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Clustered DNA damage, when multiple lesions are generated in close proximity, has various biological consequences, including cell death, chromosome aberrations, and mutations. It is generally perceived as a hallmark of ionizing radiation. The enhanced mutagenic potential of lesions within a cluster has been suggested to result, at least in part, from the selection of the strand with the mutagenic lesion as the preferred template strand, and that this process is relevant to the tolerance of persistent single-strand breaks generated during an attempted repair. Using a plasmid-based assay in Escherichia coli, we examined how the strand bias is affected in mutant strains deficient in different DNA polymerase I activities. Our study revealed that the strand-displacement and 5'-flap endonuclease activities are required for this process, while 3'-to-5' exonuclease activity is not. We also found the strand template that the mutagenic lesion was located on, whether lagging or leading, had no effect on this strand bias. Our results imply that an unknown pathway operates to repair/tolerate the single-strand break generated at a bi-stranded clustered damage site, and that there exist different backup pathways, depending on which DNA polymerase I activity is compromised.

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The genome-the source of life and platform of evolution-is continuously exposed to harmful factors, both extra- and intra-cellular. Their activity causes different types of DNA damage, with approximately 80 different types of lesions having been identified so far. In this paper, the influence of a clustered DNA damage site containing imidazolone (Iz) or oxazolone (Oz) and 7,8-dihydro-8-oxo-2'-deoxyguanosine (OXOdG) on the charge transfer through the double helix as well as their electronic properties were investigated. To this end, the structures of oligo-Iz, d[A1Iz2A3OXOG4A5]*d[T5C4T3C2T1], and oligo-Oz, d[A1Oz2A3OXOG4A5]*d[T5C4T3C2T1], were optimized at the M06-2X/6-D95**//M06-2X/sto-3G level of theory in the aqueous phase using the ONIOM methodology; all the discussed energies were obtained at the M06-2X/6-31++G** level of theory. The non-equilibrated and equilibrated solvent-solute interactions were taken into consideration. The following results were found: (A) In all the discussed cases, OXOdG showed a higher predisposition to radical cation formation, and B) the excess electron migration toward Iz and Oz was preferred. However, in the case of oligo-Oz, the electron transfer from Oz2 to complementary C4 was noted during vertical to adiabatic anion relaxation, while for oligo-Iz, it was settled exclusively on the Iz2 moiety. The above was reflected in the charge transfer rate constant, vertical/adiabatic ionization potential, and electron affinity energy values, as well as the charge and spin distribution. It can be postulated that imidazolone moiety formation within the CDL ds-oligo structure and its conversion to oxazolone can significantly influence the charge migration process, depending on the C2 carbon hybridization sp2 or sp3. The above can confuse the single DNA damage recognition and removal processes, cause an increase in mutagenesis, and harm the effectiveness of anticancer therapy.

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The genome is continuously exposed to a variety of harmful factors that result in a significant amount of DNA damage. This article examines the influence of a multi-damage site containing oxidized imino-allantoin (OXIa) and 7,8-dihydro-8-oxo-2'-deoxyguanosine (OXOdG) on the spatial geometry, electronic properties, and ds-DNA charge transfer. The ground stage of a d[A1OXIa2A3OXOG4A5]*d[T5C4T3C2T1] structure was obtained at the M06-2X/6-D95**//M06-2X/sto-3G level of theory in the condensed phase, with the energies obtained at the M06-2X/6-31++G** level. The non-equilibrated and equilibrated solvent-solute interactions were also considered. Theoretical studies reveal that the radical cation prefers to settle on the OXOG moiety, irrespective of the presence of OXIa in a ds-oligo. The lowest vertical and adiabatic ionization potential values were found for the OXOG:::C base pair (5.94 and 5.52 [eV], respectively). Conversely, the highest vertical and adiabatic electron affinity was assigned for OXIaC as follows: 3.15 and 3.49 [eV]. The charge transfers were analyzed according to Marcus' theory. The highest value of charge transfer rate constant for hole and excess electron migration was found for the process towards the OXOGC moiety. Surprisingly, the values obtained for the driving force and activation energy of electro-transfer towards OXIa2C4 located this process in the Marcus inverted region, which is thermodynamically unfavorable. Therefore, the presence of OXIa can slow down the recognition and removal processes of other DNA lesions. However, with regard to anticancer therapy (radio/chemo), the presence of OXIa in the structure of clustered DNA damage can result in improved cancer treatment outcomes.

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Genetic information stored in a DNA base sequence is continuously exposed to harmful factors. It has been determined that 9 × 104 different DNA damage events occur in a single human cell every 24 h. Of these, 7,8-dihydro-8-oxo-guanosine (OXOG) is one of the most abundant and can undergo further transformations towards spirodi(iminohydantoin) (Sp). Sp is highly mutagenic in comparison to its precursor if not repaired. In this paper, the influence of both Sp diastereomers 4R and 4S as well as their anti and syn conformers on charge transfer through the double helix was taken into theoretical consideration. In addition, the electronic properties of four modelled double-stranded oligonucleotides (ds-oligos) were also discussed, i.e., d[A1Sp2A3oxoG4A5] * [T5C4T3C2T1]. Throughout the study, the M06-2X/6-31++G** level theory was used. Solvent-solute non-equilibrated and equilibrated interactions were also considered. The subsequent results elucidated that the 7,8-dihydro-8-oxo-guanosine:cytidine (OXOGC) base pair is the settled point of a migrated radical cation in each of the discussed cases, due to its low adiabatic ionization potential, i.e., ~5.55 [eV]. The opposite was noted for excess electron transfer through ds-oligos containing anti (R)-Sp or anti (S)-Sp. The radical anion was found on the OXOGC moiety, whereas in the presence of syn (S)-Sp or syn (R)-Sp, an excess electron was found on the distal A1T5 or A5T1 base pair, respectively. Furthermore, a spatial geometry analysis of the discussed ds-oligos revealed that the presence of syn (R)-Sp in the ds-oligo caused only a slight deformation to the double helix, while syn (S)-Sp formed an almost ideal base pair with a complementary dC. The above results are in strong agreement with the final charge transfer rate constant, as calculated according to Marcus' theory. In conclusion, DNA damage such as spirodi(iminohydantoin), especially when becoming part of clustered DNA damage, can affect the effectiveness of other lesion recognition and repair processes. This can lead to the acceleration of undesired and deleterious processes such as carcinogenesis or aging. However, in terms of anticancer radio-/chemo- or combined therapy, the slowing down of the repair machinery can result in increased effectiveness. With this in mind, the influence of clustered damage on charge transfer and its subsequent effect on single-damage recognition by glycosylases justifies future investigation.

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The genetic information stored in the nucleobase sequence is continuously exposed to harmful extra- and intra-cellular factors, which can lead to different types of DNA damage, with more than 70 lesion types identified so far. In this article, the influence of a multi-damage site containing (5'R/S) 5',8-cyclo-2'-deoxyguanosine (cdG) and 7,8-dihydro-8-oxo-2'-deoxyguanosine (OXOdG) on charge transfer through ds-DNA was taken into consideration. The spatial geometries of oligo-RcdG: d[A1(5'R)cG2A3OXOG4A5]*d[T5C4T3C2T1] and oligo-ScdG: d[A1(5'S)cG2A3OXOG4A5]*d[T5C4T3C2T1] were optimized at the M06-2X/6-D95**//M06-2X/sto-3G level of theory in the aqueous phase using ONIOM methodology. For all the electronic property energies under discussion, the M06-2X/6-31++G** level of theory was used. Additionally, the non-equilibrated and equilibrated solvent-solute interactions were into consideration. The obtained results confirm the predisposition of OXOdG to radical cation formation regardless of the presence of other lesions in a ds-DNA structure. In the case of electron transfer, however, the situation is different. An excess electron migration towards (5'S)cdG was found to be preferred in the case of oligo-ScdG, while in the case of oligo-RcdG, OXOdG was favored. The above observation was confirmed by the charge transfer rate constant, vertical/adiabatic ionization potential, and electron affinity energy values, as well as the charge and spin distribution analysis. The obtained results indicate that 5',8-cyclo-2'-deoxyguanosine, depending on the C5' atom chirality, can significantly influence the charge migration process through the double helix. The above can be manifested by the slowdown of DNA lesion recognition and removal processes, which can increase the probability of mutagenesis and subsequent pathological processes. With regard to anticancer therapy (radio/chemo), the presence of (5'S)cdG in the structure of formed clustered DNA damage can lead to improvements in cancer treatment.

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Genetic information, irrespective of cell type (normal or cancerous), is exposed to a range of harmful factors, which can lead to more than 80 different types of DNA damage. Of these, oxoG and FapyG have been identified as the most abundant in normoxic and hypoxic conditions, respectively. This article considers d[AFapyGAOXOGA]*[TCTCT] (oligo-FapyG) with clustered DNA lesions (CDLs) containing both the above types of damage at the M06-2x/6-31++G** level of theory in the condensed phase. Furthermore, the electronic properties of oligo-FapyG were analysed in both equilibrated and non-equilibrated solvation-solute interaction modes. The vertical/adiabatic ionization potential (VIP, AIP) and electron affinity (VEA, AEA) of the investigated ds-oligo were found as follows in [eV]: 5.87/5.39 and -1.41/-2.09, respectively. The optimization of the four ds-DNA spatial geometries revealed that the transFapydG was energetically privileged. Additionally, CDLs were found to have little influence on the ds-oligo structure. Furthermore, for the FapyGC base-pair isolated from the discussed ds-oligo, the ionization potential and electron affinity values were higher than those assigned to OXOGC. Finally, a comparison of the influence of FapyGC and OXOGC on charge transfer revealed that, in contrast to the OXOGC base-pair, which, as expected, acted as a radical cation/anion sink in the oligo-FapyG structure, FapyGC did not significantly affect charge transfer (electron-hole and excess-electron). The results presented below indicate that 7,8-dihydro-8-oxo-2'-deoxyguanosine plays a significant role in charge transfer through ds-DNA containing CDL and indirectly has an influence on the DNA lesion recognition and repair process. In contrast, the electronic properties obtained for 2,6-diamino-4-hydroxy-5-foramido-2'deoxypyrimidine were found to be too weak to compete with OXOG to influence charge transfer through the discussed ds-DNA containing CDL. Because increases in multi-damage site formation are observed during radio- or chemotherapy, understanding their role in the above processes can be crucial for the efficiency and safety of medical cancer treatment.

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Modeling ionizing radiation interaction with biological matter is a major scientific challenge, especially for protons that are nowadays widely used in cancer treatment. That presupposes a sound understanding of the mechanisms that take place from the early events of the induction of DNA damage. Herein, we present results of irradiation-induced complex DNA damage measurements using plasmid pBR322 along a typical Proton Treatment Plan at the MedAustron proton and carbon beam therapy facility (energy 137-198 MeV and Linear Energy Transfer (LET) range 1-9 keV/µm), by means of Agarose Gel Electrophoresis and DNA fragmentation using Atomic Force Microscopy (AFM). The induction rate Mbp-1 Gy-1 for each type of damage, single strand breaks (SSBs), double-strand breaks (DSBs), base lesions and non-DSB clusters was measured after irradiations in solutions with varying scavenging capacity containing 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris) and coumarin-3-carboxylic acid (C3CA) as scavengers. Our combined results reveal the determining role of LET and Reactive Oxygen Species (ROS) in DNA fragmentation. Furthermore, AFM used to measure apparent DNA lengths provided us with insights into the role of increasing LET in the induction of highly complex DNA damage.

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Ionizing radiation causes various types of DNA damage, such as single- (SSBs) and double-strand breaks (DSBs), nucleobase lesions, abasic sites (AP sites), and cross-linking between complementary strands of DNA or DNA and proteins. DSBs are among the most harmful type of DNA damage, inducing serious genetic effects such as cell lethality and mutation. Nucleobase lesions and AP sites, on the other hand, may be less deleterious and are promptly repaired by base excision repair (BER) pathways. Recently, biochemical approaches to quantify nucleobase lesions and AP sites have revealed certain types of non-strand break lesions as harmful DNA damage, called clustered DNA damage. Such clusters can retard nucleobase excision repair enzymes, and can sometimes be converted to DSBs by BER catalysis. This unique character of clustered DNA damage strongly depends on the spatial density of ionization or excitation events occurring at the track end of initial radiation or low energy secondary electrons. In particular, the photoelectric effect of elements comprising biological molecules, followed by emission of Auger electrons, are key factors in determining the future fate of each clustered damage site. This chapter describes biological studies of clustered nucleobase lesions with SSBs or AP sites, and mechanistical studies on core level excitation and Auger relaxation giving rise to clustered DNA damage.

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Exposure to environmental ionizing radiation is prevalent, with greatest lifetime doses typically from high Linear Energy Transfer (high-LET) alpha particles via the radioactive decay of radon gas in indoor air. Particle radiation is highly genotoxic, inducing DNA damage including oxidative base lesions and DNA double strand breaks. Due to the ionization density of high-LET radiation, the consequent damage is highly clustered wherein ≥2 distinct DNA lesions occur within 1-2 helical turns of one another. These multiply-damaged sites are difficult for eukaryotic cells to resolve either quickly or accurately, resulting in the persistence of DNA damage and/or the accumulation of mutations at a greater rate per absorbed dose, relative to lower LET radiation types. The proximity of the same and different types of DNA lesions to one another is challenging for DNA repair processes, with diverse pathways often confounding or interplaying with one another in complex ways. In this context, understanding the state of the higher order chromatin compaction and arrangements is essential, as it influences the density of damage produced by high-LET radiation and regulates the recruitment and activity of DNA repair factors. This review will summarize the latest research exploring the processes by which clustered DNA damage sites are induced, detected, and repaired in the context of chromatin.

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SignificanceDNA damage causes loss of or alterations in genetic information, resulting in cell death or mutations. Ionizing radiations produce local, multiple DNA damage sites called clustered DNA damage. In this study, a complete protocol was established to analyze the damage complexity of clustered DNA damage, wherein damage-containing genomic DNA fragments were selectively concentrated via pulldown, and clustered DNA damage was visualized by atomic force microscopy. It was found that X-rays and Fe ion beams caused clustered DNA damage. Fe ion beams also produced clustered DNA damage with high complexity. Fe ion beam-induced complex DNA double-strand breaks (DSBs) containing one or more base lesion(s) near the DSB end were refractory to repair, implying their lethal effects.

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Different types of DNA lesions forming in close vicinity, create clusters of damaged sites termed as "clustered/complex DNA damage" and they are considered to be a major challenge for DNA repair mechanisms resulting in significant repair delays and induction of genomic instability. Upon detection of DNA damage, the corresponding DNA damage response and repair (DDR/R) mechanisms are activated. The inability of cells to process clustered DNA lesions efficiently has a great impact on the normal function and survival of cells. If complex lesions are left unrepaired or misrepaired, they can lead to mutations and if persistent, they may lead to apoptotic cell death. In this in silico study, and through rigorous data mining, we have identified human genes that are activated upon complex DNA damage induction like in the case of ionizing radiation (IR) and beyond the standard DNA repair pathways, and are also involved in cancer pathways, by employing stringent bioinformatics and systems biology methodologies. Given that IR can cause repair resistant lesions within a short DNA segment (a few nm), thereby augmenting the hazardous and toxic effects of radiation, we also investigated the possible implication of the most biologically important of those genes in comorbid non-neoplastic diseases through network integration, as well as their potential for predicting survival in cancer patients.

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The 5',8-cyclo-2'-deoxypurines (cdPus) affect the DNA structure. When these bulky structures are a part of clustered DNA lesions (CDL), they affect the repair of the other lesions within the cluster. Mitochondria are crucial for cell survival and have their own genome, hence, are highly interesting in the context of CDL repair. However, no studies are exploring this topic. Here, the initial stages of mitochondrial base excision repair (mtBER) were considered-the strand incision and elongation. The repair of a single lesion (apurinic site (AP site)) accompanying the cdPu within the double-stranded CDL has been investigated for the first time. The type of cdPu, its diastereomeric form, and the interlesion distance were taken into consideration. For these studies, the established experimental model of short oligonucleotides (containing AP sites located ≤7 base pairs to the cdPu in both directions) and mitochondrial extracts of the xrs5 cells were used. The obtained results have shown that the presence of cdPus influenced the processing of an AP site within the CDL. Levels of strand incision and elongation were higher for oligos containing RcdA and ScdG than for those with ScdA and RcdG. Investigated stages of mtBER were more efficient for DNA containing AP sites located on 5'-end side of cdPu than on its 3'-end side. In conclusion, the presence of cdPus in mtDNA structure may affect mtBER (processing the second mutagenic lesion within the CDL). As impaired repair processes may lead to serious biological consequences, further studies concerning the mitochondrial repair of CDL are highly demanded.

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Clusters of DNA damage, also called multiply damaged sites (MDS), are a signature of ionizing radiation exposure. They are defined as two or more lesions within one or two helix turns, which are created by the passage of a single radiation track. It has been shown that the clustering of DNA damage compromises their repair. Unresolved repair may lead to the formation of double-strand breaks (DSB) or the induction of mutation. We engineered three complex MDS, comprised of oxidatively damaged bases and a one-nucleotide (1 nt) gap (or not), in order to investigate the processing and the outcome of these MDS in yeast Saccharomyces cerevisiae. Such MDS could be caused by high linear energy transfer (LET) radiation. Using a whole-cell extract, deficient (or not) in base excision repair (BER), and a plasmid-based assay, we investigated in vitro excision/incision at the damaged bases and the mutations generated at MDS in wild-type, BER, and translesion synthesis-deficient cells. The processing of the studied MDS did not give rise to DSB (previously published). Our major finding is the extremely high mutation frequency that occurs at the MDS. The proposed processing of MDS is rather complex, and it largely depends on the nature and the distribution of the damaged bases relative to the 1 nt gap. Our results emphasize the deleterious consequences of MDS in eukaryotic cells.

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The clustered DNA lesions (CDLs) are a characteristic feature of ionizing radiation's impact on the human genetic material. CDLs impair the efficiency of cellular repair machinery, especially base excision repair (BER). When CDLs contain a lesion repaired by BER (e.g., apurinic/apyrimidinic (AP) sites) and a bulkier 5',8-cyclo-2'-deoxypurine (cdPu), which is not a substrate for BER, the repair efficiency of the first one may be affected. The cdPus' influence on the efficiency of nuclear BER in xrs5 cells have been investigated using synthetic oligonucleotides with bi-stranded CDL (containing (5'S) 5',8-cyclo-2'-deoxyadenosine (ScdA), (5'R) 5',8-cyclo-2'-deoxyadenosine (RcdA), (5'S) 5',8-cyclo-2'-deoxyguanosine (ScdG) or (5'R) 5',8-cyclo-2'-deoxyguanosine (RcdG) in one strand and an AP site in the other strand at different interlesion distances). Here, for the first time, the impact of ScdG and RcdG was experimentally tested in the context of nuclear BER. This study shows that the presence of RcdA inhibits BER more than ScdA; however, ScdG decreases repair level more than RcdG. Moreover, AP sites located ≤10 base pairs to the cdPu on its 5'-end side were repaired less efficiently than AP sites located ≤10 base pairs on the 3'-end side of cdPu. The strand with an AP site placed opposite cdPu or one base in the 5'-end direction was not reconstituted for cdA nor cdG. CdPus affect the repair of the other lesion within the CDL. It may translate to a prolonged lifetime of unrepaired lesions leading to mutations and impaired cellular processes. Therefore, future research should focus on exploring this subject in more detail.

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A clustered DNA damage site (cluster), in which two or more lesions exist within a few helical turns, is believed to be a key factor determining the fate of a living cell exposed to a DNA damaging agent such as ionizing radiation. However, the structural details of a cluster such as the number of included lesions and their proximity are unknown. Herein, we develop a method to characterize a cluster by fluorescence anisotropy measurements based on Förster resonance energy transfer (homo-FRET). Plasmid DNA (pUC19) was irradiated with 2.0 and 0.52 MeV/u 4He2+, or 0.37 MeV/u 12C5+ ion beams (linear energy transfer: ~ 70, ~ 150, ~ 760 keV/µm, respectively) and 60Co γ-rays as a standard (~ 0.2 keV/µm) in the solid state. The irradiated DNA was labeled with an aminooxyl fluorophore (Alexa Fluor 488) to the aldehyde/ketone moieties such as apurinic/apyrimidinic sites. Homo-FRET analyses provided the apparent base separation values between lesions in a cluster produced by each ion beam track as 21.1, 19.4, and 18.7 base pairs. The production frequency of a cluster increases with increasing linear energy transfer of radiation. Our results demonstrate that homo-FRET analysis has the potential to discover the qualitative and the quantitative differences of the clusters produced not only by a variety of ionizing radiation but also by other DNA damaging agents.

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Radiation therapy (RT), an integral component of curative treatment for many malignancies, can be administered via an increasing array of techniques. In this review, we summarize the properties and application of different types of RT, specifically, conventional therapy with x-rays, stereotactic body RT, and proton and carbon particle therapies. We highlight how low-linear energy transfer (LET) radiation induces simple DNA lesions that are efficiently repaired by cells, whereas high-LET radiation causes complex DNA lesions that are difficult to repair and that ultimately enhance cancer cell killing. Additionally, we discuss the immunogenicity of radiation-induced tumor death, elucidate the molecular mechanisms by which radiation mounts innate and adaptive immune responses and explore strategies by which we can increase the efficacy of these mechanisms. Understanding the mechanisms by which RT modulates immune signaling and the key players involved in modulating the RT-mediated immune response will help to improve therapeutic efficacy and to identify novel immunomodulatory drugs that will benefit cancer patients undergoing targeted RT.

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Approximately 3 × 1017 DNA damage events take place per hour in the human body. Within clustered DNA lesions, they pose a serious problem for repair proteins, especially for iron-sulfur glycosylases (MutyH), which can recognize them by the electron-transfer process. It has been found that the presence of both 5',8-cyclo-2'-deoxyadenosine (cdA) diastereomers in the ds-DNA structure, as part of a clustered lesion, can influence vertical radical cation distribution within the proximal part of the double helix, i.e., d[~oxoGcAoxoG~] (7,8-dihydro-8-oxo-2'-deoxyguaosine - oxodG). Here, the influence of cdA, "the simplest tandem lesion", on the charge transfer through ds-DNA was taken into theoretical consideration at the M062x/6-31+G** level of theory in the aqueous phase. It was shown that the presence of (5'S)- or (5'R)-cdA leads to a slowdown in the hole transfer by one order of magnitude between the neighboring dGoxodG in comparison to "native" ds-DNA. Therefore, it can be concluded that such clustered lesions can lead to defective damage recognition with a subsequent slowing down of the DNA repair process, giving rise to an increase in mutations. As a result, the unrepaired, oxodG: dA base pair prior to genetic information replication can finally result in GC TA or ATCG transversion. This type of mutation is commonly observed in human cancer cells. Moreover, because local multiple damage sites (LMSD) are effectively produced as a result of ionization factors, the presented data in this article might be useful in developing a new scheme of radiotherapy treatment against the background of DNA repair efficiency.

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The deoxyribonucleic acid (DNA) molecule damage simulations with an atom level geometric model use the traversal algorithm that has the disadvantages of quite time-consuming, slow convergence and high-performance computer requirement. Therefore, this work presents a density-based spatial clustering of applications with noise (DBSCAN) clustering algorithm based on the spatial distributions of energy depositions and hydroxyl radicals (·OH). The algorithm with probability and statistics can quickly get the DNA strand break yields and help to study the variation pattern of the clustered DNA damage. Firstly, we simulated the transportation of protons and secondary particles through the nucleus, as well as the ionization and excitation of water molecules by using Geant4-DNA that is the Monte Carlo simulation toolkit for radiobiology, and got the distributions of energy depositions and hydroxyl radicals. Then we used the damage probability functions to get the spatial distribution dataset of DNA damage points in a simplified geometric model. The DBSCAN clustering algorithm based on damage points density was used to determine the single-strand break (SSB) yield and double-strand break (DSB) yield. Finally, we analyzed the DNA strand break yield variation trend with particle linear energy transfer (LET) and summarized the variation pattern of damage clusters. The simulation results show that the new algorithm has a faster simulation speed than the traversal algorithm and a good precision result. The simulation results have consistency when compared to other experiments and simulations. This work achieves more precise information on clustered DNA damage induced by proton radiation at the molecular level with high speed, so that it provides an essential and powerful research method for the study of radiation biological damage mechanism.

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The deoxyribonucleic acid (DNA) molecule damage simulations with an atom level geometric model use the traversal algorithm that has the disadvantages of quite time-consuming, slow convergence and high-performance computer requirement. Therefore, this work presents a density-based spatial clustering of applications with noise (DBSCAN) clustering algorithm based on the spatial distributions of energy depositions and hydroxyl radicals (·OH). The algorithm with probability and statistics can quickly get the DNA strand break yields and help to study the variation pattern of the clustered DNA damage. Firstly, we simulated the transportation of protons and secondary particles through the nucleus, as well as the ionization and excitation of water molecules by using Geant4-DNA that is the Monte Carlo simulation toolkit for radiobiology, and got the distributions of energy depositions and hydroxyl radicals. Then we used the damage probability functions to get the spatial distribution dataset of DNA damage points in a simplified geometric model. The DBSCAN clustering algorithm based on damage points density was used to determine the single-strand break (SSB) yield and double-strand break (DSB) yield. Finally, we analyzed the DNA strand break yield variation trend with particle linear energy transfer (LET) and summarized the variation pattern of damage clusters. The simulation results show that the new algorithm has a faster simulation speed than the traversal algorithm and a good precision result. The simulation results have consistency when compared to other experiments and simulations. This work achieves more precise information on clustered DNA damage induced by proton radiation at the molecular level with high speed, so that it provides an essential and powerful research method for the study of radiation biological damage mechanism.

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Ionizing radiation causes various different types of DNA damage. If not repaired, DNA damage can have detrimental effects. Previous studies indicate that the spatial distribution of DNA lesions induced by ionizing radiation is highly relevant to the ensuing biological effects. Clustered DNA damage, consisting of DNA lesions in close proximity, has been studied in detail, and has enhanced mutagenic potential depending on the configuration of the lesions. However, it is not known whether clustered DNA damage affects the mutagenic potential of a sufficiently separated, isolated lesion. Using synthetic damage constructs, we investigated the mutagenic potential of an isolated 8-oxo-7,8-dihydroguanine (8-oxoG) separated by at least 7 bp from other lesions. Under the spatial distribution of DNA lesions tested in the present study, neighboring clustered DNA lesions likely retarded the processing of the isolated 8-oxoG and resulted in enhanced mutation frequency. However, the enhanced mutagenic potential was dependent on which strand the isolated 8-oxoG was located. Our results indicate that the processing of a bi-stranded cluster could affect the mutagenic outcome of a nearby isolated lesion, separated up to ∼20 bp.

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