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

Contribution of DNA repair and cell cycle checkpoint arrest to the maintenance of genomic stability

DNA damage response mechanisms encompass pathways of DNA repair, cell cycle checkpoint arrest and apoptosis. Together, these mechanisms function to maintain genomic stability in the face of exogenous and endogenous DNA damage. ATM is activated in response to double strand breaks and initiates cell cycle checkpoint arrest. Recent studies in human fibroblasts have shown that ATM also regulates a mechanism of end-processing that is required for a component of double strand break repair. Human fibroblasts rarely undergo apoptosis after ionising radiation and, therefore, apoptosis is not considered in our review. The dual function of ATM raises the question as to how the two processes, DNA repair and checkpoint arrest, interplay to maintain genomic stability. In this review, we consider the impact of ATM's repair and checkpoint functions to the maintenance of genomic stability following irradiation in G2. We discuss evidence that ATM's repair function plays little role in the maintenance of genomic stability following exposure to ionising radiation. ATM's checkpoint function has a bigger impact on genomic stability but strikingly the two damage response pathways co-operate in a more than additive manner. In contrast, ATM's repair function is important for survival post irradiation

The Maintenance of ATM Dependent G2/M Checkpoint Arrest Following Exposure to Ionizing Radiation

The G2/M checkpoint is important in preventing cells with unrepaired DNA double strand breaks (DSBs) entering mitosis, an event which is likely to result in genomic instability. We recently reported that checkpoint arrest is maintained until close to completion of DSB repair and that the duration of checkpoint arrest depends on the dose and DSB repair capacity rather than lasting for a fixed period of time. ATM leads to phosphorylation of Chk1/2 in G2 phase following exposure to ionizing radiation. These transducer kinases can phosphorylate and inhibit Cdc25 activity, which is the phosphatase regulating mitotic entry. In this study we dissect three processes that contribute to the maintenance of checkpoint arrest in irradiated G2 phase cells. First, the ATR-Chk1 pathway contributes to maintaining checkpoint arrest, although it is dispensable for the initial activation of checkpoint arrest. Second, ongoing ATM to Chk2 signalling from unrepaired DSBs contributes to checkpoint arrest. This process plays a greater role in a repair defective background. Finally, slow decay of the initially activated Chk2 also contributes to the maintenance of checkpoint arrest. 53BP1 and MDC1 defective cells show an initial checkpoint defect after low doses but are proficient in initial activation of arrest after high doses. After higher radiation doses, however, 53BP1-/- and MDC1-/- MEFs fail to maintain checkpoint arrest. Furthermore 53BP1-/- and MDC1-/- MEFs display elevated mitotic breakage even after high doses. We show that the defect in the maintenance of checkpoint arrest conferred by 53BP1 and MDC1 deficiency substantially enhances chromosome breakage

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

How cancer cells hijack DNA double-strand break repair pathways to gain genomic instability

DNA double-strand breaks (DSBs) are a significant threat to the viability of a normal cell, since they can result in loss of genetic material if mitosis or replication is attempted in their presence. Consequently, evolutionary pressure has resulted in multiple pathways and responses to enable DSBs to be repaired efficiently and faithfully. Cancer cells, which are under pressure to gain genomic instability, have a striking ability to avoid the elegant mechanisms by which normal cells maintain genomic stability. Current models suggest that in normal cells DSB repair occurs in a hierarchical manner that promotes rapid and efficient rejoining first, with the utilisation of additional steps or pathways of diminished accuracy if rejoining is unsuccessful or delayed. We evaluate the fidelity of DSB repair pathways and discuss how cancer cells promote the utilisation of less accurate processes. Homologous recombination serves to promote accuracy and stability during replication, providing a battlefield for cancer to gain instability. Non-homologous end-joining, a major DSB repair pathway in mammalian cells, usually operates with high fidelity and only switches to less faithful modes if timely repair fails. The transition step is finely tuned and provides another point of attack during tumour progression. In addition to DSB repair, a DSB signalling response activates processes such as cell cycle checkpoint arrest, which enhance the possibility of accurate DSB repair. We will consider the ways by which cancers modify and accost these processes to gain genomic instabilit

Meromorphic Modular Forms with Rational Cycle Integrals

We study rationality properties of geodesic cycle integrals of meromorphic modular forms associated to positive definite binary quadratic forms. In particular, we obtain finite rational formulas for the cycle integrals of suitable linear combinations of these meromorphic modular forms

The Maintenance of ATM Dependent G2/M Checkpoint Arrest Following Exposure to Ionizing Radiation

The G2/M checkpoint is important in preventing cells with unrepaired DNA double strand breaks (DSBs) entering mitosis, an event which is likely to result in genomic instability. We recently reported that checkpoint arrest is maintained until close to completion of DSB repair and that the duration of checkpoint arrest depends on the dose and DSB repair capacity rather than lasting for a fixed period of time. ATM leads to phosphorylation of Chk1/2 in G2 phase following exposure to ionizing radiation. These transducer kinases can phosphorylate and inhibit Cdc25 activity, which is the phosphatase regulating mitotic entry. In this study we dissect three processes that contribute to the maintenance of checkpoint arrest in irradiated G2 phase cells. First, the ATR-Chk1 pathway contributes to maintaining checkpoint arrest, although it is dispensable for the initial activation of checkpoint arrest. Second, ongoing ATM to Chk2 signalling from unrepaired DSBs contributes to checkpoint arrest. This process plays a greater role in a repair defective background. Finally, slow decay of the initially activated Chk2 also contributes to the maintenance of checkpoint arrest. 53BP1 and MDC1 defective cells show an initial checkpoint defect after low doses but are proficient in initial activation of arrest after high doses. After higher radiation doses, however, 53BP1-/- and MDC1-/- MEFs fail to maintain checkpoint arrest. Furthermore 53BP1-/- and MDC1-/- MEFs display elevated mitotic breakage even after high doses. We show that the defect in the maintenance of checkpoint arrest conferred by 53BP1 and MDC1 deficiency substantially enhances chromosome breakage

γH2AX foci analysis for monitoring DNA double-strand break repair: strengths, limitations and optimization

DNA double-strand breaks (DSBs) represent an important radiation-induced lesion and impaired DSB repair provides the best available correlation with radiosensitivity. Physical techniques for monitoring DSB repair require high, non-physiological doses and cannot reliably detect subtle defects. One outcome from extensive research into the DNA damage response is the observation that H2AX, a variant form of the histone H2A, undergoes extensive phosphorylation at the DSB, creating gammaH2AX foci that can be visualized by immunofluorescence. There is a close correlation between gammaH2AX foci and DSB numbers and between the rate of foci loss and DSB repair, providing a sensitive assay to monitor DSB repair in individual cells using physiological doses. However, gammaH2AX formation can occur at single-stranded DNA regions which arise during replication or repair and thus does not solely correlate with DSB formation. Here, we present and discuss evidence that following exposure to ionizing radiation, gammaH2AX foci analysis can provide a sensitive monitor of DSB formation and repair and describe techniques to optimize the analysis. We discuss the limitations and benefits of the technique, enabling the procedure to be optimally exploited but not misused