Modular architecture of the hexameric human mitochondrial DNA helicase - PubMed (original) (raw)

Modular architecture of the hexameric human mitochondrial DNA helicase

Tawn D Ziebarth et al. J Mol Biol. 2007.

Abstract

We have probed the structure of the human mitochondrial DNA helicase, an enzyme that uses the energy of nucleotide hydrolysis to unwind duplex DNA during mitochondrial DNA replication. This novel helicase shares substantial amino acid sequence and functional similarities with the bacteriophage T7 primase-helicase. We show in velocity sedimentation and gel filtration analyses that the mitochondrial DNA helicase exists as a hexamer. Limited proteolysis by trypsin results in the production of several stable fragments, and N-terminal sequencing reveals distinct N and C-terminal polypeptides that represent minimal structural domains. Physical analysis of the proteolytic products defines the region required to maintain oligomeric structure to reside within amino acid residues approximately 405-590. Truncations of the N and C termini affect differentially DNA-dependent ATPase activity, and whereas a C-terminal domain polypeptide is functional, an N-terminal domain polypeptide lacks ATPase activity. Sequence similarity and secondary structural alignments combined with biochemical data suggest that amino acid residue R609 serves as the putative arginine finger that is essential for ATPase activity in ring helicases. The hexameric conformation and modular architecture revealed in our study document that the mitochondrial DNA helicase and bacteriophage T7 primase-helicase share physical features. Our findings place the mitochondrial DNA helicase firmly in the DnaB-like family of replicative DNA helicases.

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Figures

Figure 1

Figure 1

Hydrodynamic analysis of human mtDNA helicase. (a) Glycerol gradient sedimentation. Helicase was sedimented in a 12-30% glycerol gradient (see Methods). Standard protein markers run in parallel gradients were: jack bean urease (URE, 18.6 S), bovine liver catalase (CAT, 11.3 S), and rabbit muscle lactate dehydrogenase (LDH, 7.3 S). (b) Gel filtration. Helicase was chromatographed on a Superdex 200 column. Standard protein markers used to calibrate the column were: chymotrypsinogen (CHY, Rs = 2.9 nm), hen egg ovalbumin (OVA, Rs = 3.05 nm), yeast alcohol dehydrogenase (ADH, Rs = 4.6 nm), bovine liver catalase (CAY, Rs = 5.2 nm), and bovine thyroid thyroglobulin (THY, Rs = 8.5 nm).

Figure 2

Figure 2

Proteolytic digestion produces stable fragments of human mtDNA helicase. (a) Trypsin titration. Helicase was digested with increasing concentrations of trypsin. Samples were analyzed by SDS-PAGE followed by silver staining. Ratios are given as helicase:trypsin. Lane 1, undigested helicase; lane 2-8, 1: 0.02, 1: 0.08, 1: 0.2, 1: 0.3, 1: 0.6, 1: 1.3, 1: 2.0. (b) Multiple protease digestion. Helicase was digested with five different proteases, and the products were analyzed by SDS-PAGE followed by silver staining. Ratios are given as helicase:protease. Lane 1, undigested helicase; lane 2, trypsin 1:1; lane 3 chymotrypsin 1:0.35; lane 4, thermolysin 1:1; lane 5, subtilisin 1:0.005; lane 6, papain 1:0.15.

Figure 3

Figure 3

(a) Schematic map of proteolytic fragments of human mtDNA helicase. The recombinant form of the full-length human mtDNA helicase used in this study lacks its mitochondrial targeting sequence, and comprises amino acid residues 43-684. (b). Sequence alignment of human mtDNA helicase and bacteriophage T7 helicase-primase. A secondary structure-based sequence alignment of T7 gp4 (PDB codes 1NUI and 1Q5727) and human mtDNA helicase (GenBank accession number NP068602) was produced using 3D-PSSM (

www.sbg.bio.ic.ac.uk

). In the T7 gp4 amino acid sequence, residue Y13 is displaced below the alignment and amino acid residues #44-48 (AGNED) are omitted. Barrels designate alpha helices and arrows designate beta sheets. The 5 conserved amino acid motifs in superfamily IV DNA helicases are indicated by labeled boxes. The N-terminal T28 fragment of human mtDNA helicase is shaded in blue, the C-terminal T30 fragment is shaded in yellow and the overlapping region is in green. The position of the arginine finger in the helicase domain of T7 gp4 (R522) and the corresponding amino acid residue in the human mtDNA helicase (R609) are indicated in bold.

Figure 3

Figure 3

(a) Schematic map of proteolytic fragments of human mtDNA helicase. The recombinant form of the full-length human mtDNA helicase used in this study lacks its mitochondrial targeting sequence, and comprises amino acid residues 43-684. (b). Sequence alignment of human mtDNA helicase and bacteriophage T7 helicase-primase. A secondary structure-based sequence alignment of T7 gp4 (PDB codes 1NUI and 1Q5727) and human mtDNA helicase (GenBank accession number NP068602) was produced using 3D-PSSM (

www.sbg.bio.ic.ac.uk

). In the T7 gp4 amino acid sequence, residue Y13 is displaced below the alignment and amino acid residues #44-48 (AGNED) are omitted. Barrels designate alpha helices and arrows designate beta sheets. The 5 conserved amino acid motifs in superfamily IV DNA helicases are indicated by labeled boxes. The N-terminal T28 fragment of human mtDNA helicase is shaded in blue, the C-terminal T30 fragment is shaded in yellow and the overlapping region is in green. The position of the arginine finger in the helicase domain of T7 gp4 (R522) and the corresponding amino acid residue in the human mtDNA helicase (R609) are indicated in bold.

Figure 4

Figure 4

Time course of trypsin digestion. Helicase was digested over a time course of 1.5 h. Samples were extracted at the indicated time points, and analyzed by SDS-PAGE followed by silver staining. Lane 1, undigested helicase; lanes 2-8, 1, 5, 10, 20, 30, 60, and 90 min.

Figure 5

Figure 5

Gel filtration of trypsin products T66/T57, T57/T50 and T34/T28. Helicase was subjected to limited proteolysis with trypsin and the samples were fractionated by gel filtration on a FPLC Superdex 200 column. The elution fractions were analyzed by SDS-PAGE and silver staining; all those containing staining material are shown here. (a) T66/T57. Lane 1, undigested helicase; lane 2, gel filtration load; lanes 3-7, hexameric peak fractions #38-42. (b) T57/T50. Lane 1, undigested helicase; lane 2, gel filtration load; lanes 3-6, hexameric peak fractions #39-42. (c) T34/T28. Lane 1, gel filtration load; lanes 2-4, monomeric peak fractions #65-67; lane 5, blank; lane 6, trypsin.

Figure 6

Figure 6

Time course of ATP hydrolysis by trypsin products. Full-length helicase (open circles), P66 (closed circles), P57 (open triangles), T57/T50 (4:1, closed triangles and 3:1, open squares) were assayed for ATPase activity over 0-40 min.

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