Volume 7, Issue 1, Pages 43-54 (January 2001) Crystal Structure and Deletion Analysis Show that the Accessory Subunit of Mammalian DNA Polymerase γ, PolγB, Functions as a Homodimer  José A. Carrodeguas, Karsten Theis, Daniel F. Bogenhagen, Caroline Kisker  Molecular Cell  Volume 7, Issue 1, Pages 43-54 (January 2001) DOI: 10.1016/S1097-2765(01)00153-8

Figure 1 Structure of PolγB (A) Secondary structure of PolγB colored by domains, shown together with a sequence alignment of mouse, human, and Xenopus PolγB (accession numbers AF177202, AF177201, and AF124606) and segments of the T. thermophilus glycyl-tRNA synthetase (Logan et al. 1995) that superimpose with the PolγB structure. The sequence identity between ttGRS and human PolγB is 20.2%. Cylinders refer to α helices and arrows to β strands; yellow, green, and red correspond to domains 1, 2, and 3. β strands are labeled with numbers, α helices with letters, and 310 helices are indicated. (B) Overall structure of the PolγB dimer, with one subunit colored by domains as in (A) and the other in gray. Because the crystallographic model is more complete for monomers C and D than A and B, all figures and calculations are based on the C/D dimer. The C-terminal residues present in the recombinant protein are omitted because they do not occur in the native protein and do not change PolγB's properties detectably. Figures 1B, 2, 3B, 3C were prepared with Molscript and Raster3D (Kraulis 1991; Merritt and Murphy 1994). Molecular Cell 2001 7, 43-54DOI: (10.1016/S1097-2765(01)00153-8)

Figure 2 Structural Relationship to Type IIa Aminoacyl-tRNA Synthetases (A) Superposition of PolγB and ttGRS monomers. PolγB is colored by domains as in Figure 1, and ttGRS (PDB code 1ATI) is shown in blue. (B) Superposition of domain 3 of PolγB with the C-terminal domain of threonyl-tRNA synthetase bound to its cognate tRNA (Yaremchuk et al. 2000; PDB code 1QF6). The view is from the top of PolγB (oriented as in Figure 1B) along the helical axis of the tRNA anticodon stem loop. The synthetase is shown as a ribbon in cyan, and the sugar phosphate backbone of the tRNA as a thick blue line with the anticodon nucleotides as thin blue lines. Domain 3 of PolγB is shown in red with labeled helices J, K, and M; domain 2 of the other monomer is shown in gray, with labeled helices F and H. (C) Adenylation active site of ttGRS (PDB code 1B76) with bound ATP (right) and the corresponding pocket in domain 2 of PolγB (left). Critical residues for ATP binding in ttGRS and the corresponding residues in PolγB are shown in all-bonds representation. Molecular Cell 2001 7, 43-54DOI: (10.1016/S1097-2765(01)00153-8)

Figure 3 Interface of the PolγB Homodimer (A) Dimer interface of PolγB. Left, oriented as in Figure 1B; right, rotated by 120° around the y axis to show the interface in domain 2. Red, atoms that become solvent inaccessible upon dimer formation; yellow, atoms whose accessibility decreases upon dimer formation; blue, waters that are in van der Waals distance to both monomers in the dimer; green, metal bound to main chain carbonyls at the dimer interface. Figure 3A and Figure 5 were prepared with Grasp (Nicholls et al. 1991). (B) 2Fo − Fc electron density map of the metal binding site. The octahedral coordination sphere of the metal is indicated with dotted lines. (C) Comparison of the dimerization domain of CheA (blue and red subunits, PDB code 1B3Q) with domain 2 of PolγB (green and gray monomers). Helices are shown as cylinders and strands in ball-and-stick with hydrogen bonds as dotted lines. N and C termini are indicated. Molecular Cell 2001 7, 43-54DOI: (10.1016/S1097-2765(01)00153-8)

Figure 5 Surface Features of PolγB (A) Conserved residues in PolγB from human, mouse, and Xenopus laevis. Residues of mouse PolγB strictly conserved in human and at least type-conserved in Xenopus are colored green (noncarbon atoms) or yellow (carbon atoms). Residues of mouse PolγB deleted in Xenopus PolγB are colored in magenta. (B) Surface potential of the PolγB dimer calculated at neutral pH, ionic strength 100 mM, contoured at ± 10 kT (blue, positive; red, negative). The surface potential of human PolγB (homology model, not shown) is similar. Top, front view as in Figure 1B; bottom, view of the C-terminal regions of PolγB. The arrows indicate the pocket in domain 1. Molecular Cell 2001 7, 43-54DOI: (10.1016/S1097-2765(01)00153-8)

Figure 4 Deletion Analysis of PolγB (A) Location of characterized deletions (N1, C1, I2, and I3 [Carrodeguas and Bogenhagen 2000], I4-I7 [this work]) in the structure of PolγB. Deletions are shown in black with overlapping regions in gray. The residues deleted in I5 (and also in I2) are disordered in the crystal and are shown in an arbitrary conformation as a dashed line. (B) Sedimentation analysis of PolγB. Four hundred nanograms of each wild-type recombinant PolγB (B, 53.6 kDa) and the I4 mutant (50 kDa) were separated by sedimentation in a 10%–30% glycerol gradient together with protein standards. Fractions (3–16 shown) were collected from the bottom of the gradient and analyzed by Western blotting. The sedimentation positions of protein standards are indicated according to their sedimentation coefficients. (C) Electrophoretic mobility shift assay of protein–DNA complexes. Left, complexes formed between PolγA alone or together with wild-type PolγB or PolγB derivatives. Right, relative mobilities of several protein–DNA complexes. T4 DNA polymerase (103.6 kDa monomer with a calculated net charge similar to that of PolγA (−8 versus −11)) is included as a mobility marker. A, PolγA; B, wild-type PolγB; I3–I7, deletion mutants; C, PolγB-CBD fusion protein; T4, T4 DNA polymerase. Arrows on the right point to supershifted complexes containing PolγB monomers (1) or homodimers (2). Reactions were carried out in 10 μl aliquots containing 4 nM of a 26-mer:45-mer primer:template DNA, 4 nM PolγA and wild-type PolγB, 8 nM T4 DNA polymerase, 8 nM PolγB deletion mutants and 40 nM mutant I3, respectively. Note that equimolar amounts of PolγA and wild-type PolγB in the left panel are able to supershift only part of the PolγA:DNA complex. A 2-fold excess of wild-type PolγB over PolγA (not shown) supershifts the complex in a way similar to mutants I5–I7. (D) Stimulation of PolγA activity by PolγB derivatives on poly(dA):oligo(dT). Labels are as in (C). Molecular Cell 2001 7, 43-54DOI: (10.1016/S1097-2765(01)00153-8)

Figure 6 Binding of PolγB to ssDNA Studied by Gel Electrophoresis Wild-type PolγB and several deletion mutants were tested for their ability to bind to a 129 nt ssDNA fragment derived from the human mtDNA light strand origin of replication (OL). The presence of two complexes with different mobilities might be due to multiple binding sites on the 129-mer or to complexes of fixed stoichiometry with distinct conformations. Reactions were carried out in 10 μl containing 0.5 nM DNA and 4, 12, or 40 nM protein (triangles). Molecular Cell 2001 7, 43-54DOI: (10.1016/S1097-2765(01)00153-8)