Chromosome thripsis by DNA double strand break clusters causes enhanced cell lethality, chromosomal translocations and 53BP1-recruitment

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 translocation-formation compared to single-DSBs. Translocation-formation from DSB-clusters utilizes Parp1 activity, implicating alt-EJ in their formation. Immunofluorescence experiments show that single-DSBs and DSB-clusters uniformly provoke the formation of single γ-H2AX foci, suggesting similar activation of early DNA damage response (DDR). Live-cell imaging also shows similar single-focus recruitment of the early-response protein MDC1, to single-DSBs and DSB-clusters. Notably, the late DDR protein, 53BP1 shows in live-cell imaging strikingly stronger recruitment to DSB-clusters as compared to single-DSBs. This is the first report that chromatin thripsis, in the form of engineered DSB-clusters, compromises first-line DSB-repair pathways, allowing alt-EJ to function as rescuing-backup. DSB-cluster-formation is indirectly linked to the increased biological effectiveness of high ionization-density radiations, such as the alpha-particles emitted by radon gas or the heavy-ions utilized in cancer therapy. Our observations provide the first direct mechanistic explanation for this long-known effect.

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

“…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].…”

“…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.…”

“…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.…”

“…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].…”

A Whole-Genome Association Approach for Large-scaled Inter-species Trait

“…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.…”