Spatial and temporal cellular responses to single-strand breaks in human cells

Abstract

DNA single-strand breaks (SSB) are one of the most frequent DNA lesions produced by reactive oxygen species and during DNA metabolism, but the analysis of cellular responses to SSB remains difficult due to the lack of an experimental method to produce SSB alone in cells. By using human cells expressing a foreign UV damage endonuclease (UVDE) and irradiating the cells with UV through tiny pores in membrane filters, we created SSB in restricted areas in the nucleus by the immediate action of UVDE on UV-induced DNA lesions. Cellular responses to the SSB were characterized by using antibodies and fluorescence microscopy. Upon UV irradiation, poly(ADP-ribose) synthesis occurred immediately in the irradiated area. Simultaneously, but dependent on poly(ADP-ribosyl)ation, XRCC1 was translocated from throughout the nucleus, including nucleoli, to the SSB. The BRCT1 domain of XRCC1 protein was indispensable for its poly(ADP-ribose)-dependent recruitment to the SSB. Proliferating cell nuclear antigen and the p150 subunit of chromatin assembly factor 1 also accumulated at the SSB in a detergent-resistant form, which was significantly reduced by inhibition of poly(ADP-ribose) synthesis. Our results show the importance of poly(ADP-ribosyl)ation in sequential cellular responses to SSB.

FIG. 1.

Fluorescent micrographs of XPA-UVDE and XPA-Vector cells, doubly immunolabeled for PAR and XRCC1. The columns from a to e show XPA-UVDE cells, and f shows XPA-Vector cells, before irradiation (a), 2 min after local UV irradiation with 20 J/m² (b and f), and 10 and 30 min after local UV irradiation (c and d, respectively). Cells were fixed and costained with anti-PAR antibody (upper row in green) and anti-XRCC1 antibody (middle row in red). Colocalization of PAR and XRCC1 is shown (bottom row in yellow). Column e is DIQ-treated XPA-UVDE cells fixed 2 min after local UV irradiation. Bar, 10 μm.

FIG. 2.

Nuclear distribution of XRCC1 and PARP-1 before and after local UV irradiation. (A) Double immunolabeling for nucleolin and XRCC1 in XPA-UVDE cells, XPA-Vector cells, and HeLa cells. Panels in column a are unirradiated XPA-UVDE cells, b and c are XPA-UVDE and XPA-Vector cells fixed at 2 min after local UV irradiation (20 J/m²), respectively, and panels in column d are unirradiated HeLa cells. Panels in the upper and middle rows correspond to XRCC1 (red) and nucleolin (green), respectively. Panels in the bottom row represent an overlay of the panels from the upper and middle rows. Colocalization of the two proteins appears yellow. (B) Double immunolabeling of XPA-UVDE cells for XRCC1 (red in the upper-row panels) and PARP-1 (green in middle-row panels); panels in the bottom row are the corresponding overlay; colocalization of the proteins appears yellow. Panels in column e are unirradiated cells, and those in f are cells at 2 min after local UV irradiation (20 J/m²). Bar, 10 μm.

FIG. 3.

In situ determination of XRCC1 domains necessary for its recruitment to SSB. (A) Double immunolabeling of PAR and Flag-tagged full-length XRCC1 and XRCC1 fragment after local UV irradiation (20 J/m²). Panels in the columns from a to c are f cells transfected with full-length XRCC1 tagged with Flag, and those from d to f are the cells transfected with the Flag-tagged XRCC1 fragment which comprises amino acids 242 to 403 of XRCC1. Columns a and d are unirradiated cells; b and e are cells fixed 2 min after local UV irradiation (20 J/m²); c and f are DIQ-treated cells fixed at 2 min after local UV irradiation (20 J/m²). Not all the cells express the gene. Cells were costained with anti-PAR (panels in the upper row, green) and anti-Flag epitope (panels in the middle row, red). The corresponding fluorescent images were superimposed onto the Nomarski images and are shown in the bottom row. Colocalization appears yellow. Bar, 10 μm. (B) Schematic representation of the ability to be recruited to SSB for deletion and site-specific mutations of XRCC1 polypeptides, all tagged with Flag at the NH₂ terminus. Thick lines below the schematic drawing of XRCC1 protein represent the deleted fragments; the corresponding numbers at the right-hand side are the amino acids at both ends of the polypeptides. Two mutants having amino acid replacement mutations are also shown here. The results are given at the extreme right-hand side. The reported binding proteins of XRCC1 are shown uppermost. The N-terminal domain (NTD) contains the binding site for OL β, and the BRCT domains contain the binding site for PARP-1, PARP-2, and ligase IIIα. NLS, bipartite nuclear localization signal.

FIG. 4.

In situ visualization PCNA and CAF-1 p150 at SSB. (A) Double immunolabeling for PCNA and CAF-1 p150 in asynchronous cells. Panels in column a are unirradiated XPA-UVDE cells; those in c are DIQ-treated XPA-UVDE cells fixed at 2 min after local UV irradiation (20 J/m²). Those in column b are XPA-UVDE cells fixed at 2 min after local UV irradiation (20 J/m²); those in d are XPA-Vector cells fixed at 2 min after local UV irradiation (20 J/m²). Panels in the uppermost row are cells immunolabeled for PCNA (red), whereas those in the second row are cells immunolabeled for CAF-1 p150 (green); both proteins were in the detergent-resistant form, as described in Results. Colocalization of the proteins appears yellow, as seen in the third row for the overlay of the corresponding panels. The nuclei were stained with DAPI and are shown in the bottom row. Bar, 10 μm. (B) Double immunolabeling for PCNA and CAF-1 p150 in G₁-phase cells. XPA-UVDE (column e) and XPA-Vector (column f) cells were treated with mimosine as described in Materials and Methods. Cells were exposed to local UV irradiation (20 J/m²) and incubated for 2 min, then doubly immunolabeled for PCNA (uppermost row, red) and for CAF-1 p150 (second row, green); both proteins were in the detergent-resistant form as described in Results. Colocalization of the proteins appears yellow, as seen in the third row for the overlay of the corresponding panels. The nuclei were stained with DAPI (bottom row). Bar, 10 μm.