The structural basis of XRCC1-mediated DNA repair

Abstract

Scaffold proteins play a central role in DNA repair by recruiting and organizing sets of enzymes required to perform multi-step repair processes. X-ray cross complementing group 1 protein (XRCC1) forms enzyme complexes optimized for single-strand break repair, but participates in other repair pathways as well. Available structural data for XRCC1 interactions is summarized and evaluated in terms of its proposed roles in DNA repair. Mutational approaches related to the abrogation of specific XRCC1 interactions are also discussed. Although substantial progress has been made in elucidating the structural basis for XRCC1 function, the molecular mechanisms of XRCC1 recruitment related to several proposed roles of the XRCC1 DNA repair complex remain undetermined.

Keywords: DNA ligase 3α; DNA polymerase beta; DNA repair; Single-strand break repair; XRCC1.

Figure 1

Domain structure of XRCC1. A) Domain structure of XRCC1 showing the positions of the N-terminal domain (NTD), reported REV1 interacting region (RIR), nuclear localization signal (NLS), first BRCT domain (X1BRCTa), phosphorylated FHA binding sequence (PFBS), and second BRCT domain (X1BRCTb). B) Domain pI values, determined from Expasy site: http://web.expasy.org/compute_pi/, and C) reported phosphorylated residues, taken from PhosphositePlus (http://www.phosphosite.org).

Figure 2

Interaction of the XRCC1 N-terminal domain (X1NTD) with pol β. A) Ribbon diagrams showing the complex of the pol β polymerase domain (subdomain color coding: DNA binding (cyan), catalytic (yellow), nucleotide binding (purple)) with X1NTD (green). B) Overlay of the complex shown in A with a pol β bound to a single nucleotide gapped DNA (gray, bound DNA with orange backbone). Figure B indicates the relative positions of X1NTD with pol β-bound gapped DNA.

Figure 3

Residues mediating the binding of pol β N-domain with X1NTD. The figure shows a ribbon diagram of the Val303 loop region of the pol β Nucleotide binding subdomain (purple) in complex with X1NTD (green). X1NTD residues Phe67, Glu69, Val86, Arg100, Arg109, and Tyr136 originally identified to mediate the interaction based on mutagenesis studies [39] are indicated in yellow. All of these residues are located at the pol β: interface.

Figure 4

Oxidation of X1NTD. A) A ribbon diagram showing the oxidized form of the X1NTD (green; the remodeled 40 residues at the N-terminus are yellow) in complex with the pol β N-subdomain (purple). The Pro2, Cys12, and Cys20 sidechains are in cyan. B) Stabilizing interactions of the N-terminal carbimate group with Arg7, Ser44, and Lys129. C) Initial formation of the disulfide bond leads to a dynamic structure that is then stabilized by non-enzymatic recruitment of CO₂ to form the carbimate adduct.

Figure 5

Structure of the complex formed between the XRCC1 RIR motif and the REV1 C-terminal domain. The ribbon diagram shows an overlay of the NMR-derived structure of the XRCC1 RIR peptide [12] with the crystal structure of the REV1 C-terminal domain in complex with Rev7/Rev3 (pdb: 4FJO, Wojtaszek et al., 2012).

Figure 6

Ribbon diagram of BRCTa. NMR-derived solution structure of BRCTa (green, pdb: 2D8M, Nagashima et al., 2006). The PAR-binding region identified by Pleschke et al., [17] is shown in blue. Residues Leu360, Ile361, Trp385, and Cys389 that have been mutated in functional studies are shown in yellow. The reported structure corresponds to BRCTa containing the common R399Q polymorphism (magenta). Additional disordered residues at the terminal positions are not included.

Figure 7

Complex of the PNKP FHA domain with the XRCC1 FHA-binding peptide. A) The ribbon diagram shows the FHA domain and sidechains for the major interacting residues (green) with the XRCC1 PFBS peptide (cyan): Y⁵¹⁵AG(pS)(pT)DEN (pdb: 2W3O, [20]. The structure also includes two Ca²⁺ ions (magenta) that interact with the phosphorylated residues. The ribbon diagram corresponding to the APTX FHA domain is also overlayed (pdb: 3KT9, [166], gray). Important XRCC1-binding residues are conserved in the APTX FHA domain, with the main difference being substitution of Lys38 for Arg44 (not shown).

Figure 8

BRCT dimer structures. A) Ribbon diagram showing the mX1BRCTb homodimer structure (pdb: 3PC6). The N-terminal loop and α-helices of the domains mediate the dimerization. B) Ribbon diagram of the L3BRCT homodimer (pdb: 3PC7). As with X1BRCTb, the interface similarly involves the N-terminal segment, and the first α-helix. C) Structure of the L3αBRCT:X1BRCTb heterodimer formed from the two BRCT domains, illustrating the preservation of the interface interaction motif (pdb: 3PC8, [38]). In each structure, the diagrams are color coded from violet (N-terminus) to red (C-terminus).

Figure 9

Structure of the L3BRCT:X1BRCTb heterodimer. A) The crystal structure contains two L3BRCT:X1BRCTb heterodimers in the unit cell, connected by an additional hydrophobic interface between the pair of X1BRCTb domains at the center (pdb: 3QVG). The X1BRCTb domain in this structure included residues starting at Glu⁵²⁸ which forms an extended segment that interacts with L3BRCT. B) Expanded view of the interaction of the extended X1BRCTb chain with L3BRCT, providing a basis for selecting the heterodimer over the structurally analogous homodimers that lack this additional interaction.

Figure 10

Structural implications of XRCC1 and pol β polymorphisms. A) The structure of the X1BRCTb :L3αBRCT heterodimer includes salt bridges linking XArg558 with LAsp876 and LAsp878. The rare R558W polymorphism (R560W in hX1BRCTb) (Table 1) is predicted to form a weaker heterodimer structure, possibly resulting in a functional impairment. B) The breast cancer-associated K289M mutation is predicted to exert a small effect on the pol β:X1NTD binding affinity, based on its role in positioning Gln324 which interacts with the Pro135 carbonyl.

Figure 11

Interaction of the XRCC1 repair complex with damaged DNA. The PARP-1 sensor is extensively autoPARylated, providing a large recruitment signal that interacts with the XRCC1 BRCTa domain or other PAR recognition motifs,. and weakly with several other proteins that bind to damaged DNA. The DNA substrate is traded among enzymes until it can be acted upon. Alternate XRCC1 binding partners or other repair factors (gray) may accumulate based on non-specific affinity for PAR, so that they are waiting in the wings if needed.