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Nucleoid localization of Hsp40 Mdj1 is important for its function in maintenance of mitochondrial DNA.

Ciesielski GL, Plotka M, Manicki M, Schilke BA, Dutkiewicz R, Sahi C, Marszalek J, Craig EA - Biochim. Biophys. Acta (2013)

Bottom Line: Underscoring the importance of Hsp70 chaperone activity in the maintenance of mtDNA, an Mdj1 variant having an alteration in the Hsp70-interacting J-domain does not maintain mtDNA.We found that Mdj1 has DNA binding activity and that variants retaining DNA-binding activity also retained nucleoid association.Together, our results are consistent with a model in which Mdj1, tethered to the nucleoid via DNA binding, thus driving a high local concentration of the Hsp70 machinery, is important for faithful DNA maintenance and propagation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of Gdansk, Gdansk, Poland.

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Mdj1 binds DNA in vitro. A,B) Gel mobility shift analysis of Mdj1 binding to an mtDNA fragment. Purified Mdj1 or its variants were incubated with 32P-labeled 71 bp ori5 fragment at 0.36 nM, and then subjected to electrophoresis, followed by autoradiography. A) (Top) Different concentrations of wt Mdj1 (wt): Lines 1–8: at concentrations: 0 (indicated by “−”); 0.04, 0.08, 0.16, 0.32, 0.64, 2.56 and 6.4 μM, respectively. (Bottom) Quantification of the results (apparent Kd ~ 0.21 μM). B) Mdj1 variants at 0.64 μM were analyzed: (−) no protein control; (wt) wild-type; (H89Q) Mdj1H89Q; (J) J-domain fragment Mdj1Δ190–511; (ΔC) Mdj1Δ201–429; (ΔD) Mdj1Δ430–511 ΔZ) Mdj1Δ230–288.
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f0030: Mdj1 binds DNA in vitro. A,B) Gel mobility shift analysis of Mdj1 binding to an mtDNA fragment. Purified Mdj1 or its variants were incubated with 32P-labeled 71 bp ori5 fragment at 0.36 nM, and then subjected to electrophoresis, followed by autoradiography. A) (Top) Different concentrations of wt Mdj1 (wt): Lines 1–8: at concentrations: 0 (indicated by “−”); 0.04, 0.08, 0.16, 0.32, 0.64, 2.56 and 6.4 μM, respectively. (Bottom) Quantification of the results (apparent Kd ~ 0.21 μM). B) Mdj1 variants at 0.64 μM were analyzed: (−) no protein control; (wt) wild-type; (H89Q) Mdj1H89Q; (J) J-domain fragment Mdj1Δ190–511; (ΔC) Mdj1Δ201–429; (ΔD) Mdj1Δ430–511 ΔZ) Mdj1Δ230–288.

Mentions: It was previously reported that bacterial homologs of Mdj1 are able to directly interact with DNA in a sequence nonspecific manner [41,42]. Since we had found no evidence that either the peptide binding cleft or the zinc finger-like region was important for either mtDNA maintenance or nucleoid association, we decided to test whether Mdj1 also possesses DNA binding ability and, if so, whether it might be important for nucleoid association. To test for Mdj1-DNA interaction, we used a band shift assay using a radio-labeled short fragment of DNA (71 bp) derived from a mitochondrial DNA origin, ori5. At higher Mdj1 concentrations, the DNA fragment shifted to the top of the gel, indicating that Mdj1 does bind DNA in vitro (Fig. 6A). This binding appears to be nonspecific, as Mdj1 also bound DNA fragments derived from sources other than mtDNA (data not shown). To determine whether DNA binding ability correlated with the association of Mdj1 with the nucleoid, we tested the Mdj1 variants described in the sections above in the DNA binding assays, with the exception of the variants having alterations in the peptide-binding cleft as they were prone to aggregation. The variants were lacking the zinc finger-like region (Mdj1∆Z), lacking the dimerization domain (Mdj1∆D) or carrying the inactive H89Q J-domain bound DNA. However, Mdj1∆C and Mdj1Δ190–511, the larger deletion retaining only the J-domain and glycine-phenylalanine rich (GF) region, did not (Fig. 6B). Thus, the results of in vitro DNA binding tests correlated with the results of in vivo nucleoid localization, as variants able to bind DNA were found associated with the mitochondrial nucleoid, whereas variants unable to bind DNA were found in the fractions having soluble proteins.


Nucleoid localization of Hsp40 Mdj1 is important for its function in maintenance of mitochondrial DNA.

Ciesielski GL, Plotka M, Manicki M, Schilke BA, Dutkiewicz R, Sahi C, Marszalek J, Craig EA - Biochim. Biophys. Acta (2013)

Mdj1 binds DNA in vitro. A,B) Gel mobility shift analysis of Mdj1 binding to an mtDNA fragment. Purified Mdj1 or its variants were incubated with 32P-labeled 71 bp ori5 fragment at 0.36 nM, and then subjected to electrophoresis, followed by autoradiography. A) (Top) Different concentrations of wt Mdj1 (wt): Lines 1–8: at concentrations: 0 (indicated by “−”); 0.04, 0.08, 0.16, 0.32, 0.64, 2.56 and 6.4 μM, respectively. (Bottom) Quantification of the results (apparent Kd ~ 0.21 μM). B) Mdj1 variants at 0.64 μM were analyzed: (−) no protein control; (wt) wild-type; (H89Q) Mdj1H89Q; (J) J-domain fragment Mdj1Δ190–511; (ΔC) Mdj1Δ201–429; (ΔD) Mdj1Δ430–511 ΔZ) Mdj1Δ230–288.
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f0030: Mdj1 binds DNA in vitro. A,B) Gel mobility shift analysis of Mdj1 binding to an mtDNA fragment. Purified Mdj1 or its variants were incubated with 32P-labeled 71 bp ori5 fragment at 0.36 nM, and then subjected to electrophoresis, followed by autoradiography. A) (Top) Different concentrations of wt Mdj1 (wt): Lines 1–8: at concentrations: 0 (indicated by “−”); 0.04, 0.08, 0.16, 0.32, 0.64, 2.56 and 6.4 μM, respectively. (Bottom) Quantification of the results (apparent Kd ~ 0.21 μM). B) Mdj1 variants at 0.64 μM were analyzed: (−) no protein control; (wt) wild-type; (H89Q) Mdj1H89Q; (J) J-domain fragment Mdj1Δ190–511; (ΔC) Mdj1Δ201–429; (ΔD) Mdj1Δ430–511 ΔZ) Mdj1Δ230–288.
Mentions: It was previously reported that bacterial homologs of Mdj1 are able to directly interact with DNA in a sequence nonspecific manner [41,42]. Since we had found no evidence that either the peptide binding cleft or the zinc finger-like region was important for either mtDNA maintenance or nucleoid association, we decided to test whether Mdj1 also possesses DNA binding ability and, if so, whether it might be important for nucleoid association. To test for Mdj1-DNA interaction, we used a band shift assay using a radio-labeled short fragment of DNA (71 bp) derived from a mitochondrial DNA origin, ori5. At higher Mdj1 concentrations, the DNA fragment shifted to the top of the gel, indicating that Mdj1 does bind DNA in vitro (Fig. 6A). This binding appears to be nonspecific, as Mdj1 also bound DNA fragments derived from sources other than mtDNA (data not shown). To determine whether DNA binding ability correlated with the association of Mdj1 with the nucleoid, we tested the Mdj1 variants described in the sections above in the DNA binding assays, with the exception of the variants having alterations in the peptide-binding cleft as they were prone to aggregation. The variants were lacking the zinc finger-like region (Mdj1∆Z), lacking the dimerization domain (Mdj1∆D) or carrying the inactive H89Q J-domain bound DNA. However, Mdj1∆C and Mdj1Δ190–511, the larger deletion retaining only the J-domain and glycine-phenylalanine rich (GF) region, did not (Fig. 6B). Thus, the results of in vitro DNA binding tests correlated with the results of in vivo nucleoid localization, as variants able to bind DNA were found associated with the mitochondrial nucleoid, whereas variants unable to bind DNA were found in the fractions having soluble proteins.

Bottom Line: Underscoring the importance of Hsp70 chaperone activity in the maintenance of mtDNA, an Mdj1 variant having an alteration in the Hsp70-interacting J-domain does not maintain mtDNA.We found that Mdj1 has DNA binding activity and that variants retaining DNA-binding activity also retained nucleoid association.Together, our results are consistent with a model in which Mdj1, tethered to the nucleoid via DNA binding, thus driving a high local concentration of the Hsp70 machinery, is important for faithful DNA maintenance and propagation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of Gdansk, Gdansk, Poland.

Show MeSH