PDBsum entry 3bq1

Transferase/DNA

PDB id: 3bq1

Contents

Protein chain

344 a.a.

DNA/RNA

Ligands

DG3

Metals

_CA

Waters ×41

References listed in PDB file

Key reference

Title

Structural insights into the generation of single-Base deletions by the y family DNA polymerase dbh.

Authors

R.C.Wilson, J.D.Pata.

Ref.

Mol Cell, 2008, 29, 767-779. [DOI no: 10.1016/j.molcel.2008.01.014]

PubMed id

18374650

Abstract

Dbh is a Y family translesion DNA polymerase that accurately bypasses some damaged forms of deoxyguanosine, but also generates single-base deletion errors at frequencies of up to 50%, in specific hot spot sequences. We describe preinsertion binary, insertion ternary, and postinsertion binary crystal structures of Dbh synthesizing DNA after making a single-base deletion. The skipped template base adopts an extrahelical conformation stabilized by interactions with the C-terminal domain of the enzyme. DNA translocation and positioning of the next templating base at the active site, with space opposite to accommodate incoming nucleotide, occur independently of nucleotide binding, incorporation, and pyrophosphate release. We also show that Dbh creates single-base deletions more rapidly when the skipped base is located two or three bases upstream of the nascent base pair than when it is directly adjacent to the templating base, indicating that Dbh predominantly creates single-base deletions by template slippage rather than by dNTP-stabilized misalignment.

Figure 1.
Figure 1. Crystal Structure of Dbh Extending DNA Synthesis beyond a Single-Base Deletion in a Repetitive Sequence
(A) Ternary complex structure. The polymerase and primer-template DNA are shown in ribbons representation, the incoming dNTP is shown as a ball-and-stick model, and the Ca^2+ ion (bound in the metal B position) is shown as a sphere (green). Residues Arg333 and Tyr249 that interact with the bulged template cytosine at position −3 are shown in ball-and-stick representation. Coloring is as follows: palm (magenta), thumb (green), fingers (blue), C-terminal domain (orange), linker between polymerase and C-terminal domains (pale yellow); and DNA (white). The template strand single-base deletion hot spot sequence (5′-GCCC-3′) is labeled and highlighted in red.
(B) Protein-DNA interactions. Hydrogen bonds and van der Waals contacts are indicated by dotted lines; residues that contact the DNA with side-chain atoms are labeled in boldface; other residues that contact main-chain atoms are labeled in plain type. Residues are colored according to domain, as in (A). Nucleotides are numbered relative to the templating base (position 0). The complete sequence of the DNA oligonucleotides used is shown; the template-strand hot spot sequence is highlighted in red. Unpaired nucleotides at the 5′ and 3′ ends of the template strand (positions +2 and −12) were not clearly visible in the electron density maps and were thus not modeled. Residues in the flexible loop that are close enough to contact the backbone of the template in positions −1, −2, and −3 are boxed and labeled in italic font; precise contacts are not identified, because of the weak electron density in this part of the protein. The Ca^2+ ion bound at the active site in the metal B position is shown as a circle labeled B.
(C–E) The active sites and primer-template DNA sequences are shown for (C) preinsertion binary complex, (D) insertion ternary complex, and (E) postinsertion binary complex. For each structure, a final refined 2F[o] − F[c] electron density map, calculated using the same resolution limits (Table 1) as used for refining each structure and contoured at 1.2 σ (gray mesh), is shown for the DNA substrates, calcium, and Dbh residues Asp7, Asp105, Glu106, Tyr10, Tyr48, and Arg51 and is superimposed on the final refined model. The incoming nucleotide (ddGTP), DNA, and selected residues in the active site pocket are shown in stick representation; other portions of the protein are shown in ribbons representation. The 3′ primer terminus (dG) of each structure is labeled. Atoms are colored by element: oxygen (red), nitrogen (blue), phosphate (yellow), carbon (white), and calcium (green). Coordination of the Ca^2+ ion in the ternary complex is shown with dotted lines (dark gray).

Figure 3.
Figure 3. Orientation of the C-Terminal Domains of Dbh and Dpo4 and Their Interactions with DNA
(A) Comparison of the structures of apo-Dbh (left; PDB code 1K1Q, chain A [Silvian et al., 2001]), Dbh ternary complex (middle), and Dpo4 ternary complex (right; Ab-2A, PDB code 1S0O, chain B [Ling et al., 2004]). Arrows indicate direction and magnitude of movement needed to move the C-terminal domain of one structure to the orientation found in the Dbh ternary complex structure. Rotation axes are shown as black lines. Structures were aligned based on the polymerase domains; Dbh (apo) and Dbh (ternary), rmsd 1.2 Å, 232 Cα atoms; Dbh(ternary) and Dpo4 (Ab-2A ternary), rmsd 1.7 Å, 240 Cα atoms.
(B) Residues of the C-terminal domain of Dbh that contact the DNA. Atoms in the C-terminal domain of Dbh that are located within 3.8 Å of the DNA are highlighted in white.
(C) Comparison of DNA binding by the C-terminal domains in the ternary complexes of the Dbh (top) and Dpo4 Ab-2a (bottom). The bulged base in Dbh (C-3) and the bulged ribose in Dpo4 (Ab-2A) are highlighted in red. The structures are oriented identically, by superimposition of the polymerase domains as in (A), and are viewed from the same direction. For clarity, only the DNA and C-terminal domains are shown. Structures are colored as in Figure 1.

The above figures are reprinted by permission from Cell Press: Mol Cell (2008, 29, 767-779) copyright 2008.