Abstract:Chromosome translocations are hallmark of cancer and of radiation-induced cell killing, reflecting joining of incongruent DNA-ends that alter the genome. Translocation-formation requires DNA end-joining mechanisms and incompletely characterized, permissive chromatin conditions. We show that chromatin destabilization by clusters of DNA double-strand-breaks (DSBs) generated by the I-SceI meganuclease at multiple, appropriately engineered genomic sites, compromises c-NHEJ and markedly increases cell killing and t… Show more
“…In the case of repair deficient cells MO59J compared to their repair proficient counterpart MO59K cells, we detect interesting enough a high increase of colocalization between these two DSB proteins especially after 8 h post-irradiation with maximum at 24 h, suggesting possibly active repair resistant sites [7].…”
Section: Damage Clusteringmentioning
confidence: 69%
“…The most accepted theory is that when the phosphorylated H2AX (c-H2AX) forms megabase-size foci at a DSB this leads to the recruitment of various downstream DNA damage response (DDR) factors including 53BP1 (as a DSB sensor), BRCA1 and NSB1 enabling correct repair of DNA damage [51]. There are indications that there is a preferential enrolment of 53BP1 in persistent DSBs [52], late repair like for example in heterochromatic regions [51] or DSB clusters [7] and there is an increase of c-irradiated cells with 53BP1 foci with dose [43]. Our results for MO59J (Figure 4(c)) certainly agree with this hypothesis since we also observe an increase of c-H2AX/53BP1 colocalization at late repair times, suggesting active repair and recruitment of 53BP1 in these sites.…”
Section: Discussionmentioning
confidence: 99%
“…This possibility is high especially when the two non-DSB lesions are located in a distance of up to 10 bp [56]. The inaccurate repair of such "hybrid clusters", consisting of DSBs and non-DSBs, can be attributed to the error-prone DNA repair pathways implicated in this case like NHEJ and its backup subpathways alt-EJ (or B-NHEJ) and BER, respectively [7,8,37]. Previous studies by Peddi et al [57] have shown, using an adaptation of PFGE and different types and levels of DNA-PK deficiency, that these deficient cells not only exhibit as expected DSB repair problems, but also the non-DSB oxidative clustered DNA lesions processing is significantly impeded.…”
Section: Discussionmentioning
confidence: 99%
“…Such types of lesions can occur separately or closely to each other (a few bp) resulting in complex or clustered DNA damage. Despite the fact that DSBs are considered to be the most critical lesions relating to cell survival or other late cellular effects, non-DSB clusters, that is the combination of two or more DNA lesions that do not form a DSB [6] adds to damage complexity of DSBs, significantly affecting their repair [7,8]. One major hypothesis is that this temporal simultaneous activation of different repair pathways like non-homologous end joining (NHEJ) for DSBs, base excision repair (BER) for non-DSB lesions, signifies a "stress" genome region [9] or a DNA repair center [10,11].…”
Detrimental effects of ionizing radiation (IR) are correlated to the varying efficiency of IR to induce complex DNA damage. A double strand break (DSB) can be considered the simpler form of complex DNA damage. These types of damage can consist of DSBs, single strand breaks (SSBs) and/or non-DSB lesions such as base damages and apurinic/apyrimidinic (AP; abasic) sites in different combinations. Enthralling theoretical (Monte Carlo simulations) and experimental evidence suggests an increase in the complexity of DNA damage and therefore repair resistance with linear energy transfer (LET). In this study, we have measured the induction and processing of DSB and non-DSB oxidative clusters using adaptations of immunofluorescence. Specifically, we applied foci colocalization approaches as the most current methodologies for the in situ detection of clustered DNA lesions in a variety of human normal (FEP18-11-T1) and cancerous cell lines of varying repair efficiency (MCF7, HepG2, A549, MO59K/J) and radiation qualities of increasing LET, that is c-, X-rays 0.3-1 keV/lm, a-particles 116 keV/lm and 36 Ar ions 270 keV/lm. Using c-H2AX or 53BP1 foci staining as DSB probes, we calculated a DSB apparent rate of 5-16 DSBs/cell/Gy decreasing with LET. A similar trend was measured for non-DSB oxidized base lesions detected using antibodies against the human repair enzymes 8-oxoguanine-DNA glycosylase (OGG1) or AP endonuclease (APE1), that is damage foci as probes for oxidized purines or abasic sites, respectively. In addition, using colocalization parameters previously introduced by our groups, we detected an increasing clustering of damage for DSBs and non-DSBs. We also make correlations of damage complexity with the repair efficiency of each cell line and we discuss the biological importance of these new findings with regard to the severity of IR due to the complex nature of its DNA damage.
ARTICLE HISTORY
“…In the case of repair deficient cells MO59J compared to their repair proficient counterpart MO59K cells, we detect interesting enough a high increase of colocalization between these two DSB proteins especially after 8 h post-irradiation with maximum at 24 h, suggesting possibly active repair resistant sites [7].…”
Section: Damage Clusteringmentioning
confidence: 69%
“…The most accepted theory is that when the phosphorylated H2AX (c-H2AX) forms megabase-size foci at a DSB this leads to the recruitment of various downstream DNA damage response (DDR) factors including 53BP1 (as a DSB sensor), BRCA1 and NSB1 enabling correct repair of DNA damage [51]. There are indications that there is a preferential enrolment of 53BP1 in persistent DSBs [52], late repair like for example in heterochromatic regions [51] or DSB clusters [7] and there is an increase of c-irradiated cells with 53BP1 foci with dose [43]. Our results for MO59J (Figure 4(c)) certainly agree with this hypothesis since we also observe an increase of c-H2AX/53BP1 colocalization at late repair times, suggesting active repair and recruitment of 53BP1 in these sites.…”
Section: Discussionmentioning
confidence: 99%
“…This possibility is high especially when the two non-DSB lesions are located in a distance of up to 10 bp [56]. The inaccurate repair of such "hybrid clusters", consisting of DSBs and non-DSBs, can be attributed to the error-prone DNA repair pathways implicated in this case like NHEJ and its backup subpathways alt-EJ (or B-NHEJ) and BER, respectively [7,8,37]. Previous studies by Peddi et al [57] have shown, using an adaptation of PFGE and different types and levels of DNA-PK deficiency, that these deficient cells not only exhibit as expected DSB repair problems, but also the non-DSB oxidative clustered DNA lesions processing is significantly impeded.…”
Section: Discussionmentioning
confidence: 99%
“…Such types of lesions can occur separately or closely to each other (a few bp) resulting in complex or clustered DNA damage. Despite the fact that DSBs are considered to be the most critical lesions relating to cell survival or other late cellular effects, non-DSB clusters, that is the combination of two or more DNA lesions that do not form a DSB [6] adds to damage complexity of DSBs, significantly affecting their repair [7,8]. One major hypothesis is that this temporal simultaneous activation of different repair pathways like non-homologous end joining (NHEJ) for DSBs, base excision repair (BER) for non-DSB lesions, signifies a "stress" genome region [9] or a DNA repair center [10,11].…”
Detrimental effects of ionizing radiation (IR) are correlated to the varying efficiency of IR to induce complex DNA damage. A double strand break (DSB) can be considered the simpler form of complex DNA damage. These types of damage can consist of DSBs, single strand breaks (SSBs) and/or non-DSB lesions such as base damages and apurinic/apyrimidinic (AP; abasic) sites in different combinations. Enthralling theoretical (Monte Carlo simulations) and experimental evidence suggests an increase in the complexity of DNA damage and therefore repair resistance with linear energy transfer (LET). In this study, we have measured the induction and processing of DSB and non-DSB oxidative clusters using adaptations of immunofluorescence. Specifically, we applied foci colocalization approaches as the most current methodologies for the in situ detection of clustered DNA lesions in a variety of human normal (FEP18-11-T1) and cancerous cell lines of varying repair efficiency (MCF7, HepG2, A549, MO59K/J) and radiation qualities of increasing LET, that is c-, X-rays 0.3-1 keV/lm, a-particles 116 keV/lm and 36 Ar ions 270 keV/lm. Using c-H2AX or 53BP1 foci staining as DSB probes, we calculated a DSB apparent rate of 5-16 DSBs/cell/Gy decreasing with LET. A similar trend was measured for non-DSB oxidized base lesions detected using antibodies against the human repair enzymes 8-oxoguanine-DNA glycosylase (OGG1) or AP endonuclease (APE1), that is damage foci as probes for oxidized purines or abasic sites, respectively. In addition, using colocalization parameters previously introduced by our groups, we detected an increasing clustering of damage for DSBs and non-DSBs. We also make correlations of damage complexity with the repair efficiency of each cell line and we discuss the biological importance of these new findings with regard to the severity of IR due to the complex nature of its DNA damage.
ARTICLE HISTORY
“…Therefore, for the survive of individuals with different chromosome number, a mechanism which can evade apoptosis under high level of DSB is required. The critical role of DSBs during cancer development [29,30], peculiar the most recent advances in the relation of DSBs and chromothripsis [31], has offered some indirect evidences for identifying the role of DSBs on molecular mechanism about the changes of chromosomes number in evolution.…”
1Genome wide association studies (GWAS) have provided an avenue for the association between 2 common genetic variants and complex traits. However, using SNP as a genetic marker, GWAS has 3 been confined to detect genetic basis traits only for within species but not for the large-scale 4 inter-species traits. Here, we propose a practical statistical approach that is using kmer frequencies 5 as the genetic markers to associate genetic variants with large scale inter-species traits. We applied 6 this new approach to the trait of chromosome number in 96 mammalian proteomes, and we 7 prioritized 130 genes including TP53 and BAD, of which 6 were candidate genes. These genes 8 were proved to be associated with cellular reaction of DNA double-strand breaks caused by 9 chromosome fission/fusion. Our study provides a new effective genomic strategy to perform 1 0 association studies for large-scaled inter-species traits, using the chromosome number as a case. 1 1We hope this approach could provide exploration for broadly widely traits. 1 2 1 3
Whereas most endogenous and exogenous DNA damaging agents typically generate lesions that are relatively isolated and can be repaired easily, ionizing radiation (IR) also induces clustered lesions causing DNA double strand breaks (DSBs). Moreover, forms of IR characterized by high linear energy transfer (LET) induce not only isolated DSBs but also DSB clusters - multiple DSBs in close proximity -that pose increased risks for the cell. DSB clusters can destabilize chromatin locally and compromise processing of individual DSBs within the cluster. Since the discovery of chromothripsis, a phenomenon whereby multiple DSBs locally generated by a catastrophic event causes genomic rearrangements that feed carcinogenesis, DSB clusters receive increased attention also in the field of cancer. While formation of DSB clusters after exposure to high LET is a direct and inherent consequence of the spatial distribution of the constituting energy deposition events, also called track structure, the sources of local genomic shattering underpinning chromothripsis are under investigation. Notably, many consequences of DSB clusters in the affected genome reflect processing by pathways that have evolved to repair DSBs, but which operate with widely different degrees of fidelity. The molecular underpinnings and the basis of the underlying repair pathway choices that ultimately lead to the observed consequences from DSB clusters remain unknown. We developed a tractable model of DSB clustering that allows direct analysis in cells of the consequences of certain configurations of DSB clusters. We outline the rationale for the development of this model and describe its key characteristics. We summarize results suggesting that DSB clusters compromise the first-line DSB-processing pathways of c-NHEJ and HRR, increasing as a consequence the contribution of alt-EJ, which has high propensity of generating chromosomal rearrangements. The results suggest a mechanism for the increased toxicity of high LET radiation and the extensive genomic rearrangements associated with chromothripsis.
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