PRISM :: Browsing by Author "Lees-Miller, Susan P."

Browsing by Author "Lees-Miller, Susan P."

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Open Access
An Analysis of Human Exposure to Alpha Particle Radiation
(2018-12-17) Stanley, Fintan; Goodarzi, Aaron A.; Lees-Miller, Susan P.; Cobb, Jennifer A.
High linear energy transfer (LET) ionizing radiation (IR) is the predominant source of IR humans are exposed to. Radon gas, which emits a high energy alpha-particle, represents the greatest single lifetime source, but also remains comparatively understudied versus low LET IR sources such as x-rays. The inhalation radon (222Rn) gas from indoor air exposes lung tissue to alpha particle radiation, damaging DNA and increasing the lifetime risk of lung cancer. Buildings can concentrate radioactive radon (222Rn) gas to harmful levels. To enable cancer prevention, I examined how Canadian Prairie radon exposure is modified by environmental design and human behavior and evaluated different radon test modalities. I also developed a high-throughput, benchtop alpha-particle irradiation system to facilitate future research into the biological consequences of high LET radiation exposure. Initially, I examined 90+ day radon test results from 2,382 residential homes from an area encompassing 82.5% of the Southern Alberta population. Remediated homes were retested to determine efficacy of radon reduction techniques in this region. Subsequently, 11,726 Alberta and Saskatchewan homes were radon tested, coupled to geographic, design and behavior metrics. Canadian Prairie homes contained 140 Bq/m3 average radon (min <15 Bq/m3; max 7,199 Bq/m3) and 17.8% were ≥ 200 Bq/m3. Geostatistical analysis indicates significant variation between regions. More recently constructed homes contain higher radon versus older. Finally, I also designed and validated a benchtop, 96 well plate-based 241Am irradiation system to expose cultured eukaryotic cells to alpha particles in a controlled environment. My validation of this novel setup includes quantification of nuclear alpha particle-induced DNA damage signalling (γH2AX) using a purpose-designed 3D analysis method, physical readouts of alpha particle-induced DNA damage by alkaline comet assay, and an investigation of cellular viability after alpha particle exposure. This method brings significant advances over existing techniques in its ease of setup and use, affordability, accessibility and flexibility and should enable future alpha particle radiation biology. Collectively, my work demonstrates that radon is a genuine public health concern in the Canadian Prairies, legitimatizes efforts to understand the consequences of radon exposure to the public, and suggest that radon testing and mitigation is likely to be an impactful cancer prevention strategy.
Open Access
ATM and the cellular response to ionizing radiation-induced DNA damage
(2005) Goodarzi, Aaron A.; Lees-Miller, Susan P.
Open Access
Characterization of the BRCT domain of DNA polymerase in non-homologous end-joining
(2009) Knight, Christopher Paul; Lees-Miller, Susan P.
Open Access
Characterization of the interaction of nuclear factors associated with double-stranded RNA with the DNA-dependent protein Kinase
(2002) Knorr, Laina M.; Lees-Miller, Susan P.
Open Access
DNA damage induced phosphorylation of artemis
(2008) Hiebert, Stephanie E.; Lees-Miller, Susan P.
Open Access
DNA damage signaling in response to etoposide
(2005) Siponen, Marina I.; Lees-Miller, Susan P.
Open Access
Estrogen increases radiation resistance and induces DNA damage in an estrogen receptor alpha-dependent manner
(2011) Williamson, Laura Michelle; Lees-Miller, Susan P.
Open Access
Exploiting the synthetic lethal interaction of ATM and PARP to target ATM-deficient malignancy
(2011) Williamson, Christopher Thomas; Lees-Miller, Susan P.
Open Access
Identification and characterization of proteins that interact with the human DNA dependent protein kinase (DNA-PK)
(1999) Ting, Nicholas S. Y.; Lees-Miller, Susan P.
Open Access
Identifying physiological substrates of DNA-dependent protein kinase (DNA-PK)
(2004) Bader, Mohamed; Lees-Miller, Susan P.
The DNA-dependent protein kinase (DNA-PK) is required for the repair of DNA double strand breaks (DSB) in human cells. DNA-PK is a serine/threonine protein kinase composed of a 469 kDa catalytic subunit (DNA-PKcs) and a DNA targeting subunit, Ku. Previous studies have demonstrated a requirement of the protein kinase activity of DNA-PK during non homologous end joining-mediated repair of DNA DSBs and during variable (diversity) joining [V(D)J] recombination. However, its physiological substrates are not well defined. Here we have utilized a broad-based proteomics approach to attempt to identify DNA-PK-dependent phosphorylation events. Utilizing a more targeted approach involving immunoprecipitation of DNA-PKcs, an ionizing radiation-dependent interaction between DNA-PKcs and p53 transcription factor was identified in human lymphoblastoid cells.
Open Access
Integrative Structural Model of DNA-PKcs in the Initial Steps of Non-Homologous End Joining
(2020-06-04) Hepburn, Morgan Rose; Schriemer, David C.; Lees-Miller, Susan P.; Ng, Kenneth Kai Sing; Williams, Gareth J.; Huang, Lan
Non-homologous end joining (NHEJ) performs untemplated repair of DNA double strand breaks (DSBs). Despite lack of a template, intricate repair, coordinated by the core NHEJ factors, can repair breaks with minimal to no alterations. Initiating repair, Ku70/80 binds to the free DNA ends, and interacts with the large protein kinase, DNA dependent protein kinase catalytic subunit (DNA-PKcs), forming the holoenzyme DNA-PK. Holoenzymes can synapse across the break to tether the DNA ends. Assembly of the initial synaptic complex and its role in NHEJ is poorly understood, as final ligation requires a structural rearrangement of this initial complex. To better understand DNA-PKcs’ role in NHEJ, an integrative structural model of DNA-PKcs in the initial stages of NHEJ was developed using mass spectrometry (MS) techniques. Due to technical challenges working with DNA-PKcs, each of the MS techniques were optimized for the system. Hydrogen deuterium exchange (HX) methods were optimized on a nano-spray HX system, allowing for differential HX analysis of bead bound DNA-PKcs complexes with high sequence coverage, and 5X improvement in protein consumption. Reversible crosslinking and peptide fingerprinting (RCAP) was optimized to allow for direct detection of DNA binding peptides, using a single sample. Finally, given the benefits of DNA-PKcs complex assembly on beads to limit heterogeneity, an on-bead crosslinking method was developed. Mass Spec Studio was used to accurately identify many crosslinks, which can be utilized for a label free quantitation comparison of states. Using HX-MS to explore DNA-PKcs conformational changes from binding to activation of the kinase, an allosteric pathway was identified in DNA-PKcs connecting DNA-binding with the kinase domain. Nucleotide loading of the kinase domain revealed that DNA-PK occupies a tensed state when active. From integrative structural modelling, with the XL-MS restraints, a model with a precision of 13.5Å was reported, revealing a symmetric DNA-PK dimer, with head-to-head interactions. In our synaptic model, the DNA ends are positioned with a large offset, protected by a previously uncharacterized plug domain of DNA-PKcs. We propose the initial formation of the synaptic complex allows for a hierarchical processing of DNA ends and assembly of a core NHEJ scaffold.
Embargo
Investigating radiosensitivity and DNA repair in different neural cell types
(2020-05-12) Berger, Nelson Daniel; Chan, Jennifer A. W.; Goodarzi, Aaron A.; Lees-Miller, Susan P.; Yong, Voon Wee
Cranial radiotherapy (CRT) is an important and effective treatment for childhood brain cancers, but is also strongly associated with persistent neurocognitive decline, a heightened risk of secondary radiation-induced malignancies and altered white matter developmental trajectory. These late effects of CRT increase in severity the younger a patient is treated. Despite substantial advances in radiotherapy planning and treatment, late effects remain an issue to the ever-increasing group of childhood cancer survivors. The cellular and molecular mechanisms of these late effects are largely unknown, and the subtleties of how a developing, pediatric brain interacts with the ionizing radiation (IR) requires further investigation. This study aimed to characterize how distinct neural lineages present in a developing brain respond to IR, and how these cells repair DNA damage incurred by IR. Cells of the oligodendrocyte lineage, and particularly, oligodendrocyte progenitor cells (OPCs), exhibit marked radiosensitivity relative to other neural cell types, both post-mitotic and replicative. These OPCs are sensitive to a single dose of IR as low as 0.25 Gy, and display impaired DNA double-strand break (DSB) repair kinetics with a high baseline level of accumulated DSBs. While high in reactive oxygen species, modulation of the oxidative stress levels of OPCs is ineffective in rescuing radiosensitivity or DSB levels at rest. Interestingly, OPCs are profoundly sensitive to small molecules that compromise replication fork stability, and have other indicators consistent with aberrant collapsed replication fork processing and repair. Though the reason for this replication phenomenon remains elusive, it may be related to a baseline difficulty engaging HR-directed fork repair and restart mechanisms. Altogether, these data provide cellular mechanisms for the sensitivity of the developing brain to IR. Highly radiosensitive OPCs, more abundant and proliferative in the pediatric brain, might be ablated by IR at a young age. This may contribute in part to the clinical late effects of pediatric cranial radiotherapy. Further understanding of this sensitive cell type might provide an opportunity to protect the developing brain from IR, decrease the severity of late effects of childhood CRT, and provide the safest, most effective treatment for childhood brain cancers.
Open Access
Nepenthesin from monkey cups for hydrogen/deuterium exchange mass spectrometry.
(MOLECULAR & CELLULAR PROTEOMICS, 2013-02) Rey, Martial; Yang, Menglin; Burns, Kyle M.; Yu, Yaping; Lees-Miller, Susan P.; Schriemer, David C.
Open Access
Physical and genetic interactions involving the nonhomologous end-joining factor XLF/Nej1
(2013-07-09) Mahaney, Brandi Lynn; Lees-Miller, Susan P.; Cobb, Jennifer A.
DNA double-strand breaks (DSBs) are potentially toxic lesions. Maintenance of genomic stability requires the proper repair of DSBs by either the nonhomologous end-joining (NHEJ) pathway or the homologous recombination (HR) pathway. The XRCC4-like factor (XLF) and the NHEJ defective protein 1 (Nej1), stimulate the repair of DSBs by NHEJ in humans and Saccharomyces cerevisiae, respectively. The precise mechanism(s) through which XLF/Nej1 functions remain poorly understood. I show that the C-terminal region (CTR) of XLF is required for DNA binding and contains a conserved phenylalanine that is important for its interaction with Ku70/80 and for recruitment of XLF to DSBs in vivo. Surprisingly, I find that loss of the XLF-Ku interaction does not affect repair as measured by γ-H2AX foci resolution in vivo. In contrast, I show that while the CTR of the XLF homolog in S. cerevisiae, Nej1, is not required for recruitment to DSBs in vivo, it is critical for NHEJ in vivo. Previous studies by other groups have demonstrated a crucial role for the interaction between XLF and X-ray repair cross-complementing protein 4 (XRCC4) for NHEJ in vitro and in vivo. Similarly, I find that the interaction between Nej1 and the S. cerevisiae homolog of XRCC4, ligase interacting factor 1 (Lif1), correlates with NHEJ activity in vivo. These results bolster the current model of XLF function in which XLF/Nej1 stimulates NHEJ through its interaction with XRCC4/Lif1. In addition, my results uncover important differences in how XLF and Nej1 provide this function and provide useful tools for future investigation of the precise structural mechanism underlying their stimulation of NHEJ. I have also uncovered novel genetic interactions between an uncharacterized yeast gene, maintenance of telomere capping 5 (MTC5), the HR factor RAD52, and NHEJ factors including NEJ1. I show that MTC5 does not regulate NHEJ, but does regulate telomere length and dNTP levels. Based on a recent report and previously reported genetic interactions involving MTC5, I propose that the telomere length defects observed in mtc5Δ cells and the genetic interactions between MTC5, RAD52, and NHEJ factors may result from decreased dNTP levels and defects in amino acid uptake.
Open Access
Preventing the Emergence of Temozolomide Resistance in Glioblastoma by PARP Inhibition
(2019-05-15) Yuan, Alice; Cairncross, John Gregory; Lees-Miller, Susan P.; Roldán, Gloria B.
Temozolomide (TMZ) is active against the subset of glioblastomas (GBMs) in which the O6-methylguanine DNA-methyltransferase (MGMT) gene is silenced by promoter methylation. Unfortunately, virtually all MGMT-methylated tumors acquire TMZ resistance and regrow in patients. In a preliminary study, we showed that inhibition of the DNA repair enzymes poly (ADP-ribose) polymerase-1 and -2 (collectively, ‘PARP’) reverses TMZ resistance in patient-derived GBM and oligodendroglioma cell lines (Chapter 3). While this suggests that PARP inhibitors may be useful for treating TMZ-resistant cases, we predict that greater clinical benefit can be derived from their use in TMZ-sensitive cases, where inhibition may prevent the emergence of TMZ-resistant cell populations. To test this hypothesis, we developed a model of acquired TMZ resistance in the MGMT- methylated GBM cell line U251N (Chapter 4). In this system, prolonged treatment of U251N with TMZ induces TMZ-resistant colonies of cells that frequently harbor mutations in and reduced expression of DNA mismatch repair genes, and less frequently re-express MGMT. In Chapter 5, we observed that co-treatment of the parent U251N line with TMZ and the PARP inhibitor ABT-888 prevents such resistant colonies from emerging. Moreover, co-treatment causes established resistant colonies to regress and may also enhance cell kill in TMZ-sensitive cellular subpopulations. For some colonies, however, sensitivity to PARP inhibition diminishes with increasing exposure to TMZ. Collectively, these observations suggest that PARP inhibitors may be optimally used early in newly-diagnosed, MGMT-methylated GBMs to prevent or delay progression to a TMZ-refractory disease state by targeting both TMZ-resistant and sensitive tumour subpopulations.
Open Access
Purification and biochemical characterization of human DNA-PK and ATM
(1999) Chan, Doug Duk Wah; Lees-Miller, Susan P.
Open Access
Replication protein A and the DNA-dependent protein kinase: targets and regulators of the DNA damage response
(2005) Block, Wesley D.; Lees-Miller, Susan P.
Open Access
Role of Cdk5rap2 in Cell Senescence and CENP-A-mediated Centromeric Chromatin Integrity
(2020-04-22) Wang, Xidi; Lee, Kee-Young; Mains, Paul E.; Lees-Miller, Susan P.; Riabowol, Karl T.
Cyclin-dependent kinase 5 (Cdk5) regulatory subunit-associated protein 2 (Cdk5rap2, also known as MCPH3) is one of the genes mutant in primary microcephaly (MCPH), an autosomal recessive neurodevelopmental disorder characterized by small brain and cognitive deficit. In fact, Cdk5rap2 is most abundant at the luminal surface of the brain’s ventricular zone, particularly in cells lining the ventricular wall where neural stem and progenitor cells reside. However, since Cdk5rap2 is also expressed in other tissues, it is not surprising that loss-of-function mutations in Cdk5rap2 are further associated with cochlear and retinal developmental defects as well as primordial dwarfism, a developmental disorder associated with small body size and other growth abnormalities. The molecular mechanisms by which Cdk5rap2 loss-of-function mutations cause these developmental disorders remain obscure. In this study, I show that loss of Cdk5rap2 induces cell senescence through activation of p53 and subsequent downregulation of the wild-type p53-induced phosphatase 1 (WIP1). Using BJ-5ta human foreskin fibroblast cells, I show that Cdk5rap2 loss causes increased (i) senescence-associated heterochromatin foci (SAHF), which colocalize with that SAHF marker, heterochromatin protein 1γ (HP1γ), (ii) SA-β-gal staining, (iii) p53 activation as measured by increased phosphorylation at Ser15, (iv) p16INK4a and p21CIP1, and (v) G0/G1 population, resulting in reduced cell proliferation. Interestingly, increased phosphorylation of p53 at Ser15 does not correlate with activation of the p53 Ser15 kinases, Ataxia Telangiectasia Mutated (ATM), checkpoint kinase 1 (Chk1) and checkpoint kinase 2 (Chk2), but correlates with decreased level of the p53 phosphatase, WIP1. Ectopic expression of WIP1 reverses the senescent phenotypes observed in cells depleted of Cdk5rap2, indicating that cell senescence due to loss of Cdk5rap2 is linked to down-regulation of WIP1. Since the WIP1 promoter contains an NF-κB binding site and β-catenin-associated NF-κB affects the expression of NF-κB target genes, I tested whether loss of Cdk5rap2 that reduces WIP1 level influences β-catenin expression. Indeed, I found that loss of Cdk5rap2 significantly reduces nuclear β-catenin level. Because β-catenin phosphorylation by GSK3β results in its degradation through the ubiquitin-proteasome pathway, I further examined whether Cdk5rap2 interacts with GSK3β and affects GSK3β activity. My studies show that Cdk5rap2 interacts with GSK3β and such interaction causes increased GSK3β phosphorylation and subsequent decrease in GSK3β activity. Consistently, Cdk5rap2-depleted cells exhibit decreased GSK3β phosphorylation and increased GSK3β activity. Depletion of GSK3β increases β-catenin and WIP1 levels while depletion of β-catenin inhibits WIP1 expression. These findings suggest that GSK3β activation due to Cdk5rap2 loss causes β-catenin phosphorylation and degradation, and subsequent downregulation of WIP1. The potent GSK3β inhibitor, TWS119, which increases β-catenin level and upregulates WIP1 expression, recues cell senescence due to Cdk5rap2 loss. TWS119 together with the potent WIP1 inhibitor, GSK2830371, causes recurrence of senescence due to Cdk5rap2 loss, suggesting that GSK3β activity controls the β-catenin/WIP1-mediated senescence due to Cdk5rap2 loss. Promoter bashing analysis by transfecting a luciferase reporter vector carrying wt WIP1 promoter (pGL3-WIP1) into cells depleted of β-catenin or Cdk5rap2 reveal reduced luciferase activity compared to cells that are not depleted of β-catenin or Cdk5rap2. Conversely, transfection with NF-κB binding site-deleted WIP1 (pGL3-WIP1-ΔκB) show no difference in luciferase activity in β-catenin- or Cdk5rap2-depleted and non-depleted cells. Reduced luciferase activity in Cdk5rap2-depleted cells transfected with pGL3-WIP1 is reversed by TWS119, suggesting that β-catenin/WIP1-mediated senescence due to Cdk5rap2 loss is controlled by GSK3β through regulation of the WIP1 promoter via β-catenin. Potentially, cell senescence contributes to the developmental disorders associated with loss-of-function of Cdk5rap2. In previous studies, loss of Cdk5rap2 or CENtromeric Protein A (CENP-A) was shown to trigger the occurrence of lagging chromosomes, leading me to investigate a possible link between these two proteins. I demonstrate that Cdk5rap2 loss causes reduced CENP-A expression while ectopic Cdk5rap2 expression in cells depleted of endogenous Cdk5rap2 restores CENP-A expression. In this regard, I found that Cdk5rap2 is a nuclear protein that acts as a positive transcriptional regulator of CENP-A. Cdk5rap2 interacts with the CENP-A promoter and upregulates CENP-A transcription. Loss of Cdk5rap2 particularly causes reduced centromeric CENP-A, which is known to induce kinetochore recruitment. I found that aside from manifesting lagging chromosomes, cells lacking Cdk5rap2, and thus CENP-A, show increased micronuclei and chromatin bridge formation, indicating that loss of Cdk5rap2 compromises centromeric chromatin integrity through downregulation of CENP-A.
Open Access
Role of cyclin-dependent kinase 5 (Cdk5) in mitochondrial permeability transition pore (mPTP) opening and intracellular Ca2+ dynamics
(2020-06-25) Navaneetha Krishnan, Saranya; Lee, Ki-Young; Riabowol, Karl T.; Lees-Miller, Susan P.; Shutt, Timothy; Braun, Andrew P.; Eitzen, Gary A.
Cyclin-dependent kinase 5 (Cdk5), which plays a role in the development and progression of many human cancers, localizes in the mitochondria, a key determinant of apoptotic cell death. Cdk5 is upregulated in breast cancer cells and Cdk5 loss increases cancer cell sensitivity to chemotherapeutic drugs. However, the molecular mechanism by which Cdk5 loss promotes cell death remains unclear. I hypothesized that Cdk5 loss activates the intrinsic apoptotic pathway in breast cancer cells. I demonstrated that Cdk5-deficient breast cancer cells exhibit increased mitochondrial depolarization, mitochondrial reactive oxygen species (mtROS) levels, and mitochondrial fragmentation that is associated with an increase in both intracellular Ca2+ level and calcineurin activity, and dynamin related protein 1 (DRP1) Ser637 dephosphorylation. To define mitochondria-mediated apoptotic pathway, I utilized various inhibitors of mitochondrial function. Apoptosis is completely prevented by mitochondrial permeability transition pore (mPTP) inhibition, almost fully inhibited by blocking ROS and unaffected by inhibition of mitochondrial fission, suggesting that apoptosis in breast cancer cells due to Cdk5 loss occurs via a novel mPTP-dependent mechanism that acts primarily through ROS increase. Since calcium is the major regulator of mPTP opening, I hypothesized that Cdk5 regulates intracellular calcium homeostasis. Using primary mouse embryonic fibroblasts (MEFs) isolated from Cdk5?/? mouse embryos, I showed that loss of Cdk5 increases inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release from internal stores. Cdk5 associates with and phosphorylates the IP3R1 Ca2+ channel at Ser421 and such phosphorylation controls IP3R1-mediated Ca2+ release as loss of Cdk5, and thus loss of IP3R1 Ser421 phosphorylation, triggers an increase in IP3R1-mediated Ca2+ release in Cdk5?/? MEFs. Analysis of subcellular fractions of MEFs demonstrates that Cdk5 localizes in the mitochondria-associated endoplasmic reticulum membrane (MAM) and Cdk5 loss causes increased ER-mitochondria tethering, a process required for Ca2+ transfer from the ER to the mitochondria. Inhibition of ER Ca2+ release or mitochondrial Ca2+ uptake in Cdk5?/? MEFs prevents mPTP opening, indicating that mPTP opening in Cdk5?/? MEFs is due to increased Ca2+ transfer from the ER to the mitochondria. Altogether, our findings suggest that Cdk5 regulates IP3R1-mediated calcium release and mitochondrial Ca2+ homeostasis that are disturbed upon Cdk5 loss, which lead to mPTP opening.
Open Access
XRCC4 Protein Interactions with XRCC4-like Factor (XLF) Create an Extended Grooved Scaffold for DNA Ligation and Double Strand Break Repair
(Journal of Biological Chemistry, 2011-09-16) Hammel, Michal; Rey, Martial; Yu, Yaping; Mani, Rajam S.; Classen, Scott; Liu, Mona; Pique, Michael E.; Fang, Shujuan; Mahaney, Brandi L.; Weinfeld, Michael; Schriemer, David C.; Lees-Miller, Susan P.; Tainer, John A.
The XRCC4-like factor (XLF)-XRCC4 complex is essential for nonhomologous end joining, the major repair pathway for DNA double strand breaks in human cells. Yet, how XLF binds XRCC4 and impacts nonhomologous end joining functions has been enigmatic. Here, we report the XLF-XRCC4 complex crystal structure in combination with biophysical and mutational analyses to define the XLF-XRCC4 interactions. Crystal and solution structures plus mutations characterize alternating XRCC4 and XLF head domain interfaces forming parallel super-helical filaments. XLF Leu-115 ("Leu-lock") inserts into a hydrophobic pocket formed by XRCC4 Met-59, Met-61, Lys-65, Lys-99, Phe-106, and Leu-108 in synergy with pseudo-symmetric β-zipper hydrogen bonds to drive specificity. XLF C terminus and DNA enhance parallel filament formation. Super-helical XLF-XRCC4 filaments form a positively charged channel to bind DNA and align ends for efficient ligation. Collective results reveal how human XLF and XRCC4 interact to bind DNA, suggest consequences of patient mutations, and support a unified molecular mechanism for XLF-XRCC4 stimulation of DNA ligation.