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

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Arthroscopic Repair of a Posterior Bony Bankart Lesion

Posterior bony defects of the glenoid rim, particularly those associated with instability, are often a frustrating challenge for arthroscopists because of the defects' inaccessibility from standard portals. This challenge is enhanced when the lesion is chronic and fibrous malunion of the fragment makes mobilization difficult. We present our technique for arthroscopic repair of the relatively uncommon chronic posterior bony Bankart lesion. By use of lateral positioning and a standard anterior viewing portal and posterior working portal, as well as a strategically placed posterolateral accessory portal, the lesion is first freed from its malreduced position and ultimately repaired using suture anchor fixation of the bony fragment along with its associated labrum directly to the remaining glenoid rim. This technique, facilitated by precise portal placement, results in satisfactory fragment reduction, appropriate capsular tension, and restoration of anatomy

Role of ATM and the Damage Response Mediator Proteins 53BP1 and MDC1 in the Maintenance of G2/M Checkpoint Arrest▿ †

ATM-dependent initiation of the radiation-induced G2/M checkpoint arrest is well established. Recent results have shown that the majority of DNA double-strand breaks (DSBs) in G2 phase are repaired by DNA nonhomologous end joining (NHEJ), while ∼15% of DSBs are slowly repaired by homologous recombination. Here, we evaluate how the G2/M checkpoint is maintained in irradiated G2 cells, in light of our current understanding of G2 phase DSB repair. We show that ATM-dependent resection at a subset of DSBs leads to ATR-dependent Chk1 activation. ATR-Seckel syndrome cells, which fail to efficiently activate Chk1, and small interfering RNA (siRNA) Chk1-treated cells show premature mitotic entry. Thus, Chk1 significantly contributes to maintaining checkpoint arrest. Second, sustained ATM signaling to Chk2 contributes, particularly when NHEJ is impaired by XLF deficiency. We also show that cells lacking the mediator proteins 53BP1 and MDC1 initially arrest following radiation doses greater than 3 Gy but are subsequently released prematurely. Thus, 53BP1−/− and MDC1−/− cells manifest a checkpoint defect at high doses. This failure to maintain arrest is due to diminished Chk1 activation and a decreased ability to sustain ATM-Chk2 signaling. The combined repair and checkpoint defects conferred by 53BP1 and MDC1 deficiency act synergistically to enhance chromosome breakage

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

AP endonuclease paralogues with distinct activities in DNA repair and bacterial pathogenesis.

Oxidative stress is a principal cause of DNA damage, and mechanisms to repair this damage are among the most highly conserved of biological processes. Oxidative stress is also used by phagocytes to attack bacterial pathogens in defence of the host. We have identified and characterised two apurinic/apyrimidinic (AP) endonuclease paralogues in the human pathogen Neisseria meningitidis. The presence of multiple versions of DNA repair enzymes in a single organism is usually thought to reflect redundancy in activities that are essential for cellular viability. We demonstrate here that these two AP endonuclease paralogues have distinct activities in DNA repair: one is a typical Neisserial AP endonuclease (NApe), whereas the other is a specialised 3′-phosphodiesterase Neisserial exonuclease (NExo). The lack of AP endonuclease activity of NExo is shown to be attributable to the presence of a histidine side chain, blocking the abasic ribose-binding site. Both enzymes are necessary for survival of N. meningitidis under oxidative stress and during bloodstream infection. The novel functional pairing of NExo and NApe is widespread among bacteria and appears to have evolved independently on several occasions