Marked contribution of alternative end-joining to chromosome-translocation-formation by stochastically induced DNA double-strand-breaks in G2-phase human cells

Soni, Aashish; Siemann, Maria; Pantelias, Gabriel E.; Iliakis, George

doi:10.1016/j.mrgentox.2015.07.002

Cited by 33 publications

(34 citation statements)

References 66 publications

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“…Previous reports have suggested that MMEJ is a slow DSB repair process, which is stimulated during the G2 phase of the cell cycle (37,38). IR-induced genome damage causes arrest of dividing human cells in the G2/M phase (39).…”

Section: Resultsmentioning

confidence: 99%

Microhomology-mediated end joining is activated in irradiated human cells due to phosphorylation-dependent formation of the XRCC1 repair complex

et al. 2016

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Microhomology-mediated end joining (MMEJ), an error-prone pathway for DNA double-strand break (DSB) repair, is implicated in genomic rearrangement and oncogenic transformation; however, its contribution to repair of radiation-induced DSBs has not been characterized. We used recircularization of a linearized plasmid with 3΄-P-blocked termini, mimicking those at X-ray-induced strand breaks, to recapitulate DSB repair via MMEJ or nonhomologous end-joining (NHEJ). Sequence analysis of the circularized plasmids allowed measurement of relative activity of MMEJ versus NHEJ. While we predictably observed NHEJ to be the predominant pathway for DSB repair in our assay, MMEJ was significantly enhanced in preirradiated cells, independent of their radiation-induced arrest in the G2/M phase. MMEJ activation was dependent on XRCC1 phosphorylation by casein kinase 2 (CK2), enhancing XRCC1's interaction with the end resection enzymes MRE11 and CtIP. Both endonuclease and exonuclease activities of MRE11 were required for MMEJ, as has been observed for homology-directed DSB repair (HDR). Furthermore, the XRCC1 co-immunoprecipitate complex (IP) displayed MMEJ activity in vitro, which was significantly elevated after irradiation. Our studies thus suggest that radiation-mediated enhancement of MMEJ in cells surviving radiation therapy may contribute to their radioresistance and could be therapeutically targeted.

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Section: Resultsmentioning

confidence: 99%

Microhomology-mediated end joining is activated in irradiated human cells due to phosphorylation-dependent formation of the XRCC1 repair complex

et al. 2016

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“…One main reaction is the phosphorylation of the histone variant H2AX at serine 139 (S139) to obtain γH2AX through kinases such as ATM, ATR and DNA-PK1. The γH2AX domains occur in mega-base-pair (Mbp) large regions of the chromatin around DSB23456 and can be visualized as so-called ionizing radiation induced foci (IRIF)7. The recruitment and activation of proteins due to damage induction can later on lead to the repair of DSB.…”

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confidence: 99%

“…The cell has different repair mechanisms to properly rejoin the ends of a DSB, including the possibly error-prone non-homologous end joining (NHEJ)8 and the in most cases error-free homologous recombination (HR)9. HR is limited to the S/G2 cell cycle phase, due to the fact that a homologous sister chromatin is needed in close vicinity to the DSB as a template to repair the damaged chromatin234. As a backup pathway for failed NHEJ in G1 an alternative end-joining pathway (alt-EJ) has previously been identified, which works as a last resort, when the other pathways fail8.…”

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confidence: 99%

“…After the first reactions to DSB induction, such as phosphorylation of H2AX (γH2AX), the recruitment of downstream repair proteins starts for NHEJ as well as for HR. One of the key proteins accumulating in Mbp regions around the damage is the mediator protein 53BP1234, which can in most cell lines be found at all damage regions marked by γH2AX and plays a major role in NHEJ. When end-resection of damaged sites starts, NHEJ is impaired and proteins responsible for HR are recruited.…”

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Chromatin organization revealed by nanostructure of irradiation induced γH2AX, 53BP1 and Rad51 foci

Reindl

Girst

Walsh

et al. 2017

Sci Rep

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The spatial distribution of DSB repair factors γH2AX, 53BP1 and Rad51 in ionizing radiation induced foci (IRIF) in HeLa cells using super resolution STED nanoscopy after low and high linear energy transfer (LET) irradiation was investigated. 53BP1 and γH2AX form IRIF with same mean size of (540 ± 40) nm after high LET irradiation while the size after low LET irradiation is significantly smaller. The IRIF of both repair factors show nanostructures with partial anti-correlation. These structures are related to domains formed within the chromatin territories marked by γH2AX while 53BP1 is mainly situated in the perichromatin region. The nanostructures have a mean size of (129 ± 6) nm and are found to be irrespective of the applied LET and the labelled damage marker. In contrast, Rad51 shows no nanostructure and a mean size of (143 ± 13) nm independent of LET. Although Rad51 is surrounded by 53BP1 it strongly anti-correlates meaning an exclusion of 53BP1 next to DSB when decision for homologous DSB repair happened.Ionizing radiation induces a variety of different types of damage when targeted to living cells. Severe damage, which can influence cell survival or lead to carcinogenesis, occurs due to ionizing events in the DNA molecule itself. The most lethal of these types of DNA damages are the double-strand breaks (DSB), as they may lead to genetic alterations which in turn can be responsible for cell death or carcigonesis. Mammalian cells react with a variety of complex response mechanisms to DSB induction. One main reaction is the phosphorylation of the histone variant H2AX at serine 139 (S139) to obtain γ H2AX through kinases such as ATM, ATR and DNA-PK 1 . The γ H2AX domains occur in mega-base-pair (Mbp) large regions of the chromatin around DSB 2-6 and can be visualized as so-called ionizing radiation induced foci (IRIF) 7 . The recruitment and activation of proteins due to damage induction can later on lead to the repair of DSB. The cell has different repair mechanisms to properly rejoin the ends of a DSB, including the possibly error-prone non-homologous end joining (NHEJ) 8 and the in most cases error-free homologous recombination (HR) 9 . HR is limited to the S/G2 cell cycle phase, due to the fact that a homologous sister chromatin is needed in close vicinity to the DSB as a template to repair the damaged chromatin 2-4 . As a backup pathway for failed NHEJ in G1 an alternative end-joining pathway (alt-EJ) has previously been identified, which works as a last resort, when the other pathways fail 8 . Recent work analyzed the clustering of DSB repair factors in detail using high resolution microscopy 10-12 and nanoscopy 11,[13][14][15][16] in combination with state of the art correlation and clustering analysis methods. With these methods it is possible to gain a deeper understanding of the functionality of DSB repair proteins and their interactions. After the first reactions to DSB induction, such as phosphorylation of H2AX (γ H2AX), the recruitment of downstream repair proteins starts for NHEJ as well as f...

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“…Interestingly, however, it was recently proposed that the mechanisms of chromosomal translocation vary between species, such that translocations are predominantly formed by alternative end-joining (a-NHEJ or alt-EJ) pathways such as MMEJ in mouse cells and by c-NHEJ in human cells [54]. Nevertheless, subsequent studies have shown that a-NHEJ is a major source of chromosome translocations in human cells [55] and that CtIP in particular can promote the formation of chromosomal rearrangements in human cells [20]. Conceivably, the repair pathways that generate translocations may differ depending on tissue type rather than species.…”

Section: Discussionmentioning

confidence: 99%

The DNA resection protein CtIP promotes mammary tumorigenesis

et al. 2016

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Many DNA repair factors act to suppress tumor formation by preserving genomic stability. Similarly, the CtIP protein, which interacts with the BRCA1 tumor suppressor, is also thought to have tumor suppression activity. Through its role in DNA end resection, CtIP facilitates DNA double-strand break (DSB) repair by homologous recombination (DSBR-HR) and microhomology-mediated end joining (MMEJ). In addition, however, CtIP has also been implicated in the formation of aberrant chromosomal rearrangements in an MMEJ-dependent manner, an activity that could potentially promote tumor development by increasing genome instability. To clarify whether CtIP acts in vivo to suppress or promote tumorigenesis, we have examined its oncogenic potential in mouse models of human breast cancer. Surprisingly, mice heterozygous for a null Ctip allele did not display an increased susceptibility to tumor formation. Moreover, mammary-specific biallelic CtIP ablation did not elicit breast tumors in a manner reminiscent of BRCA1 loss. Instead, CtIP inactivation dramatically reduced the kinetics of mammary tumorigenesis in mice bearing mammary-specific lesions of the p53 gene. Thus, unlike other repair factors, CtIP is not a tumor suppressor, but has oncogenic properties that can promote tumorigenesis, consistent with its ability to facilitate MMEJ-dependent chromosomal instability. Consequently, inhibition of CtIP-mediated MMEJ may prove effective against tumor types, such as human breast cancer, that display MMEJ-dependent chromosomal rearrangements.

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Marked contribution of alternative end-joining to chromosome-translocation-formation by stochastically induced DNA double-strand-breaks in G2-phase human cells

Cited by 33 publications

References 66 publications

Microhomology-mediated end joining is activated in irradiated human cells due to phosphorylation-dependent formation of the XRCC1 repair complex

Microhomology-mediated end joining is activated in irradiated human cells due to phosphorylation-dependent formation of the XRCC1 repair complex

Chromatin organization revealed by nanostructure of irradiation induced γH2AX, 53BP1 and Rad51 foci

The DNA resection protein CtIP promotes mammary tumorigenesis

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