Chromosome breakage after G2 checkpoint release

DNA double-strand break (DSB) repair and checkpoint control represent distinct mechanisms to reduce chromosomal instability. Ataxia telangiectasia (A-T) cells have checkpoint arrest and DSB repair defects. We examine the efficiency and interplay of ATM's G2 checkpoint and repair functions. Artemis cells manifest a repair defect identical and epistatic to A-T but show proficient checkpoint responses. Only a few G2 cells enter mitosis within 4 h after irradiation with 1 Gy but manifest multiple chromosome breaks. Most checkpoint-proficient cells arrest at the G2/M checkpoint, with the length of arrest being dependent on the repair capacity. Strikingly, cells released from checkpoint arrest display one to two chromosome breaks. This represents a major contribution to chromosome breakage. The presence of chromosome breaks in cells released from checkpoint arrest suggests that release occurs before the completion of DSB repair. Strikingly, we show that checkpoint release occurs at a point when approximately three to four premature chromosome condensation breaks and approximately 20 gammaH2AX foci remain

An imperfect G2M checkpoint contributes to chromosome instability following irradiation of S and G2 phase cells

DNA double strand break (DSB) repair and checkpoint control represent two major mechanisms that function to reduce chromosomal instability following ionising irradiation (IR). Ataxia telangiectasia (A-T) cells have long been known to have defective checkpoint responses. Recent studies have shown that they also have a DSB repair defect following IR raising the issue of how ATM’s repair and checkpoint functions interplay to maintain chromosomal stability. A-T and Artemis cells manifest an identical and epistatic repair defect throughout the cell cycle demonstrating that ATM’s major repair defect following IR represents Artemis-dependent end-processing. Artemis cells show efficient G2/M checkpoint induction and a prolonged arrest relative to normal cells. Following irradiation of G2 cells, this checkpoint is dependent on ATM and A-T cells fail to show checkpoint arrest. In contrast, cells irradiated during S phase initiate a G2/M checkpoint which is independent of ATM and, significantly, both Artemis and A-T cells show a prolonged arrest at the G2/M checkpoint likely reflecting their repair defect. Strikingly, the G2/M checkpoint is released before the completion of repair when approximately 10-20 DSBs remain both for S phase and G2 phase irradiated cells. This defined sensitivity level of the G2/M checkpoint explains the prolonged arrest in repair-deficient relative to normal cells and provides a conceptual framework for the co-operative phenotype between checkpoint and repair functions in maintaining chromosomal stability

ATM and Artemis promote homologous recombination of radiation-induced DNA double-strand breaks in G2

Homologous recombination (HR) and non‐homologous end joining (NHEJ) represent distinct pathways for repairing DNA double‐strand breaks (DSBs). Previous work implicated Artemis and ATM in an NHEJ‐dependent process, which repairs a defined subset of radiation‐induced DSBs in G1‐phase. Here, we show that in G2, as in G1, NHEJ represents the major DSB‐repair pathway whereas HR is only essential for repair of ∼15% of X‐ or γ‐ray‐induced DSBs. In addition to requiring the known HR proteins, Brca2, Rad51 and Rad54, repair of radiation‐induced DSBs by HR in G2 also involves Artemis and ATM suggesting that they promote NHEJ during G1 but HR during G2. The dependency for ATM for repair is relieved by depleting KAP‐1, providing evidence that HR in G2 repairs heterochromatin‐associated DSBs. Although not core HR proteins, ATM and Artemis are required for efficient formation of single‐stranded DNA and Rad51 foci at radiation‐induced DSBs in G2 with Artemis function requiring its endonuclease activity. We suggest that Artemis endonuclease removes lesions or secondary structures, which inhibit end resection and preclude the completion of HR or NHEJ

CtIP and MRN promote non-homologous end-joining of etoposide-induced DNA double-strand breaks in G1

Topoisomerases class II (topoII) cleave and re-ligate the DNA double helix to allow the passage of an intact DNA strand through it. Chemotherapeutic drugs such as etoposide target topoII, interfere with the normal enzymatic cleavage/re-ligation reaction and create a DNA double-strand break (DSB) with the enzyme covalently bound to the 5′-end of the DNA. Such DSBs are repaired by one of the two major DSB repair pathways, non-homologous end-joining (NHEJ) or homologous recombination. However, prior to repair, the covalently bound topoII needs to be removed from the DNA end, a process requiring the MRX complex and ctp1 in fission yeast. CtIP, the mammalian ortholog of ctp1, is known to promote homologous recombination by resecting DSB ends. Here, we show that human cells arrested in G0/G1 repair etoposide-induced DSBs by NHEJ and, surprisingly, require the MRN complex (the ortholog of MRX) and CtIP. CtIP's function for repairing etoposide-induced DSBs by NHEJ in G0/G1 requires the Thr-847 but not the Ser-327 phosphorylation site, both of which are needed for resection during HR. This finding establishes that CtIP promotes NHEJ of etoposide-induced DSBs during G0/G1 phase with an end-processing function that is distinct to its resection function

Glioma-amplified sequence KUB3 influences double-strand break repair after ionizing radiation.

Human glioblastomas are characterized by frequent DNA amplifications most often at chromosome regions 7p11.2 and 12q13-15. Although amplification is a well-known hallmark of glioblastoma genetics the function of most amplified genes in glioblastoma biology is not understood. Previously, we cloned Ku70-binding protein 3 (KUB3) from the amplified domain at 12q13-15. Here, we report that glioblastoma cell cultures with endogenous KUB3 gene amplification and with elevated KUB3 protein expression show an efficient double-strand break (DSB) repair after being irradiated with 1 Gy. A significantly less efficient DSB repair was found in glioma cell cultures without KUB3 amplification and expression. Furthermore, we found that a siRNA-mediated reduction of the endogenous KUB3 expression in glioblastoma cells resulted in a reduction of the repair efficiency. HeLa cells transfected with KUB3 showed an increased DSB repair in comparison to untreated HeLa cells. In addition, KUB3 seems to influence DSB efficiency via the DNA-PK-dependent repair pathway as shown by simultaneous inhibition of KUB3 and DNA-PK. The data provide the first evidence for a link between the level of KUB3 amplification and expression in glioma and DSB repair efficiency

Elevated radiation-induced γH2AX foci in G2 phase heterozygous BRCA2 fibroblasts.

BACKGROUND AND PURPOSE: About 5-10% of all breast cancer cases are associated with heterozygous germ-line mutations in the genes encoding BRCA1 and BRCA2. Carriers of such mutations are highly predisposed for developing breast or ovarian cancer and, thus, are advised to undergo regular radio-diagnostic examinations. BRCA1 and BRCA2 are involved in multiple cellular processes including the repair of ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) and different studies addressing the DSB repair capacity of BRCA1+/- or BRCA2+/- cells led to contradictory results. MATERIALS AND METHODS: Using the sensitive method of γH2AX foci analysis in combination with cell cycle markers, we specifically measured DSB repair in confluent G0 as well as in exponentially growing G1 and G2 phase primary WT, BRCA1+/- and BRCA2+/- fibroblasts. RESULTS: Both BRCA1+/- and BRCA2+/- cells displayed normal DSB repair in G0 and in G1. In contrast, in G2, BRCA2+/- but not BRCA1+/- cells exhibited a decreased DSB repair capacity which was in between that of WT and that of a hypomorphic BRCA2-/- cell line. CONCLUSIONS: The residual amount of normal BRCA1 seems to be sufficient for efficient DSB repair in all cell cycle phases, while the decreased DSB repair capacity of heterozygous BRCA2 mutations suggests gene dosage effects in G2

Glioma-amplified sequence KUB3 influences double-strand break repair after ionizing radiation

Human glioblastomas are characterized by frequent DNA amplifications most often at chromosome regions 7p11.2 and 12q13-15. Although amplification is a well-known hallmark of glioblastoma genetics the function of most amplified genes in glioblastoma biology is not understood. Previously, we cloned Ku70-binding protein 3 (KUB3) from the amplified domain at 12q13-15. Here, we report that glioblastoma cell cultures with endogenous KUB3 gene amplification and with elevated KUB3 protein expression show an efficient double-strand break (DSB) repair after being irradiated with 1 Gy. A significantly less efficient DSB repair was found in glioma cell cultures without KUB3 amplification and expression. Furthermore, we found that a siRNA-mediated reduction of the endogenous KUB3 expression in glioblastoma cells resulted in a reduction of the repair efficiency. HeLa cells transfected with KUB3 showed an increased DSB repair in comparison to untreated HeLa cells. In addition, KUB3 seems to influence DSB efficiency via the DNA-PK-dependent repair pathway as shown by simultaneous inhibition of KUB3 and DNA-PK. The data provide the first evidence for a link between the level of KUB3 amplification and expression in glioma and DSB repair efficiency