Crystal Structure of a DinB Lesion Bypass DNA Polymerase Catalytic Fragment Reveals a Classic Polymerase Catalytic Domain  Bo-Lu Zhou, Janice D. Pata, Thomas A. Steitz  Molecular Cell  Volume 8, Issue 2, Pages 427-437 (August 2001) DOI: 10.1016/S1097-2765(01)00310-0

Figure 4 Conserved Sequences in the UmuC/DinB Bypass Polymerase Family Sequence alignment of representative members of each UmuC/DinB subfamily. Motifs are as defined in Kulaeva et al. (1996). Residues that are identical in the majority of the sequences shown are highlighted in yellow. Residues of T7 DNAP that structurally align with Dbh are also shown. The catalytic residues and steric gate residue are marked with asterisks. The secondary structure of Dbh is represented by arrows (sheet) and bars (helix), which are colored as in Figure 1A. A gap is introduced to account for the different positions of bulged residues (A102 in Dbh and M649 in T7 DNAP) found in the β strands containing these residues Molecular Cell 2001 8, 427-437DOI: (10.1016/S1097-2765(01)00310-0)

Figure 1 Overall Structure and Active Site of Dbh (A) Schematic ribbons representation of the overall structure with helices designated with letters and β strands by numbers. Secondary structure elements were defined using PROCHECK and are as follows: 1 (3–8), A (11–19), B (21–23), 2 (28–33), 3 (41–46), C (48–51), D (61–67), 4 (72–75), E (78–93), 5 (98–103), 6 (106–110), F (119–137), 7 (141–146), G (149–158), 8 (164–166), H (172–177), I (181–183), and J (189–197). Helices B and I are 310 helices. Residues 206–216 are completely disordered. Residues 34–39 (called the flexible loop) are poorly ordered and are indicated with a dashed line. Five of the six His6-tag residues (−3 to 1) are visible. The fingers (residues 20–75), palm (residues 1–19 and 76–169), and thumb (residues 170–205) domains are indicated. The diagram is colored according to sequence motifs (see Figure 4) that are conserved across the entire UmuC/DinB family (Kulaeva et al., 1996): I (3–30, red), II (43–58, yellow), III (77–111, green), IV (125–161, blue), and V (174–205, magenta). A sulfate ion bound at the active site is shown in ball-and-stick representation (black). (B) The experimental density-modified electron density map calculated at 2.5 Å resolution and contoured at 2.0 sigma (blue) and 6.0 sigma (green) is shown superimposed on the final model that was refined to 2.3 Å resolution. Some of the conserved residues around the active site are indicated. (C) Stereo Cα backbone diagram of Dbh. Conserved residues discussed in the text are shown in ball-and-stick representation, as is the bound sulfate. Coloring is as described previously Molecular Cell 2001 8, 427-437DOI: (10.1016/S1097-2765(01)00310-0)

Figure 2 Homology Modeling of DNA from the T7 DNAP Ternary Complex onto the Molecular Surface of Dbh (A) Superposition of the palm domains of Dbh (colored as in Figure 1A) and T7 DNAP (colored in dark gray). The T7 DNAP catalytic aspartates (D475 and D654) and the residues that function as the steric gate (E480) and bind the 3′ terminal phosphorous of the primer strand (H704) are shown in stick representation, as are the corresponding residues from Dbh (D7, D105, F12, and K153). The Mg2+ ions from the T7 DNAP ternary complex are shown as yellow spheres. Cα atoms used in the superposition were 1–21, 77–95, 97–112, 118–137, and 138–153 from Dbh and 469–487, 614–632, 646–661, 662–681, and 689–704 from T7 DNAP ternary complex (Protein Data Bank code 1T7P; Doublié et al., 1998). Only the structurally aligned core of the palm domains is shown. (B) View looking down onto the Dbh active site. Primer and template DNA (light and dark gray, respectively), incoming nucleotide, and magnesium ions (yellow spheres) from the T7DNAP ternary complex structure (Doublié et al., 1998) were modeled onto the molecular surface of Dbh. Substrate molecules from the T7 DNAP ternary complex structure were positioned onto the Dbh surface based on the superposition of the palm domains shown in (A). The flexible loop and N-terminal His6-tag are not shown in surface representation and are, instead, shown with transparent and gray lines, respectively. The molecular surface of Dbh was calculated using a 1.4 Å radius probe in SPOCK (Christopher, 1998) and is colored according to conserved motifs I-V as in Figure 1A. (C) Closer view of the active site with surface colored according to electrostatic potential, with positively charged areas in blue and negatively charged areas in red. The thumb domain has been omitted for clarity, and the molecule has been rotated slightly to show the surface of the nascent base pair binding pocket more clearly. Surface areas contributed by some of the residues discussed in the text are labeled Molecular Cell 2001 8, 427-437DOI: (10.1016/S1097-2765(01)00310-0)

Figure 3 Polymerase Active Site of Dbh Residues discussed in the text are shown in stick representation together with a ribbons diagram of Dbh. Coloring of conserved motifs is as described in Figure 1A. Hydrogen bonds discussed are indicated with dotted lines Molecular Cell 2001 8, 427-437DOI: (10.1016/S1097-2765(01)00310-0)

Figure 5 Bypass Polymerase Activity and Processivity of Dbh (A) Primer template combinations used to assay Dbh activity. The two templates differ only in the first templating position, which is a guanosine (shown in bold) in the “undamaged” template and is a deoxyribose (dSpacer, Glen Research; shown as “_”) in the abasic-site template. Asterisks indicate position of 33P radioactive label. (B and C) The activity of full-length (DBH, lane 3) and N-terminal fragment (DBH-216, lane 4) Dbh polymerase on undamaged and abasic-site primer/templates, respectively. Enzyme and DNA substrate concentrations were 1 μM each, and reactions were incubated for 20 min at 37°C. The activity of Klenow fragment (KF, lane 2) on these templates is shown for comparison, and reactions without any enzyme added are shown as a control (none, lane 1). (D) Processivity of full-length Dbh on undamaged primer/template. When heparin was included in the reaction, it was added either during the preincubation of Dbh with DNA (1 μM each) to test the efficiency of the trap (pre-inc, lane 1) or at the same time as the reaction was initiated by the addition of Mg2+ and dNTPs to measure the processivity of Dbh (trap, lane 2). The processivity of Dbh was tested in an alternate way, without the use of heparin by decreasing the concentration of enzyme (1, 0.3, 0.01, 0.03, and 0.01 μM, lanes 3–7) while the concentration of substrate DNA was kept constant. In all reactions, the primer/template DNA concentration was 1 μM, and the reactions were incubated for 20 min at 37°C. Lane 3 also serves as a comparison for lanes 1 and 2, showing the activity of Dbh in the absence of heparin. Lengths of markers (M) in nucleotides are indicated alongside (B) and (D) Molecular Cell 2001 8, 427-437DOI: (10.1016/S1097-2765(01)00310-0)