Deciphering repair pathways of clustered DNA damage in human TK6 cells: insights from atomic force microscopy direct visualization

Abstract

Ionizing radiation induces various types of DNA damage, and the reparability and lethal effects of DNA damage differ depending on its spatial density. Elucidating the structure of radiation-induced clustered DNA damage and its repair processes will enhance our understanding of the lethal impact of ionizing radiation and advance progress toward precise therapeutics. Previously, we developed a method to directly visualize DNA damage using atomic force microscopy (AFM) and classified clustered DNA damage into simple base damage clusters (BDCs), complex BDCs and complex double-strand breaks (DSBs). This study investigated the repair of each type of damage in DNA-repair-deficient human TK6 cells and elucidated the association between each type of clustered DNA damage and the pathway responsible for its repair postirradiation with low linear energy transfer (LET) radiation (X-rays) and high-LET radiation (Fe-ion beams) in cells. We found that base excision repair and, surprisingly, nucleotide excision repair restored simple and complex BDCs. In addition, the number of complex DSBs in wild-type cells increases 1 h postirradiation, which was most likely caused by BDC cleavage initiated with DNA glycosylases. Furthermore, complex DSBs, which are likely associated with lethality, are repaired by homologous recombination with little contribution from nonhomologous-end joining.