Mechanisms of ATM Activation.

The ataxia-telangiectasia mutated (ATM) protein kinase is a master regulator of the DNA damage response, and it coordinates checkpoint activation, DNA repair, and metabolic changes in eukaryotic cells in response to DNA double-strand breaks and oxidative stress. Loss of ATM activity in humans results in the pleiotropic neurodegeneration disorder ataxia-telangiectasia. ATM exists in an inactive state in resting cells but can be activated by the Mre11-Rad50-Nbs1 (MRN) complex and other factors at sites of DNA breaks. In addition, oxidation of ATM activates the kinase independently of the MRN complex. This review discusses these mechanisms of activation, as well as the posttranslational modifications that affect this process and the cellular factors that affect the efficiency and specificity of ATM activation and substrate phosphorylation. I highlight functional similarities between the activation mechanisms of ATM, phosphatidylinositol 3-kinases (PI3Ks), and the other PI3K-like kinases, as well as recent structural insights into their regulation.

C1QBP Promotes Homologous Recombination by Stabilizing MRE11 and Controlling the Assembly and Activation of MRE11/RAD50/NBS1 Complex.

MRE11 nuclease forms a trimeric complex (MRN) with RAD50 and NBS1 and plays a central role in preventing genomic instability. When DNA double-strand breaks (DSBs) occur, MRN is quickly recruited to the damage site and initiates DNA end resection; accordingly, MRE11 must be tightly regulated to avoid inefficient repair or nonspecific resection. Here, we show that MRE11 and RAD50 form a complex (MRC) with C1QBP, which stabilizes MRE11/RAD50, while inhibiting MRE11 nuclease activity by preventing its binding to DNA or chromatin. Upon DNA damage, ATM phosphorylates MRE11-S676/S678 to quickly dissociate the MRC complex. Either excess or insufficient C1QBP impedes the recruitment of MRE11 to DSBs and impairs the DNA damage response. C1QBP is highly expressed in breast cancer and positively correlates with MRE11 expression, and the inhibition of C1QBP enhances tumor regression with chemotherapy. By influencing MRE11 at multiple levels, C1QBP is, thus, an important player in the DNA damage response.

TOP2ß-Dependent Nuclear DNA Damage Shapes Extracellular Growth Factor Responses via Dynamic AKT Phosphorylation to Control Virus Latency.

The mTOR pathway integrates both extracellular and intracellular signals and serves as a central regulator of cell metabolism, growth, survival, and stress responses. Neurotropic viruses, such as herpes simplex virus-1 (HSV-1), also rely on cellular AKT-mTORC1 signaling to achieve viral latency. Here, we define a novel genotoxic response whereby spatially separated signals initiated by extracellular neurotrophic factors and nuclear DNA damage are integrated by the AKT-mTORC1 pathway. We demonstrate that endogenous DNA double-strand breaks (DSBs) mediated by Topoisomerase 2ß-DNA cleavage complex (TOP2ßcc) intermediates are required to achieve AKT-mTORC1 signaling and maintain HSV-1 latency in neurons. Suppression of host DNA-repair pathways that remove TOP2ßcc trigger HSV-1 reactivation. Moreover, perturbation of AKT phosphorylation dynamics by downregulating the PHLPP1 phosphatase led to AKT mis-localization and disruption of DSB-induced HSV-1 reactivation. Thus, the cellular genome integrity and environmental inputs are consolidated and co-opted by a latent virus to balance lifelong infection with transmission.

Single-Molecule Imaging Reveals How Mre11-Rad50-Nbs1 Initiates DNA Break Repair.

DNA double-strand break (DSB) repair is essential for maintaining our genomes. Mre11-Rad50-Nbs1 (MRN) and Ku70-Ku80 (Ku) direct distinct DSB repair pathways, but the interplay between these complexes at a DSB remains unclear. Here, we use high-throughput single-molecule microscopy to show that MRN searches for free DNA ends by one-dimensional facilitated diffusion, even on nucleosome-coated DNA. Rad50 binds homoduplex DNA and promotes facilitated diffusion, whereas Mre11 is required for DNA end recognition and nuclease activities. MRN gains access to occluded DNA ends by removing Ku or other DNA adducts via an Mre11-dependent nucleolytic reaction. Next, MRN loads exonuclease 1 (Exo1) onto the free DNA ends to initiate DNA resection. In the presence of replication protein A (RPA), MRN acts as a processivity factor for Exo1, retaining the exonuclease on DNA for long-range resection. Our results provide a mechanism for how MRN promotes homologous recombination on nucleosome-coated DNA.

The DNA damage sensor ATM kinase interacts with the p53 mRNA and guides the DNA damage response pathway.

BACKGROUND: The ATM kinase constitutes a master regulatory hub of DNA damage and activates the p53 response pathway by phosphorylating the MDM2 protein, which develops an affinity for the p53 mRNA secondary structure. Disruption of this interaction prevents the activation of the nascent p53. The link of the MDM2 protein-p53 mRNA interaction with the upstream DNA damage sensor ATM kinase and the role of the p53 mRNA in the DNA damage sensing mechanism, are still highly anticipated. METHODS: The proximity ligation assay (PLA) has been extensively used to reveal the sub-cellular localisation of the protein-mRNA and protein-protein interactions. ELISA and co-immunoprecipitation confirmed the interactions in vitro and in cells. RESULTS: This study provides a novel mechanism whereby the p53 mRNA interacts with the ATM kinase enzyme and shows that the L22L synonymous mutant, known to alter the secondary structure of the p53 mRNA, prevents the interaction. The relevant mechanistic roles in the DNA Damage Sensing pathway, which is linked to downstream DNA damage response, are explored. Following DNA damage (double-stranded DNA breaks activating ATM), activated MDMX protein competes the ATM-p53 mRNA interaction and prevents the association of the p53 mRNA with NBS1 (MRN complex). These data also reveal the binding domains and the phosphorylation events on ATM that regulate the interaction and the trafficking of the complex to the cytoplasm. CONCLUSION: The presented model shows a novel interaction of ATM with the p53 mRNA and describes the link between DNA Damage Sensing with the downstream p53 activation pathways; supporting the rising functional implications of synonymous mutations altering secondary mRNA structures.

Interior modification of Macrobrachium rosenbergii nodavirus-like particle enhances encapsulation of VP37-dsRNA against shrimp white spot syndrome infection.

BACKGROUND: Application of a virus-like particle (VLP) as a nanocontainer to encapsulate double stranded (ds)RNA to control viral infection in shrimp aquaculture has been extensively reported. In this study, we aimed at improving VLP's encapsulation efficiency which should lead to a superior fighting weapon with disastrous viruses. RESULTS: We constructed 2 variants of chimeric Macrobrachium rosenbergii nodavirus (MrNV)-like particles (V1- and V2-MrN-VLPs) and tested their efficiency to encapsulate VP37 double stranded RNA as well as WSSV protection in P. vannamei. Two types of short peptides, RNA-binding domain (RBD) and deca-arginine (10R) were successfully engineered into the interior surface of VLP, the site where the contact with VP37-dsRNA occurs. TEM and dynamic light scattering (DLS) analyses revealed that the chimeric VLPs remained their assembling property to be an icosahedral symmetric particle with a diameter of about 30 nm, similar to the original MrN-VLP particle. The superior encapsulation efficiency of VP37-dsRNA into V2-MrN-VLP was achieved, which was slightly better than that of V1-MrN-VLP but far better (1.4-fold) than its parental V0-MrN-VLP which the mole ratio of 7.5-10.5 for all VLP variants. The protection effect against challenging WSSV (as gauged from the level of VP37 gene and the remaining viral copy number in shrimp) was significantly improved in both V1- and V2-MrN-VLP compared with an original V0-MrN-VLP template. CONCLUSION: MrN-VLP (V0-) were re-engineered interiorly with RBD (V1-) and 10R (V2-) peptides which had an improved VP37-dsRNA encapsulation capability. The protection effect against WSSV infection through shrimp administration with dsRNA + V1-/V2-MrN VLPs was experimentally evident.

MRI findings correlate with difficult dissection during proximal hamstring repair and with postoperative sciatica.

OBJECTIVE: This study examines the correlation between MRI findings and difficult dissection during proximal primary hamstring repair and postoperative sciatica. MATERIALS AND METHODS: A total of 32 cases of surgically repaired hamstring tendon tears that underwent preoperative and postoperative MRI were divided into sciatica (n = 12) and control (n = 20) groups based on the presence or absence of postoperative sciatica. Cases were scored by two blinded musculoskeletal radiologists for imaging features associated with difficult surgical dissection and the development of subsequent sciatica. Intra- and interrater agreements, as well as correlation of MRI findings with symptoms (odds ratio, OR), were calculated. RESULTS: On preoperative MRI, diffuse hamstring muscle edema pattern suggestive of active denervation (OR 9.4-13.6), and greater sciatic perineural scar circumference (OR 1.9-2) and length (OR 1.2-1.3) were significantly correlated with both difficult dissection and postoperative sciatica. Preoperatively, a greater number of tendons torn (OR 3.3), greater tear cross-sectional area (CSA, OR 1.03), and increased nerve T2-weighted signal (OR 3.2) and greater perineural scar thickness (OR 1.7) were also associated with difficult dissection, but not postoperative sciatica. On postoperative MRI, hamstring denervation, sciatic nerve tethering to the hamstring tendon, and development of perineural scar and greater perineural scar extent were all significantly correlated with postoperative sciatica. CONCLUSION: Preoperative hamstring MRI demonstrates findings predictive of difficult sciatic nerve dissection; careful MRI evaluation of the nerve and for the presence and extent of perineural scar is important for preoperative planning. Preoperative and postoperative MRI both depict findings that correlate with postoperative sciatica.

Peripheral nerve involvement in hereditary spastic paraplegia characterized by quantitative magnetic resonance neurography.

BACKGROUND AND OBJECTIVES: Hereditary spastic paraplegias (HSPs) are heterogenous genetic disorders. While peripheral nerve involvement is frequent in spastic paraplegia 7 (SPG7), the evidence of peripheral nerve involvement in SPG4 is more controversial. We aimed to characterize lower extremity peripheral nerve involvement in SPG4 and SPG7 by quantitative magnetic resonance neurography (MRN). METHODS: Twenty-six HSP patients carrying either the SPG4 or SPG7 mutation and 26 age-/sex-matched healthy controls prospectively underwent high-resolution MRN with large coverage of the sciatic and tibial nerve. Dual-echo turbo-spin-echo sequences with spectral fat-saturation were utilized for T2-relaxometry and morphometric quantification, while two gradient-echo sequences with and without an off-resonance saturation rapid frequency pulse were applied for magnetization transfer contrast (MTC) imaging. HSP patients additionally underwent detailed neurologic and electroneurographic assessments. RESULTS: All microstructural (proton spin density [ρ], T2-relaxation time, magnetization transfer ratio) and morphometric (cross-sectional area) quantitative MRN markers were decreased in SPG4 and SPG7 indicating chronic axonopathy. ρ was superior in differentiating subgroups and identifying subclinical nerve damage in SPG4 and SPG7 without neurophysiologic signs of polyneuropathy. MRN markers correlated well with clinical scores and electroneurographic results. CONCLUSIONS: MRN characterizes peripheral nerve involvement in SPG4 and SPG7 as a neuropathy with predominant axonal loss. Evidence of peripheral nerve involvement in SPG4 and SPG7, even without electroneurographically manifest polyneuropathy, and the good correlation of MRN markers with clinical measures of disease progression, challenge the traditional view of the existence of HSPs with isolated pyramidal signs and suggest MRN markers as potential progression biomarkers in HSP.

Multi-echo in steady-state acquisition improves MRI image quality and lumbosacral radiculopathy diagnosis efficacy compared with T2 fast spin-echo sequence.

PURPOSE: This study compares the performance of a 4-min multi-echo in steady-state acquisition (MENSA) with a 6-min fast spin echo with variable flip angle (CUBE) protocol for the assessment of lumbosacral plexus nerve root lesions. METHODS: Seventy-two subjects underwent MENSA and CUBE sequences on a 3.0-T MRI scanner. Two musculoskeletal radiologists independently assessed the images for quality and diagnostic capability. A qualitative assessment scoring system for image quality and quantitative nerve signal-to-noise ratio (SNR) and iliac vein and muscle contrast-to-noise ratios (CNR) was applied. Using surgical reports as the reference, sensitivity, specificity, accuracy, and area under the receiver operating characteristic curves (AUC) were evaluated. Intraclass correlation coefficients (ICC) and weighted kappa were used to calculate reliability. RESULTS: MENSA image quality rating (3.679 ± 0.47) was higher than for CUBE images (3.038 ± 0.68), and MENSA showed higher mean nerve root SNR (36.935 ± 8.33 vs. 27.777 ± 7.41), iliac vein CNR (24.678 ± 6.63 vs. 5.210 ± 3.93), and muscle CNR (19.414 ± 6.07 vs. 13.531 ± 0.65) than CUBE (P < 0.05). Weighted kappa and ICC values indicated good reliability. Sensitivity, specificity, and accuracy of diagnosis based on MENSA images were 96.23%, 89.47%, and 94.44%, respectively, and AUC was 0.929, compared with 92.45%, 84.21%, 90.28%, and 0.883 for CUBE images. The two correlated ROC curves were not significantly different. Weighted kappa values for intraobserver (0.758) and interobserver (0.768-0.818) reliability were substantial to perfect. CONCLUSION: A time-efficient 4-min MENSA protocol exhibits superior image quality and high vascular contrast with the potential to produce high-resolution lumbosacral nerve root images.

The emerging role of MRI neurography in the diagnosis of chronic inguinal pain.

INTRODUCTION: Chronic pain is a frequent and notable complication after inguinal hernia repair, it has been extensively studied, but its management and diagnosis are still difficult. The cause of chronic pain following inguinal hernia surgery is usually multifactorial. This case series highlights the utility of MRI neurography (MRN) in evaluating the damage to inguinal nerves after a hernia repair, with surgical confirmation of the preoperative imaging findings. MATERIALS AND METHODS: A retrospective review was performed on patients who underwent inguinal mesh removal and triple denervation of the groin. Inclusion criteria included MRI neurography. All patients underwent surgical exploration of the inguinal canal for partial or complete mesh removal and triple denervation of the groin by the same senior surgeon. RESULTS: A total of nine patients who underwent triple denervation were included in this case series. MRN was then performed on 100% of patients. The postoperative mean VAS score adjusted for all patients was 1.6 (SD p), resulting in a 7.5 score difference compared to the preoperative VAS score (p). Since chronic groin pain can be a severely debilitating condition, diagnosis, and treatment become imperative. CONCLUSION: MRN can detect direct and indirect signs of neuropathy even in the absence of a detectable compressive cause aids in management and diagnosis by finding the precise site of injury, and grading nerve injury to aid pre-operative assessment for the nerve surgeon. Thus, it is a valuable diagnostic tool to help with the diagnosis of nerve injuries in the setting of post-inguinal hernia groin pain.

Dynamic Properties of the DNA Damage Response Mre11/Rad50 Complex.

DNA double-strand breaks (DSBs) are a significant threat to cell viability due to the induction of genome instability and the potential loss of genetic information. One of the key players for early DNA damage response is the conserved Mre11/Rad50 Nbs1/Xrs2 (MRN/X) complex, which is quickly recruited to the DNA's ruptured ends and is required for their tethering and their subsequent repair via different pathways. The MRN/X complex associates with several other proteins to exert its functions, but it also exploits sophisticated internal dynamic properties to orchestrate the several steps required to address the damage. In this review, we summarize the intrinsic molecular features of the MRN/X complex through biophysical, structural, and computational analyses in order to describe the conformational transitions that allow for this complex to accomplish its multiple functions.

Drosophila telomere capping protein HOAP interacts with DSB sensor proteins Mre11 and Nbs.

In eukaryotes, specific DNA-protein structures called telomeres exist at linear chromosome ends. Telomere stability is maintained by a specific capping protein complex. This capping complex is essential for the inhibition of the DNA damage response (DDR) at telomeres and contributes to genome integrity. In Drosophila, the central factors of telomere capping complex are HOAP and HipHop. Furthermore, a DDR protein complex Mre11-Rad50-Nbs (MRN) is known to be important for the telomere association of HOAP and HipHop. However, whether MRN interacts with HOAP and HipHop, and the telomere recognition mechanisms of HOAP and HipHop are poorly understood. Here, we show that Nbs interacts with Mre11 and transports the Mre11-Rad50 complex from the cytoplasm to the nucleus. In addition, we report that HOAP interacts with both Mre11 and Nbs. The N-terminal region of HOAP is essential for its co-localization with HipHop. Finally, we reveal that Nbs interacts with the N-terminal region of HOAP.

A gene dosage-dependent effect unveils NBS1 as both a haploinsufficient tumour suppressor and an essential gene for SHH-medulloblastoma.

AIMS: Inherited or somatic mutations in the MRE11, RAD50 and NBN genes increase the incidence of tumours, including medulloblastoma (MB). On the other hand, MRE11, RAD50 and NBS1 protein components of the MRN complex are often overexpressed and sometimes essential in cancer. In order to solve the apparent conundrum about the oncosuppressive or oncopromoting role of the MRN complex, we explored the functions of NBS1 in an MB-prone animal model. MATERIALS AND METHODS: We generated and analysed the monoallelic or biallelic deletion of the Nbn gene in the context of the SmoA1 transgenic mouse, a Sonic Hedgehog (SHH)-dependent MB-prone animal model. We used normal and tumour tissues from these animal models, primary granule cell progenitors (GCPs) from genetically modified animals and NBS1-depleted primary MB cells, to uncover the effects of NBS1 depletion by RNA-Seq, by biochemical characterisation of the SHH pathway and the DNA damage response (DDR) as well as on the growth and clonogenic properties of GCPs. RESULTS: We found that monoallelic Nbn deletion increases SmoA1-dependent MB incidence. In addition to a defective DDR, Nbn+/- GCPs show increased clonogenicity compared to Nbn+/+ GCPs, dependent on an enhanced Notch signalling. In contrast, full NbnKO impairs MB development both in SmoA1 mice and in an SHH-driven tumour allograft. CONCLUSIONS: Our study indicates that Nbn is haploinsufficient for SHH-MB development whereas full NbnKO is epistatic on SHH-driven MB development, thus revealing a gene dosage-dependent effect of Nbn inactivation on SHH-MB development.

Chronic arsenic exposure suppresses ATM pathway activation in human keratinocytes.

An estimated 220 million people worldwide are chronically exposed to inorganic arsenic (iAs) primarily as a result of drinking iAs-contaminated water. Chronic iAs exposure is associated with a plethora of human diseases including skin lesions and multi-organ cancers. iAs is a known clastogen, inducing DNA double strand breaks (DSBs) in both exposed human populations and in vitro. However, iAs does not directly interact with DNA, suggesting that other mechanisms, such as inhibition of DNA repair and DNA Damage Response (DDR) signaling, may be responsible for iAs-induced clastogenesis. Recent RNA-sequencing data from human keratinocytes (HaCaT cells) indicate that mRNAs for phosphatases important for resolution of DDR signaling are induced as a result of chronic iAs exposure prior to epithelial to mesenchymal transition. Here, we report that phosphorylation of ataxia telengectasia mutated (ATM) protein at a critical site (pSer1981) important for DDR signaling, and downstream CHEK2 activation, are significantly reduced in two human keratinocyte lines as a result of chronic iAs exposure. Moreover, RAD50 expression is reduced in both of these lines, suggesting that suppression of the MRE11-RAD50-NBS1 (MRN) complex may be responsible for reduced ATM activation. Lastly, we demonstrate that DNA double strand break accumulation and DNA damage is significantly higher in human keratinocytes with low dose iAs exposure. Thus, inhibition of the MRN complex in iAs-exposed cells may be responsible for reduced ATM activation and reduced DSB repair by homologous recombination (HR). As a result, cells may favor error-prone DSB repair pathways to fix damaged DNA, predisposing them to chromosomal instability (CIN) and eventual carcinogenesis often seen resulting from chronic iAs exposure.

Non-canonical ATM/MRN activities temporally define the senescence secretory program.

Senescent cells display senescence-associated (SA) phenotypic programs such as stable proliferation arrest (SAPA) and a secretory phenotype (SASP). Senescence-inducing persistent DNA double-strand breaks (pDSBs) cause an immediate DNA damage response (DDR) and SAPA, but the SASP requires days to develop. Here, we show that following the immediate canonical DDR, a delayed chromatin accumulation of the ATM and MRN complexes coincides with the expression of SASP factors. Importantly, histone deacetylase inhibitors (HDACi) trigger SAPA and SASP in the absence of DNA damage. However, HDACi-induced SASP also requires ATM/MRN activities and causes their accumulation on chromatin, revealing a DNA damage-independent, non-canonical DDR activity that underlies SASP maturation. This non-canonical DDR is required for the recruitment of the transcription factor NF-κB on chromatin but not for its nuclear translocation. Non-canonical DDR further does not require ATM kinase activity, suggesting structural ATM functions. We propose that delayed chromatin recruitment of SASP modulators is the result of non-canonical DDR signaling that ensures SASP activation only in the context of senescence and not in response to transient DNA damage-induced proliferation arrest.

Development of magnetic resonance imaging of brachial plexus neuralgia.

As the incidence of peripheral neurological diseases increases, the precise display of nerves becomes important in imaging examinations. Among them, the pain caused by brachial plexus neuropathy is very prominent, and the magnetic resonance imaging of nerve is quite complex and messy. This paper will systematically elaborate from the aspects of brachial plexus neuropathy, morphological and functional imaging, and post-processing.

Prospective pre-operative 3-T MR neurography peripheral nerve mapping of upper extremity amputations implanted with FAST-LIFE electrode interfaces of robotic hands: technical report.

BACKGROUND AND PURPOSE: Fascicular targeting of longitudinal intrafascicular electrode (FAST-LIFE) interface enables hand dexterity with exogenous electrical microstimulation for sensory restoration, custom neural recording hardware, and deep learning-based artificial intelligence for motor intent decoding. The purpose of this technical report from a prospective pilot study was to illustrate magnetic resonance neurography (MRN) mapping of hand and nerve anatomy in amputees and incremental value of MRN over electrophysiology findings in pre-surgical planning of FAST-LIFE interface (robotic hand) patients. MATERIALS AND METHODS: After obtaining informed consent, patients with upper extremity amputations underwent pre-operative 3-T MRN, X-rays, and electrophysiology. MRN findings were correlated with electrophysiology reports. Descriptive statistics were performed. RESULTS: Five patients of ages 21-59 years exhibited 3/5 partial hand amputations, and 2/5 transradial amputations on X-rays. The median and ulnar nerve end bulb neuromas measured 10.1 ± 3.04 mm (range: 5.5-14 mm, median: 10.5 mm) and 10.9 ± 7.64 mm (2-22 mm, 9.75 mm), respectively. The ADC of median and ulnar nerves were increased at 1.64 ± 0.1 × 10-3 mm2/s (range: 1.5-1.8, median: 1.64 × 10-3 mm2/s) and 1.70 ± 0.17 × 10-3 mm2/s (1.49-1.98 × 10-3 mm2/s, 1.65 × 10-3 mm2/s), respectively. Other identified lesions were neuromas of superficial branch of the radial nerve and anterior interosseous nerve. On electrophysiology, 2/5 reports were unremarkable, 2/5 showed mixed motor-sensory neuropathies of median and ulnar nerves along with radial sensory neuropathy, and 1/5 showed sensory neuropathy of lateral cutaneous nerve of the forearm. All patients regained naturalistic sensations and motor control of digits. CONCLUSION: 3-T MRN allows excellent demonstration of forearm and hand nerve anatomy, altered diffusion characteristics, and their neuromas despite unremarkable electrophysiology for pre-surgical planning of the FAST-LIFE (robotic hand) interfaces.

Neuropathy Score Reporting and Data System (NS-RADS): MRI Reporting Guideline of Peripheral Neuropathy Explained and Reviewed.

A standardized guideline and scoring system should be used for the MR imaging diagnosis of peripheral neuropathy. The MR imaging-based Neuropathy Score Reporting and Data System (NS-RADS) is a newly devised classification system (in press in AJR) that can be used to communicate both type and severity of peripheral neuropathy in the light of clinical history and examination findings. The spectrum of neuropathic conditions and peripheral nerve disorders covered in this system includes nerve injury, entrapment, neoplasm, diffuse neuropathy, and post-interventional states. This classification system also describes the temporal MR imaging appearances of regional muscle denervation changes. This review article is based on the multicenter validation study pre-published in American journal of Roentgenology and discusses technical considerations of optimal MR imaging for peripheral nerve evaluation and discusses the NS-RADS classification and its severity scales with illustration of conditions that fall under each classification. The readers can gain knowledge of the NS-RADS classification system and learn to apply it in their practices for improved inter-disciplinary communications and timely patient management.

HDAC Inhibitor Sodium Butyrate Attenuates the DNA Repair in Transformed but Not in Normal Fibroblasts.

Many cancer therapy strategies cause DNA damage leading to the death of tumor cells. The DNA damage response (DDR) modulators are considered as promising candidates for use in combination therapy to enhance the efficacy of DNA-damage-mediated cancer treatment. The inhibitors of histone deacetylases (HDACis) exhibit selective antiproliferative effects against transformed and tumor cells and could enhance tumor cell sensitivity to genotoxic agents, which is partly attributed to their ability to interfere with DDR. Using the comet assay and host-cell reactivation of transcription, as well as γH2AX staining, we have shown that sodium butyrate inhibited DNA double-strand break (DSB) repair of both endo- and exogenous DNA in transformed but not in normal cells. According to our data, the dysregulation of the key repair proteins, especially the phosphorylated Mre11 pool decrease, is the cause of DNA repair impairment in transformed cells. The inability of HDACis to obstruct DSB repair in normal cells shown in this work demonstrates the advantages of HDACis in combination therapy with genotoxic agents to selectively enhance their cytotoxic activity in cancer cells.

Relations between Structure and Zn(II) Binding Affinity Shed Light on the Mechanisms of Rad50 Hook Domain Functioning and Its Phosphorylation.

The metal binding at protein-protein interfaces is still uncharted territory in intermolecular interactions. To date, only a few protein complexes binding Zn(II) in an intermolecular manner have been deeply investigated. The most notable example of such interfaces is located in the highly conserved Rad50 protein, part of the Mre11-Rad50-Nbs1 (MRN) complex, where Zn(II) is required for homodimerization (Zn(Rad50)2). The high stability of Zn(Rad50)2 is conserved not only for the protein derived from the thermophilic archaeon Pyrococcus furiosus (logK12 = 20.95 for 130-amino-acid-long fragment), which was the first one studied, but also for the human paralog studied here (logK12 = 19.52 for a 183-amino-acid-long fragment). As we reported previously, the extremely high stability results from the metal-coupled folding process where particular Rad50 protein fragments play a critical role. The sequence-structure-stability analysis based on human Rad50 presented here separates the individual structural components that increase the stability of the complex, pointing to amino acid residues far away from the Zn(II) binding site as being largely responsible for the complex stabilization. The influence of the individual components is very well reflected by the previously published crystal structure of the human Rad50 zinc hook (PDB: 5GOX). In addition, we hereby report the effect of phosphorylation of the zinc hook domain, which exerts a destabilizing effect on the domain. This study identifies factors governing the stability of metal-mediated protein-protein interactions and illuminates their molecular basis.