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Biochemical and structural characterization of Cren7, a novel chromatin protein conserved among Crenarchaea.

Guo L, Feng Y, Zhang Z, Yao H, Luo Y, Wang J, Huang L - Nucleic Acids Res. (2007)

Bottom Line: A small, basic, methylated and abundant protein, Cren7 displays a higher affinity for double-stranded DNA than for single-stranded DNA, constrains negative DNA supercoils and is associated with genomic DNA in vivo.It interacts with duplex DNA through a beta-sheet and a long flexible loop, presumably resulting in DNA distortions through intercalation of conserved hydrophobic residues into the DNA structure.These data suggest that the crenarchaeal kingdom in the Archaea shares a common strategy in chromatin organization.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, 3A Datun Road, Beijing 100101, PR China.

ABSTRACT
Archaea contain a variety of chromatin proteins consistent with the evolution of different genome packaging mechanisms. Among the two main kingdoms in the Archaea, Euryarchaeota synthesize histone homologs, whereas Crenarchaeota have not been shown to possess a chromatin protein conserved at the kingdom level. We report the identification of Cren7, a novel family of chromatin proteins highly conserved in the Crenarchaeota. A small, basic, methylated and abundant protein, Cren7 displays a higher affinity for double-stranded DNA than for single-stranded DNA, constrains negative DNA supercoils and is associated with genomic DNA in vivo. The solution structure and DNA-binding surface of Cren7 from the hyperthermophilic crenarchaeon Sulfolobus solfataricus were determined by NMR. The protein adopts an SH3-like fold. It interacts with duplex DNA through a beta-sheet and a long flexible loop, presumably resulting in DNA distortions through intercalation of conserved hydrophobic residues into the DNA structure. These data suggest that the crenarchaeal kingdom in the Archaea shares a common strategy in chromatin organization.

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Identification of the binding target of Cren7 in the cell. (A) Chromosomal fractionation. S. solfataricus cells were resuspended in Bugbuster™ protein extraction reagent. Samples were treated with RNase A or DNase I. After centrifugation, the pellets were resuspended and subjected to polyacrylamide gel electrophoresis. Cren7 was detected by immunoblotting using anti-Cren7 antibodies. (B) Co-immunoprecipitation. S. solfataricus cells were irradiated with UV light or left untreated. After cell lysis, extracts were subjected to co-immunoprecipitation with anti-Cren7 antibodies. Nucleic acids co-precipitated with Cren7 were treated with either DNase I or RNase A + T1, and electrophoresed in agarose. (C) Abundance of Cren7 in S. solfataricus. S. solfataricus was grown at 78°C. Samples were taken at different stages during the growth, centrifuged and subjected to SDS–PAGE. Cren7 was detected by immunoblotting using anti-Cren7 antibodies.
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Figure 3: Identification of the binding target of Cren7 in the cell. (A) Chromosomal fractionation. S. solfataricus cells were resuspended in Bugbuster™ protein extraction reagent. Samples were treated with RNase A or DNase I. After centrifugation, the pellets were resuspended and subjected to polyacrylamide gel electrophoresis. Cren7 was detected by immunoblotting using anti-Cren7 antibodies. (B) Co-immunoprecipitation. S. solfataricus cells were irradiated with UV light or left untreated. After cell lysis, extracts were subjected to co-immunoprecipitation with anti-Cren7 antibodies. Nucleic acids co-precipitated with Cren7 were treated with either DNase I or RNase A + T1, and electrophoresed in agarose. (C) Abundance of Cren7 in S. solfataricus. S. solfataricus was grown at 78°C. Samples were taken at different stages during the growth, centrifuged and subjected to SDS–PAGE. Cren7 was detected by immunoblotting using anti-Cren7 antibodies.

Mentions: As a small, basic and methylated protein capable of efficient binding to dsDNA and constraining negatively DNA supercoils, Cren7 may be a chromatin protein in vivo. To verify this possibility, we examined the association of Cren7 with nucleic acids in the S. solfataricus cell by using two approaches. In the first approach, we performed chromatin fractionation as described previously (10). As shown in Figure 3A, the bulk of Cren7 was in the insoluble chromatin-containing fraction. Treatment of the pellet with DNase I, but not RNase A, released a significant proportion of Cren7, indicating that the protein was associated with chromosomal DNA in vivo. In the second approach, we irradiated S. solfataricus cells with UV light to induce the formation of protein–nucleic acid cross-links, lyzed the cells and subjected the lysates to immunoprecipitation with antibodies against Cren7. Nucleic acids co-immunoprecipitated with Cren7 were treated with either DNase I or RNase A + T1. In both cross-linked and untreated samples, DNA, but not RNA, was co-immunoprecipitated with Cren7 (Figure 3B). These data indicate that Cren7 is a chromatin DNA-binding protein.Figure 3.


Biochemical and structural characterization of Cren7, a novel chromatin protein conserved among Crenarchaea.

Guo L, Feng Y, Zhang Z, Yao H, Luo Y, Wang J, Huang L - Nucleic Acids Res. (2007)

Identification of the binding target of Cren7 in the cell. (A) Chromosomal fractionation. S. solfataricus cells were resuspended in Bugbuster™ protein extraction reagent. Samples were treated with RNase A or DNase I. After centrifugation, the pellets were resuspended and subjected to polyacrylamide gel electrophoresis. Cren7 was detected by immunoblotting using anti-Cren7 antibodies. (B) Co-immunoprecipitation. S. solfataricus cells were irradiated with UV light or left untreated. After cell lysis, extracts were subjected to co-immunoprecipitation with anti-Cren7 antibodies. Nucleic acids co-precipitated with Cren7 were treated with either DNase I or RNase A + T1, and electrophoresed in agarose. (C) Abundance of Cren7 in S. solfataricus. S. solfataricus was grown at 78°C. Samples were taken at different stages during the growth, centrifuged and subjected to SDS–PAGE. Cren7 was detected by immunoblotting using anti-Cren7 antibodies.
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Figure 3: Identification of the binding target of Cren7 in the cell. (A) Chromosomal fractionation. S. solfataricus cells were resuspended in Bugbuster™ protein extraction reagent. Samples were treated with RNase A or DNase I. After centrifugation, the pellets were resuspended and subjected to polyacrylamide gel electrophoresis. Cren7 was detected by immunoblotting using anti-Cren7 antibodies. (B) Co-immunoprecipitation. S. solfataricus cells were irradiated with UV light or left untreated. After cell lysis, extracts were subjected to co-immunoprecipitation with anti-Cren7 antibodies. Nucleic acids co-precipitated with Cren7 were treated with either DNase I or RNase A + T1, and electrophoresed in agarose. (C) Abundance of Cren7 in S. solfataricus. S. solfataricus was grown at 78°C. Samples were taken at different stages during the growth, centrifuged and subjected to SDS–PAGE. Cren7 was detected by immunoblotting using anti-Cren7 antibodies.
Mentions: As a small, basic and methylated protein capable of efficient binding to dsDNA and constraining negatively DNA supercoils, Cren7 may be a chromatin protein in vivo. To verify this possibility, we examined the association of Cren7 with nucleic acids in the S. solfataricus cell by using two approaches. In the first approach, we performed chromatin fractionation as described previously (10). As shown in Figure 3A, the bulk of Cren7 was in the insoluble chromatin-containing fraction. Treatment of the pellet with DNase I, but not RNase A, released a significant proportion of Cren7, indicating that the protein was associated with chromosomal DNA in vivo. In the second approach, we irradiated S. solfataricus cells with UV light to induce the formation of protein–nucleic acid cross-links, lyzed the cells and subjected the lysates to immunoprecipitation with antibodies against Cren7. Nucleic acids co-immunoprecipitated with Cren7 were treated with either DNase I or RNase A + T1. In both cross-linked and untreated samples, DNA, but not RNA, was co-immunoprecipitated with Cren7 (Figure 3B). These data indicate that Cren7 is a chromatin DNA-binding protein.Figure 3.

Bottom Line: A small, basic, methylated and abundant protein, Cren7 displays a higher affinity for double-stranded DNA than for single-stranded DNA, constrains negative DNA supercoils and is associated with genomic DNA in vivo.It interacts with duplex DNA through a beta-sheet and a long flexible loop, presumably resulting in DNA distortions through intercalation of conserved hydrophobic residues into the DNA structure.These data suggest that the crenarchaeal kingdom in the Archaea shares a common strategy in chromatin organization.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, 3A Datun Road, Beijing 100101, PR China.

ABSTRACT
Archaea contain a variety of chromatin proteins consistent with the evolution of different genome packaging mechanisms. Among the two main kingdoms in the Archaea, Euryarchaeota synthesize histone homologs, whereas Crenarchaeota have not been shown to possess a chromatin protein conserved at the kingdom level. We report the identification of Cren7, a novel family of chromatin proteins highly conserved in the Crenarchaeota. A small, basic, methylated and abundant protein, Cren7 displays a higher affinity for double-stranded DNA than for single-stranded DNA, constrains negative DNA supercoils and is associated with genomic DNA in vivo. The solution structure and DNA-binding surface of Cren7 from the hyperthermophilic crenarchaeon Sulfolobus solfataricus were determined by NMR. The protein adopts an SH3-like fold. It interacts with duplex DNA through a beta-sheet and a long flexible loop, presumably resulting in DNA distortions through intercalation of conserved hydrophobic residues into the DNA structure. These data suggest that the crenarchaeal kingdom in the Archaea shares a common strategy in chromatin organization.

Show MeSH
Related in: MedlinePlus