Central role for the XRCC1 BRCT I domain in mammalian DNA single-strand break repair

Abstract

The DNA single-strand break repair (SSBR) protein XRCC1 is required for genetic stability and for embryonic viability. XRCC1 possesses two BRCA1 carboxyl-terminal (BRCT) protein interaction domains, denoted BRCT I and II. BRCT II is required for SSBR during G(1) but is dispensable for this process during S/G(2) and consequently for cell survival following DNA alkylation. Little is known about BRCT I, but this domain has attracted considerable interest because it is the site of a genetic polymorphism that epidemiological studies have associated with altered cancer risk. We report that the BRCT I domain comprises the evolutionarily conserved core of XRCC1 and that this domain is required for efficient SSBR during both G(1) and S/G(2) cell cycle phases and for cell survival following treatment with methyl methanesulfonate. However, the naturally occurring human polymorphism in BRCT I supported XRCC1-dependent SSBR and cell survival after DNA alkylation equally well. We conclude that while the BRCT I domain is critical for XRCC1 to maintain genetic integrity and cell survival, the polymorphism does not impact significantly on this function and therefore is unlikely to impact significantly on susceptibility to cancer.

FIG. 1.

(A) XRCC1 protein-protein interaction domains. The location of the NTD and the two BRCT domains within XRCC1 are indicated. The location of mutations in BRCT II previously employed (36, 46, 47) to disrupt activity of this domain, and the analogous BRCT I mutations employed in this study, are shown. Also shown is the location of the common human genetic polymorphism in BRCT I, R399Q. (B) CD alignment (NCBI) of the pfam00533 consensus BRCT domain with BRCT domains present in XRCC1, Lig3α, and BRCA1. Boxed residues denote regions of hydrophobicity (with hydrophobic residues in boldface) that are conserved in the BRCT family, with those in dark grey denoting those that are most conserved. The position of secondary structure, based on the crystal structure of the BRCT II domain of XRCC1 (57), is shown at the top and bottom. The amino acids mutated to aspartate (D) in this study, and the position of the common human polymorphism (Q) is also indicated.

FIG. 2.

Characterization of EM9 cells expressing wild-type or mutant XRCC1. (A) XRCC1 protein was examined in the indicated cell lines by indirect immunofluorescence, using the primary anti-XRCC1 MAb 33-2-5 and a fluorescein isothiocyanate-conjugated rabbit antimouse secondary antibody (DAKO). Nuclei were visualized with the DNA stain 4′,6′-diamidino-2-phenylindole. Photographs were taken under ×400 magnification. (B) XRCC1 protein levels were examined in cell extracts (20 μg of total protein) from the indicated cell lines (XH, EM9-XH; 360/361, EM9-XH^LI360/361DD; 385, EM9-XH^W385D; V, EM9-V) by immunoblotting, using the anti-XRCC1 MAb 33-2-5. (C) The cell lines indicated (see panel A for an explanation of symbols) were plated in six-well dishes (200 cells/well) and treated with the indicated concentrations of MMS for 1 h. After a wash, cells were incubated in drug-free medium for 7 to 10 days to allow formation of macroscopic colonies. The fraction of cells surviving MMS treatment was calculated by dividing the number of colonies in treated wells by the number in untreated wells. Results are the mean ± 1 standard deviation of three independent experiments.

FIG. 3.

Measurement of MMS-induced SSBs in asynchronous populations of transfected EM9 cells. (A) EM9-V cells were treated for 15 min with the indicated concentrations of MMS, and the level of SSBs was quantified by alkaline agarose gel electrophoresis (comet assay). SSB levels are expressed as the tail moment, an arbitrary unit reflecting the product of the amount of DNA present in the “comet” tail and the tail length, after electrophoresis. (B) SSBs were quantified in the indicated EM9 transfectants before treatment for 15 min with 0.3 mg of MMS/ml (U, untreated), immediately after MMS treatment (T, treated), or after a subsequent repair incubation in drug-free medium for 20 min (R₂₀) or 3 h (R₁₈₀). Results are the average from four independent experiments (±1 standard deviation). The asterisk denotes the absence of a data point for a 20-min repair incubation in EM9-XH^LI360/361DD cells.

FIG. 4.

Measurement of MMS-induced SSBs in synchronized populations of EM9 transfectants. (A) DNA content of EM9-V cells synchronized in G₁ (top panel) or S/G₂ (bottom panel) by serum starvation and mimosine as described previously (36, 39, 46). Similar synchrony was observed with other EM9 transfectants (data not shown). The respective arrows denote peak positions of cells with G₁ and G₂ DNA content. (B) SSBs were quantified in the indicated EM9 transfectants in G₁ (top panel) or S/G₂ (bottom panel) before treatment for 15 min with 0.3 mg of MMS/ml (U, untreated), immediately after MMS treatment (T, treated), or after a subsequent repair incubation in drug-free medium for 3 h (R₁₈₀). Results are the mean (±1 standard deviation) from three independent experiments.

FIG. 5.

Presence of Lig3 and Polβ in affinity-purified XRCC1 protein complexes. Total cellular protein (20 μg) from the cell lines indicated on the left was subjected to metal chelate affinity chromatography (nickel agarose; Qiagen) to purify histidine-tagged XRCC1 protein complexes. Aliquots of the recovered complexes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with anti-XRCC1 MAb (33-2-5), anti-Lig3 Pab (TL-25), or anti-Polβ MAb (Clone 18S; Lab Vision).

FIG. 6.

MMS sensitivity and SSBR capacity of EM9 cells harboring the human genetic polymorphism XRCC1^R399Q. (A) The indicated cell lines (pooled populations of >100 independent transfectants) were plated in six-well dishes (200 cells/plate) and treated with the indicated concentration of MMS for 1 h. After a wash, cells were incubated in drug-free medium for 7 to 10 days to allow formation of macroscopic colonies, and the fraction of surviving cells was calculated as described in the legend for Fig. 2. (B) Total cellular protein (20 μg) from the indicated cell lines was fractionated by SDS-PAGE and immunoblotted with the anti-XRCC1 MAb, 33-2-5. (C) SSBs (expressed as tail moments) were quantified in the indicated EM9 transfectants before (−MMS) or immediately after (+MMS) treatment with 0.3 mg of MMS/ml. (D) The percentage of the MMS-induced SSBs shown in panel C remaining after a 3-h repair incubation in drug-free medium (calculated from the tail moment present after the repair incubation). Symbols are as in panel C. Results are the mean ± 1 standard deviation for three independent experiments.

FIG. 7.

Evolutionary conservation of XRCC1 protein domains. Human (Hu), D. melanogaster (Dm), and A. thaliana (At) XRCC1 homologues were aligned using MACAW software, with vertical bars denoting conserved residues and enlarged boxes denoting regions of extensive conservation. The dotted box denotes the position of the BRCT II domain present in mammalian XRCC1 (represented here by the human protein). The domains identified by this alignment are the NTD, which contains the binding site for Polβ, and the two BRCT domains, which contain the binding sites for PARP-1, poly(ADP-ribose), and Lig3α. The Hu and Dm BRCT I domains, and the Hu and At BRCT I domains, share 54.5 and 52.8% identity, respectively.