Oxidation state of the XRCC1 N-terminal domain regulates DNA polymerase beta binding affinity

Abstract

Formation of a complex between the XRCC1 N-terminal domain (NTD) and DNA polymerase beta (Pol beta) is central to base excision repair of damaged DNA. Two crystal forms of XRCC1-NTD complexed with Pol beta have been solved, revealing that the XRCC1-NTD is able to adopt a redox-dependent alternate fold, characterized by a disulfide bond, and substantial variations of secondary structure, folding topology, and electrostatic surface. Although most of these structural changes occur distal to the interface, the oxidized XRCC1-NTD forms additional interactions with Pol beta, enhancing affinity by an order of magnitude. Transient disulfide bond formation is increasingly recognized as an important molecular regulatory mechanism. The results presented here suggest a paradigm in DNA repair in which the redox state of a scaffolding protein plays an active role in organizing the repair complex.

Fig. 1.

Structure of the XRCC1-NTD complex with Pol β catalytic domain. (A) Structure of reduced XRCC1-NTD (magenta ribbon representation with yellow β-strands), bound to Pol β CD (cyan ribbon representation with yellow β-strands). (B) The structure shown in A superimposed with the SAXS-derived model of the complex (surface representation).

Fig. 2.

Reduced and oxidized XRCC1-NTD. (A) (Left) XRCC1-NTD from the reduced complex shown as a ribbon diagram with yellow β-strands; (Center) topology diagram (10); (Right) surface representation color-coded by electrostatic potential (blue positive, red negative), relative to the left panel, the view has been rotated +90° about a vertical axis. (B) (Left) XRCC1-NTD from oxidized complex shown as a magenta ribbon with yellow β-strands; (Center) topology diagram; (Right) surface representation colored by electrostatic potential (blue positive, red negative), relative to the left panel, the view has been rotated +90° about a vertical axis.

Fig. 3.

Protein interface region showing structural differences between the reduced and oxidized complex of XRCC1-NTD with Pol β. In the reduced/oxidized complex, XRCC1-NTD is shown in brown/magenta and Pol β CD in gray/cyan. Water molecules are shown as red spheres and hydrogen bonds are represented by black dashed lines. Several of the important residue interactions are illustrated for the reduced complex in yellow ball and stick representations, and for the oxidized complex in gray ball and stick representations.

Fig. 4.

Close-up views of the XRCC1-NTD/Pol β PT interface. (A and C) oxidized complex, (B and D) reduced complex; hydrogen bonds less than 3.4 Å are indicated with black dashed lines; hydrogen bonds greater than 3.4 Å are indicated with red dashed lines, and water molecules are shown as red spheres. XRCC1-NTD shown in magenta tones with Pol β CD shown as a cyan ribbon.

Fig. 5.

Overlay of the reduced XRCC1-NTD-Pol β complex with the gapped DNA-Pol β complex. The structures of the two complexes were aligned based on the catalytic domain of Pol β, and the overlapping Pol β catalytic domain from the complex with XRCC1-NTD has been omitted for clarity. The XRCC1-NTD is shown in magenta; in the gapped DNA-Pol β complex (PDB code 1BPX), the DNA is shown as an orange ribbon, and the domains of Pol β, shown in various shades of blue, are indicated. As is apparent from the figure, there are no direct contacts between the XRCC1-NTD and the gapped DNA.