼105–107 times faster than an ordered one (25). To further provide evidence that DMP12 targets the DNA-binding region on Neisseria HU protein, we performed a limited trypsin digestion assay (Figure 5). The results showed Neisseria HU protein is sensitive to trypsin in the absence of DMP12 or dsDNA (Figure 5; lane 1), whereas DMP12 (lane 3) and dsDNA (lane 4) both reduced the rate of HU protein degradation. This protection effect is dose-dependent (Supplementary Figure S6). Taken together, these results suggest that DMP12 binding promotes and stabilizes the formation of DNA-binding β-ribbon arms on Neisseria HU in a similar way as does DNA binding. The biological significance of this protecting effect is not clear, but the growth rate of DMP12-expressing E. coli is noticeably higher (∼1.85-fold after the addition of IPTG for 4 h) than the control bacteria (Supplementary Figure S7). This suggests DMP12 protein may benefit bacterial replication.Figure 5.


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)

Neisseria HU protein is protected from trypsin digestion in the presence of DMP12 or DNA. Because C-terminal–tagged Neisseria HU protein and DMP12 both have similar molecular weight, His–pull-down was used to purify the uncleaved C-terminal–tagged Neisseria HU protein. The uncleaved C-terminal–tagged Neisseria HU proteins were analyzed using a 12% Bis-Tris SDS–PAGE.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt201-F5: Neisseria HU protein is protected from trypsin digestion in the presence of DMP12 or DNA. Because C-terminal–tagged Neisseria HU protein and DMP12 both have similar molecular weight, His–pull-down was used to purify the uncleaved C-terminal–tagged Neisseria HU protein. The uncleaved C-terminal–tagged Neisseria HU proteins were analyzed using a 12% Bis-Tris SDS–PAGE.
Mentions: The DNA-binding β-ribbon arms of bacterial HU protein are disordered in the absence of DNA (23,24). A disordered region is expected to undergo protease digestion ∼105–107 times faster than an ordered one (25). To further provide evidence that DMP12 targets the DNA-binding region on Neisseria HU protein, we performed a limited trypsin digestion assay (Figure 5). The results showed Neisseria HU protein is sensitive to trypsin in the absence of DMP12 or dsDNA (Figure 5; lane 1), whereas DMP12 (lane 3) and dsDNA (lane 4) both reduced the rate of HU protein degradation. This protection effect is dose-dependent (Supplementary Figure S6). Taken together, these results suggest that DMP12 binding promotes and stabilizes the formation of DNA-binding β-ribbon arms on Neisseria HU in a similar way as does DNA binding. The biological significance of this protecting effect is not clear, but the growth rate of DMP12-expressing E. coli is noticeably higher (∼1.85-fold after the addition of IPTG for 4 h) than the control bacteria (Supplementary Figure S7). This suggests DMP12 protein may benefit bacterial replication.Figure 5.

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