The dynamics of Ku70/80 and DNA-PKcs at DSBs induced by ionizing radiation is dependent on the complexity of damage

Pamela Reynolds,Jane V Harper,Anthony W Parker,Jennifer A Anderson,Peter O’Neill,Mark A Hill,Stanley W Botchway

doi:10.1093/nar/gks879

Abstract

DNA double-strand breaks (DSBs) are biologically one of the most important cellular lesions and possess varying degrees of chemical complexity. The notion that the repairability of more chemically complex DSBs is inefficient led to the concept that the extent of DSB complexity underlies the severity of the biological consequences. The repair of DSBs by non-homologous end joining (NHEJ) has been extensively studied but it remains unknown whether more complex DSBs require a different sub-set of NHEJ protein for their repair compared with simple DSBs. To address this, we have induced DSBs in fluorescently tagged mammalian cells (Ku80-EGFP, DNA-PKcs-YFP or XRCC4-GFP, key proteins in NHEJ) using ultra-soft X-rays (USX) or multi-photon near infrared (NIR) laser irradiation. We have shown in real-time that simple DSBs, induced by USX or NIR microbeam irradiation, are repaired rapidly involving Ku70/80 and XRCC4/Ligase IV/XLF. In contrast, DSBs with greater chemical complexity are repaired slowly involving not only Ku70/80 and XRCC4/Ligase IV/XLF but also DNA-PKcs. Ataxia telangiectasia-mutated inhibition only retards repair of the more chemically complex DSBs which require DNA-PKcs. In summary, the repair of DSBs by NHEJ is highly regulated with pathway choice and kinetics of repair dependent on the chemical complexity of the DSB.

Highlights

DNA double-strand breaks (DSBs) are biologically one of the most important lesions and may be induced endogenously by reactive oxygen species or exogenously through ionizing radiation and various DNA damaging chemicals
The notion that the repairability of more chemically complex DSBs is less efficient, has led to the concept that the extent of DSB complexity underlies the severity of the biological consequences [1,2,3,9,19]
Differences in the fraction of DSBs of varying chemical complexity induced by ultra-soft X-rays (USX) [7] or near infrared (NIR) microbeam radiation [45] used here confirmed our findings from real-time recruitment and loss of non-homologous end joining (NHEJ) proteins to DSBs

Summary

Introduction

DNA double-strand breaks (DSBs) are biologically one of the most important lesions and may be induced endogenously by reactive oxygen species or exogenously through ionizing radiation and various DNA damaging chemicals. Insights into the structure and chemical complexity of DSBs [12,13,14,15] were first revealed from analysis of the chemical composition of radioactive-iodine-induced DSB ends, which are complex [14] Many of these DSBs possess single-stranded overhangs of variable length and a high frequency of oxidized base modifications and abasic sites directly upstream of the DSB ends. This chemical and structural complexity of DSBs is in addition to the generally formed 30 blocking ends of DSBs, e.g. 30-phosphate or 30-phosphoglycolate moieties [12,14,16,17,18]

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