Novel Biological Approaches for Testing the Contributions of Single DSBs and DSB Clusters to the Biological Effects of High LET Radiation

Abstract

The adverse biological effects of ionizing radiation (IR) are commonly attributed to the generation of DNA double-strand breaks (DSBs). IR-induced DSBs are generated by clusters of ionizations, bear damaged terminal nucleotides, and frequently comprise base damages and single-strand breaks in the vicinity generating a unique DNA damage-clustering effect that increases DSB “complexity.” The number of ionizations in clusters of different radiation modalities increases with increasing linear energy transfer (LET), and is thought to determine the long-known LET-dependence of the relative biological effectiveness (RBE). Multiple ionizations may also lead to the formation of DSB clusters, comprising two or more DSBs that destabilize chromatin further and compromise overall processing. DSB complexity and DSB-cluster formation are increasingly considered in the development of mathematical models of radiation action, which are then “tested” by fitting available experimental data. Despite a plethora of such mathematical models the ultimate goal, i.e., the “a priori” prediction of the radiation effect, has not yet been achieved. The difficulty partly arises from unsurmountable difficulties in testing the fundamental assumptions of such mathematical models in defined biological model systems capable of providing conclusive answers. Recently, revolutionary advances in methods allowing the generation of enzymatic DSBs at random or in well-defined locations in the genome, generate unique testing opportunities for several key assumptions frequently fed into mathematical modeling – including the role of DSB clusters in the overall effect. Here, we review the problematic of DSB-cluster formation in radiation action and present novel biological technologies that promise to revolutionize the way we address the biological consequences of such lesions. We describe new ways of exploiting the I-SceI endonuclease to generate DSB-clusters at random locations in the genome and describe the possible utility of Zn-finger nucleases and of TALENs in generating DSBs at defined genomic locations. Finally, we describe ways to harness the revolution of CRISPR/Cas9 technology to advance our understanding of the biological effects of DSBs. Collectively, these approaches promise to improve the focus of mathematical modeling of radiation action by providing testing opportunities for key assumptions on the underlying biology. They are also likely to further strengthen interactions between experimental radiation biologists and mathematical modelers.

Measurement of complex DNA damage induction and repair in human cellular systems after exposure to ionizing radiations of varying linear energy transfer (LET)

“…It also accepted that $30% of DSBs contain additional lesions following exposure to low-energy electrons but this fraction can increase up to 70% at the same dose of a-particles [32], therefore increasing the damage complexity and DSB clustering [33]. Our results presented in Figure 2 for DSB induction and the response of a variety of mammalian cells to different IRs and doses show an expected almost linear dependence of DSB with dose for low-LET and high-LET.…”

Triterpenoid CDDO-Me induces ROS generation and up-regulates cellular levels of antioxidative enzymes without induction of DSBs in human peripheral blood mononuclear cells

“…Commonly, biological effects of ionizing radiation are assigned to the generation of various DNA damages such as single-strand breaks (SSB), inter-DNA crosslinks, base lesions or DSB, with the latter representing the most momentous effect due to the initiation of error-prone DNA damage repair (DDR) mechanisms (Goodhead et al 1993 ; Mladenova et al 2016 ; Lassmann et al 2010 ). Any form of IR-induced DNA damage can principally be generated either by direct ionization of DNA molecules or by indirect action with other atoms or molecules via generation of highly reactive molecules (ROS, RNS).…”

In Situ Detection of Complex DNA Damage Using Microscopy: A Rough Road Ahead

“…Khan et al [94] introduced endogenously expressed tags for advanced Single Molecule Localization Microscopy (such as halotags), suggesting that the CRISPR mediated labeling (CRISPR-PALM) could have quantitative benefits. In another work, this technology has been used to study the induction and repair of DNA lesions occurring in specified genome sites, by targeting locations of the Cas9 nuclease [95].…”