After double-strand break induction by UV-A, homologous recombination and nonhomologous end joining cooperate at the same DSB if both systems are available

Abstract

After induction of DNA double-strand breaks (DSB) two pair systems, the error-prone 'nonhomologous end joining' (NHEJ) and the more accurate 'homologous recombination repair' (HRR) can compete for the same individual DSB site. In the human keratinocyte cell line, HaCaT, we have tested the spatial co-localisation and the temporal sequence of events. We used UV-A (365 nm) as a damaging agent, which can be applied in clearly defined doses and can lead to rare DSBs via propagation of clustered single-strand breaks (SSBs). DNA fragmentation and repair was measured by the Comet assay and persisting DSBs were quantified by the micronucleus assay. Direct DSB detection was performed by immunohistochemical labelling of γ-H2AX, a phosphorylated histone that is assumed to form one foci per DSB. Intra- and inter-pathway interactions were quantified by co-localisation, FRET imaging and by coimmunoprecipitation (Co-IP) of XRCC4, DNA-PK and Ku70 as representatives of NHEJ, Rad51 and Rad52 for HRR and γ-H2AX, Mre11 and Rad50 as representatives of both pathways. In G2 cells, where both systems are available, the temporal sequence after irradiation is: (1) γ-H2AX (2) Mre11 (3) DNA-PK Rad51 (4) XRCC4. That is, the first two proteins involved in both pathways 'label' the damaged site and initiate repair, followed by the NHEJ, which is temporally overlapping with HRR activity. Taking all these observations together we suggest that a cell tries to repair DSBs with a combination of both HRR and NHEJ, if available.