Figure 1.
The crystal structure of Halo-RNase H1 (PDB code 4NYN).
(A) The entire structure of molecule A (green) and a part of the structures of molecules B (blue), A′ (magenta) and A″ (orange) are shown. The side chains of the four acidic active site residues (Asp75, Glu115, Asp139, Asp189), bi-aspartate site residues (Asp197, Asp198), and quad-aspartate site residues (Asp132, Asp133, Asp173, Asp174) of molecule A are shown as green sticks, in which the oxygen atom is colored red. The side chains of the four acidic active site residues of molecules B and A″ are shown as blue and orange sticks respectively, in which the oxygen atom is colored red. The side chains of the quad-aspartate site residues of molecule A′ are shown as magenta sticks, in which the oxygen atom is colored red. Two manganese ions are shown as yellow spheres. (B) The structure of the quad-aspartate site of molecule A (green), which contacts the active site of molecule A″ (orange) through two manganese ions (Mn1 and Mn2), is superimposed onto that of molecule B (blue), which does not contact another molecule. The side chains of the four quad-aspartate site residues and four acidic active site residues, and two manganese ions are shown as in (Fig. 1A).
Figure 2.
CD spectra of Halo-RNase H1 and its mutants.
The far-UV CD spectra of 2A-RNase H1 (A), 6A-RNase H1 (B), and 8A-RNase H1 (C) measured at 25°C and pH 8.0 in the absence of salt and presence of various concentrations of MnCl2 are shown in comparison with those of Halo-RNase H1 measured in the absence of salt and divalent metal ions (grey solid thick line) and in the absence of salt and the presence of 20 mM MnCl2 (grey dashed thick line). MnCl2 concentrations: 0 mM (solid thick line); 0.2 mM (dashed-dotted thin line); 0.4 mM (dotted thick line); 1 mM (dashed thin line); 5 mM (solid thin line); 20 mM (dashed thick line).
Figure 3.
Intrinsic tryptophan fluorescence spectra of Halo-RNase H1 and its mutants.
The spectra measured in the absence of salt and divalent metal ions are shown for Halo-RNase H1 (long-dashed thick line), 2A-RNase H1 (dotted thick line), 6A-RNase H1 (solid thick line), and 8A-RNase H1 (short-dashed thick line). The spectra measured in the absence of salt and presence of 10 mM MnCl2 are shown for Halo-RNase H1 (long-dashed thin line), 2A-RNase H1 (dotted thin line), 6A-RNase H1 (solid thin line), and 8A-RNase H1 (short-dashed thin line).
Table 1.
Tm values of Halo-RNase H1 and its mutantsa.
Figure 4.
Cleavage of R12/D12 substrate with Halo-RNase H1 and 6A-RNase H1.
The 5′-end labeled R12/D12 substrate was hydrolyzed by Halo-RNase H1 or 6A-RNase H1 at 37°C for 15 min in the presence of various concentrations of MnCl2 or MgCl2 and in the presence of 50 mM (B) or 3 M (A) NaCl. The hydrolysates were separated on a 20% polyacrylamide gel containing 7 M urea. The concentration of the substrate was 1 µM. The amount of the enzyme added to the reaction mixture (10 µl) was 1 ng. The enzyme and divalent metal ions used to hydrolyze the substrate are shown above the gel together with the concentrations of the divalent metal ions. The sequence of R12 is indicated along the gel.
Figure 5.
Manganese dependencies of Halo-RNase H1 and 6A-RNase H1 activities.
The specific activities of Halo-RNase H1 in the presence of 50 mM (open diamond) and 3 M (closed circle) NaCl and those of 6A-RNase H1 in the presence of 50 mM (cross) and 3 M (closed square) NaCl, which are estimated from the gels shown in Figure 3, are plotted as a function of the MnCl2 concentration.
Figure 6.
Alignment of the amino acid sequences.
The amino acid sequences of Halo-RNase H2 (Halo), Tk-RNase H2 (Tk), Bst-RNase H2 (Bst), Tma-RNase H2 (Tma), and Aae-RNase H2 (Aae) are compared with one another. The accession numbers are AE004437 for Halo-RNase H2, AB012613 for Tk-RNase H2, BAB91155 for Bst-RNase H2, AAD35996 for Tma-RNase H2, and AAC07736 for Aae-RNase H2. The ranges of the secondary structures of Halo-RNase H2 deduced from its tertiary model are shown above the sequences. The four acidic active site residues are shaded and marked with asterisks. A GRG or GKG motif and a tyrosine residue that are responsible for recognition of a single ribonucleotide misincorporated into dsDNA are shaded. The dashes stand for the gaps. The numbers represent the positions of the amino acid residues relative to the initiator methionine for each protein. PCNA interacting peptide (PIP) motif is boxed.
Figure 7.
A tertiary model of Halo-RNase H2.
Ribbon diagram with electrostatic surface potential of Halo-RNase H2 is shown in comparison with that of Halo-RNase H1. NT and CT represent N and C-termini. The negative and positive potentials are in red and blue respectively. The electrostatic potential value ranges from −100 to +100 kT e−1. The positions of quad-aspartate site (Quad-Asp) of Halo-RNase H1 and PIP-box of Halo-RNase H2 are shown.
Figure 8.
The far-UV CD spectra of Halo-RNase H2 measured at 25°C and pH 8.0 in the absence of salt and divalent metal ions (solid thick line), in the absence of salt and the presence of 10 mM MnCl2 (solid thin line), 20 mM MnCl2 (dashed thin line), and 500 mM MgCl2 (dotted thin line), and in the presence 3 M NaCl (dotted thick line) and 4 M NaCl (dashed thick line) and the absence of divalent metal ions are shown.
Figure 9.
Cleavage of DNA15-RNA1-DNA13/DNA29 substrate with Halo-RNase H2.
The 5′-end labeled DNA15-RNA1-DNA13/DNA29 substrate was hydrolyzed by Halo-RNase H2 at 37°C for 15 min in the presence of 3 M NaCl and various concentrations of MnCl2 or MgCl2. The hydrolysates were separated on a 20% polyacrylamide gel containing 7 M urea. The concentration of the substrate was 1 µM. The amount of the enzyme added to the reaction mixture (10 µl) was 500 ng. Divalent metal ions used to hydrolyze the substrate and the concentrations of these divalent metal ions are shown above the gel. The arrows indicate the 5′-end labeled substrate (top) and 5′-end labeled 15-mer DNA (bottom).