The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form

The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. oxidative damage induced by ionizing radiation, and the repair proteins involved in their removal from the DNA, it has been necessary to study how repair systems handle these lesions using synthetic DNA damage. This review focuses on the repair process and mutagenic consequences of clustered lesions in yeast and mammalian cells. By examining the studies on synthetic clustered lesions, and the effects of low vs high LET radiation on mammalian cells or tissues, it is possible to extrapolate the potential biological relevance of these clustered lesions to the killing of tumor cells by radiotherapy and chemotherapy, and to the risk of cancer in Danicopan non-tumor cells, and this will be discussed. Keywords:clustered DNA lesions, double strand breaks, DNA repair, mutation induction, ionizing radiation == 1. Introduction == DNA damage and death due to ineffective repair are the basis of many cancer treatments. The DNA lesions produced by radiotherapy and certain chemotherapies include modifications to thymine, adenine, guanine LIN28 antibody and cytosine, sites of base loss (AP sites), single strand breaks (SSBs) and double strand breaks (DSBs) [1]. These types of damages are very similar to those produced by endogenous reactive oxygen Danicopan species and chemicals such as hydrogen peroxide, yet ionizing radiation and bleomycin sulfate require fewer DNA lesions to produce a lethal event (the dose at which 63% of the cells are killed, according to a Poisson distribution) in a mammalian cell than hydrogen peroxide. For a lethal event 1000 SSBs are produced by ionizing radiation, 150 SSBs by bleomycin sulfate, but 400,000 SSBs are required following treatment with hydrogen peroxide [2]. So why are ionizing radiation and bleomycin sulfate more lethal than hydrogen peroxide? This was the question being considered by scientists in the late 1980s and early 1990s. At this time the DSB was considered the lethal lesion, with base damage only important in mutagenesis and not cell death. However, in 1988, John Ward put forward the idea of locally multiply damaged sites (LMDS) and proposed that the difference in lethality among DNA damaging agents was due to the spatial distribution of damage and the inability of the cell to repair these clusters of lesions in the DNA molecule [2,3]. The initial evidence for the existence of ionizing radiation-induced clustered DNA lesions was provided by the biophysical modelers. Although simulations of irradiated cells predicted that clusters of damage were unlikely to be produced by a direct ionization or primary radical [4], the clusters of ionizations generated from low secondary electrons from X- and -radiation tracks had the potential to produce multiple hydroxyl radicals from the water molecules closely associated with the DNA. The clusters of hydroxyl radicals were predicted to generate multiple lesions within a helical turn of the DNA from clinically relevant doses of X- and -radiation [4,5]. Bleomycin sulfate was also found to produce multiple lesions. Bleomycin reacts preferentially with double-stranded DNA and modifies the bases in the order of T > C > A > G [6;7]. One part of the molecule is thought to intercalate into the DNA, while another section is capable of binding metal ions such as iron. Reactive oxygen species formed from the reaction of the bleomycin with iron and oxygen have been implicated in the damaging mechanism [8]. Incubation of bleomycin-treated DNAin vitrowith endonuclease III or putrescine to cleave at AP sites resulted in an increased production of DSBs [9]. This demonstrated that bleomycin forms clustered lesions consisting of SSBs and AP sites. Neocarcinostatin, another radiomimetic compound, also produces clustered lesions that consist of AP sites and SSBs two base pairs apart, and the breaks have 3′ phosphate and 5′ aldehyde termini [10]. Hydrogen peroxide, however, reacts with a metal ion in a Fenton reaction to produce hydroxyl radicals and so single lesions are produced at the sites of bound metal ions randomly distributed in the DNA [2]. Hence it was postulated that single lesions were easier to repair than the clustered lesions and agents generating clustered lesions would be more lethal to a mammalian cell. Etoposide, another cancer drug treatment, Danicopan has also since been found to introduce multiple DSBs.