Sulforaphane induces DNA single strand breaks in cultured 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.

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DNA Damage and Repair in Rodent and Human Cells after Exposure to JANUS Fission Spectrum Neutrons: A Minor Fraction of Single-strand Breaks as Revealed by Alkaline Elution is Refractory to Repair

International Journal of Radiation Biology ◽

10.1080/09553008914550811 ◽

1989 ◽

Vol 55 (5) ◽

pp. 761-772 ◽

Cited By ~ 9

Author(s):

M.J. Peak ◽

J.G. Peak ◽

B.A. Carnes ◽

C.M. Chang Liu ◽

C.K. Hill

Keyword(s):

Dna Damage ◽

Human Cells ◽

Single Strand ◽

Alkaline Elution ◽

Dna Damage And Repair ◽

Strand Breaks ◽

Minor Fraction ◽

Fission Spectrum ◽

Single Strand Breaks ◽

A Minor

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Manipulating chromosome structure and metaphase status with ultraviolet light and repair synthesis inhibitors

Journal of Cell Science ◽

10.1242/jcs.73.1.159 ◽

1985 ◽

Vol 73 (1) ◽

pp. 159-186

Author(s):

A.M. Mullinger ◽

R.T. Johnson

Keyword(s):

Simian Virus 40 ◽

Human Cells ◽

Single Strand ◽

Dna Breaks ◽

Intact Cells ◽

Repair Synthesis ◽

Single Strand Break ◽

Strand Breaks ◽

Single Strand Breaks ◽

Chromosome Decondensation

DNA repair occurs in metaphase-arrested cells in response to ultraviolet irradiation. In the presence of the repair synthesis inhibitors hydroxyurea and 1-beta-D-arabinofuranosylcytosine the chromosomes of such cells, as seen in Carnoy-fixed preparations, are decondensed. The extent of decondensation is related to both the u.v. dose and the duration of incubation in the presence of inhibitors. For any particular cell type there is a reasonable correlation between the amount of decondensation and the number of single-strand DNA breaks generated by the repair process under the same inhibitory conditions, though the chromosome changes continue after the number of single-strand breaks has reached a plateau. The dose response of chromosome decondensation varies between different cell types but is in general correlated with differences in levels of single-strand breaks accumulated under comparable inhibitory conditions. Decondensation can be detected after 0.5 Jm-2 in repair-competent human cells. In human cells defective in excision repair there is much less chromosome decondensation in response to the same u.v. dose and time of repair inhibition. However, a simian virus 40-transformed muntjac cell displays pronounced chromosome decondensation but has limited incision ability. Both chromosome decondensation and single-strand break accumulation in the presence of inhibitors are reversed when DNA precursors are provided, but reversal after higher u.v. doses and longer periods of incubation leads to recondensed chromosomes that are fragmented. Elution of the DNA from such cells through polycarbonate filters under non-denaturing conditions reveals that double-strand DNA breaks are generated during the period of incubation with inhibitors. Although the chromosomes of repair-inhibited metaphase cells are decondensed in fixed preparations, their morphology appears normal in intact cells. The cells also retain a capacity to induce prematurely condensed chromosomes (PCC) when fused with interphase cells: compared with control mitotic cells, the speed of induction is sometimes reduced but the final amount of PCC produced is similar.

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