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.DNA damage is a continual threat to genetic stability, as demonstrated by the cancer-prone phenotype of human diseases in which DNA repair is defective. Among various DNA lesions, single-strand breaks (SSB) are one of the most frequent DNA lesions produced by endogenous reactive oxygen species or generated by ionizing radiation or through base hydrolysis (37). SSB are also intermediate products in various aspects of DNA metabolism, including DNA repair, replication, and recombination. During base excision repair, SSB are produced by DNA glycosylases and apurinic/apyrimidinic (AP) endonucleases at the site of base damage. If SSB are not properly repaired, they may result in double-strand breaks in replicating DNA and may also affect transcription (7,19). In spite of such harmful effects, however, our knowledge of the cellular responses to and repair of SSB is still incomplete. One reason for this is that no experimental procedure that induces solely SSB in cells has been developed.In higher eukaryotes, it is known that SSB produced during base excision repair are processed by two alternative repair pathways, a DNA polymerase ␤ (Pol␤)-dependent pathway and a proliferating cell nuclear antigen (PCNA)-dependent pathway. Selection of these pathways is considered to be damage and/or dose dependent (12). Poly(ADP-ribosyl)ation, a posttranslational modification of proteins catalyzed by DNAdependent poly(ADP-ribose) polymerase (PARP), occurs very rapidly in response to SSB. PARP-1 has been proposed to play a key role in SSB repair by its rapid binding to SSB and subsequent activation (3). PARP-2 is also activated by SSB and participates in the repair of SSB (2, 31). XRCC1 is another protein that plays a central role in the repair of SSB. In biochemical st...

The RNA-binding Protein Fused in Sarcoma (FUS) Functions Downstream of Poly(ADP-ribose) Polymerase (PARP) in Response to DNA Damage

“…Both the SSB repair protein XRCC1 and chromatin remodeling factor CHD4 are recruited to DNA damage in a PARP-dependent manner (21,68); however, depletion of FUS did not disrupt targeting of either XRCC1 or CHD4 (data not shown). In addition, FUS knockdown had no effect on recruitment of histone deacetylase 2 (HDAC2), which also accumulates at sites of DNA damage (data not shown) (69).…”

Chromatin disassembly and reassembly during DNA repair

“…For example, the histone chaperone CAF-1 co-localizes with sites of NER in vivo [50,56]. Furthermore, budding yeast deleted for any of the three genes encoding the subunits of CAF-1, CAC1/RLF2, CAC2 or CAC3/MSI1, are highly sensitive to UV irradiation [39].…”

Alternative Excision Repair Pathways

“…To further characterize AER initiated by UVDE, XPA-UVDE cells were UV irradiated through 3-mm micropores on a filter; several spots of this diameter within a nuclei were UV irradiated, and SSBs were produced by UVDE only at the irradiated sites (Fig. 3) (Okano et al 2003). Proteins accumulating at SSBs created by UV irradiation and UVDE were identified by immunostaining or by real-time observation of expressed green fluorescent protein (GFP)-tagged repair proteins under the microscope.…”