Deep space exploration and long-term human space missions are stalled and greatly restricted by hazards, including microgravity and cosmic radiation. Cosmic radiation is among the highest-priority threats to the health of astronauts ^[1][2][3][1,2,3]. Diverse ionizing radiations existing in the complex space environment, especially those of high linear energy transfer (LET), cause cataracts [4], promote Alzheimer’s disease [5], and affect cardiac physiology [6]. Crucially, increased cancer risks have been extensively reported ^[2][7][8][2,7,8]. Ionizing radiation exposure leads to chromosomal aberrations [9], DNA damage ^[10][11][10,11], alterations in the cell cycle ^[12][13][12,13], and apoptosis ^[14][15][14,15]. However, the complex nature and small astronaut cohort have made the research on space radiation protection challenging and the results highly uncertain [16]. The elusive mechanisms of ionizing radiation (IR)-induced DNA damage and repair have made research even more difficult. Shielding materials in space crafts are not sufficient, although they are still the main protective measures used. Medicine should play a more important role in protecting against space radiation, but no radioprotectors specifically counteracting the effects of space radiation, including high LET and chronic low-dose IR exposure, have been approved by the United States Food and Drug Administration (US FDA) ^[17][18][17,18]. The current protection technology is far behind the requirements of human space exploration. Thus, improvements in medical protection approaches against space radiation through the discovery and development of efficient agents with favorable toxicity profiles are urgently needed.

Elucidating the DNA damage response (DDR) facilitates research into radioprotectors. One of the ways in which space radiation damages DNA indirectly is through oxidative stress, with the production of reactive oxygen species (ROS) ^[19][20][19,20] and free radicals [21]. Antioxidants such as MitoQ decrease mitochondrial ROS [19], CBLB502 and trace elements scavenge free radicals ^[22][23][22,23], and vitamin A inhibits the expression of inflammation factors. They have been considered important radioprotective compounds [24]. Another kind of radioprotector is protease inhibitors, including ilomasta, which promotes the recovery of immunity [25], and Bowman–Birk inhibitors (BBI), which exert anticarcinogenic and anti-inflammatory properties ^[26][27][26,27]. Gamma-tocotrienol (GT3) and coenzyme Q10 (CoQ10) are also promising radioprotectors by preventing the apoptosis of cells ^[28][29][30][28,29,30]. Herbal mixtures, such as Hong Shan Capsule (HSC) [31] and resveratrol [32], were proven to be effective against IR. These agents lack structural diversity and have major drawbacks, including limited availability, uncertain safety profiles, and ambiguous mechanisms of action.

Direct IR-induced DNA damage is caused by the interaction of charged particles with DNA molecules [21], in which DNA double-strand breaks (DSBs) are extremely cytotoxic lesions ^[33][34][33,34]. DSBs can be repaired by several organized mechanisms to maintain the stability and integrity of the genome [35], which is vital for cell survival. Classical non-homologous end joining (NHEJ), homologous recombination (HR), alternative end joining (alt-EJ), and single-strand annealing (SSA) represent distinct DSB repair mechanisms [36], of which NHEJ and HR are the most pivotal and common. Post-translational modifications (PTMs) are covalent chemical modifications of proteins that occur after translation, conferring proper activity and biological functions to these proteins. The main PTMs related to DDR are phosphorylation, ubiquitylation, acetylation, methylation, SUMOylation, and poly ADP-ribosylation (PARylation). It was revealed that a number of DNA-damage-repair-related factors are subjected to these PTMs, which play indispensable roles in chromatin structures and functions. These factors lead to the rapid initiation and efficient regulation of a variety of biological processes by modulating DDR spatiotemporal dynamics ^{[37][38][39][40]}[37,38,39,40]. Most PTMs are deposited on histones [41] and engage in the recruitment of a series of DDR proteins [38]. Targeting essential factors in the PTM of DNA DSB repair may lead to a promising strategy for developing radioprotectors for human space exploration, as the identification and verification of drug targets are the early and critical steps of drug discovery.

2. PTMs in the Choice of DNA Repair Pathways

HR is a critical pathway for the error-free repair of DNA DSBs, while NHEJ always occurs in the absence of a sister chromatid, leading to error-prone repair and more mutations ^[42][43][44][42,43,44]. NHEJ was reported to be the predominant DNA repair pathway in mammalian cells [45]. The choice of the repair pathway was found to be tightly associated with the cell cycle, as NHEJ is the default repair pathway [46] usually executed in the G1 phase of the cell cycle in a rapid and high-capacity manner ^[42][47][42,47]. Unlike NHEJ, which may occur throughout the entire cell cycle, HR is largely limited to the S/G2 phases ^[42][48][42,48] and is conducted more slowly than NHEJ [47]. The underlying mechanism is that DSB repair is executed with higher efficiency during the S phase. DSB processing and checkpoint activation are much more efficient in the G2/M phase than in the G1 phase [49]. In general, the 5′-3′ degradation of DSB ends is needed for the loading of checkpoint and recombination proteins in all HR reactions ^[50][51][50,51]. The generation of long 3′ single-strand DNA (ssDNA) overhangs mediated by DNA helicases and exonuclease in DNA end resection was proven to be an essential committed process in HR ^{[47][48][52][53][54]}[47,48,52,53,54]. In contrast, NHEJ requires DNA ends that have not been resected instead of 3′ ssDNA tails. DNA end resection is not needed, leading to the joining of two DNA ends with few references to the DNA sequence ^{[47][48][55][56]}[47,48,55,56]. Therefore, controlling DNA end resection is one of the processes affecting whether DNA repair is conducted by NHEJ or HR ^[35][40][35,40]. For example, the 53BP1-RIF1-shieldin complex cooperates with the CTC1-STN1-TEN1 (CST)/Pol α-Prim complex in regulating the generation of 3′ overhangs, which are essential for DNA end protection and switching the DSB repair mode to NHEJ ^[53][57][53,57]. In contrast, BRCA1 promotes HR and antagonizes NHEJ by stimulating end resection ^[58][59][58,59]. Several key proteins and their complexes play regulatory roles in NHEJ. For instance, the Ku70-Ku80 heterodimer is central in initiating NHEJ by recognizing DSB ends and recruiting DNA-PKcs to DSB sites ^{[51][60][61][62][63]}[51,60,61,62,63]. In addition, 53BP1 stimulates NHEJ by recruiting other DDR proteins such as ATM and inhibiting DNA end resection processing by protecting broken DNA ends with its co-factors PTIP, RIF1-shieldin, or REV7/MAD2L2 ^{[48][56][61][62][64][65][66][67]}[48,56,61,62,64,65,66,67]. In contrast, the important factors in HR mainly include BRCA1/2, EXO1, MRE11 ^{[47][48][64][68][69]}[47,48,64,68,69], and RAD51 and its paralogs ^{[43][47][69][70]}[43,47,69,70]. Among them, BRCA1 directly affects the DSB repair pathway choice by regulating the initiation of end resection ^[52][59][52,59]. The preservation of long-term resection activity requires EXO1 exonuclease [71], the deficiency of which contributes to the accumulation of unprocessed DSBs and HR failure [72]. MRE11 exonuclease activity is needed for the assembly of a series of proteins to DSB sites to mediate extended-end resection for HR [73]. HR is orchestrated by several PTMs with elaborate primary mechanisms. The first one is phosphorylation. Switching the meiotic recombination mode of HR was reported to occur by the phosphorylation of RAD54 and HED1, downregulating RAD51 activity by suppressing Rad51/Rad54 complex formation ^[74][75][74,75]. Secondly, SUMOylation is important in HR. It affects all steps in HR and exerts various regulatory functions on substrates [76]. Evidence indicated that SUMOylation induced by topoisomerase 1-binding arginine/serine-rich protein (TOPORS) was essential for the recruitment of RAD51 to the damaged sites and the support of HR repair, maintaining genomic stability [77]. On the other hand, NHEJ might be associated with phosphorylation and methylation by DNA-PKcs and 53BP1, respectively. NHEJ and HR are competitive, and their balance is finely modulated by bioprocesses that include PTMs. Ubiquitination is the most vital PTM, playing a specific role in the recruitment and enrichment of DDR factors at DSB sites in chromosomes and governing DNA repair pathway choices between NHEJ and HR. DDR proteins are mainly assembled by ubiquitin E3 ligases RNF8 and RNF168, followed by accurate repair processes [35]. The ubiquitylation-dependent DSB repair pathway choice is frequently associated with DNA end resection. For example, Cullin3-KLHL15 ubiquitin ligase participates in CtIP protein turnover through the ubiquitin–proteasome pathway, fine-tuning DNA end resection and impacting the balance between HR and NHEJ [78]. RING domain-containing E3 ligase RNF138 is involved in the ubiquitination of Ku80 during the S phase and its removal from DSB sites, stimulating DSB end resection and promoting HR initiation [79]. In addition to DNA end resection, ubiquitylation also modulates the choice of DNA repair pathways by altering the expression of specific DDR proteins. CtIP, which is a target of anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase, is downregulated during G1 and G2 phases and reduces HR [80]. CtIP ubiquitylation and upregulation are stimulated by UBE2Ds and RNF138 at DNA damage sites, promoting DNA repair by HR [81]. Moreover, ubiquitination affects the DNA repair pathway choice by regulating histone H2A at Lys15 (H2AK15ub) and initiating downstream signaling events [82]. Phosphorylation is another major PTM involved in the balance of DNA repair pathways. The phosphorylation of ubiquitin at Thr12 (pUbT12) influences DDR by regulating the activity of 53BP1 in damaged chromosomes [83], and 53BP1 inhibits excessive DNA end resection and promotes repair by NHEJ through different phosphoprotein interactions [84]. RIF1 is prominent at DSB sites in the G1 phase of the cell cycle by the ATM-associated phosphorylation of 53BP1, ensuring the dominant position of NHEJ in this phase ^[58][59][85][58,59,85]. Collectively, a variety of proteins and their complexes were revealed to act in the complicated response mechanisms to DNA lesions induced by IR, participating in distinct PTMs and coordinating NHEJ, HR, and their balance in DNA repair (Figure 1). These essential factors could be properly categorized and investigated from the view of PTMs, including phosphorylation, ubiquitylation, acetylation, and methylation. Compounds targeting these factors influence DNA repair after IR, leading to radiosensitization or radioprotective effects (Table 1). Some of them have been approved for regulating DDR, and more candidates are under development. They provide resources in the discovery of future space radioprotectors (Figure 2).