Browsing by Subject "DNA damage"

In order to maintain genome integrity cells employ a set of well conserved DNA damage checkpoints. DNA damage checkpoints are active during interphase and serve to prevent mitosis with broken DNA. Mitosis with broken DNA is associated with DNA segregation errors, genome instability and even cell death in resulting daughter cells. It has recently has been appreciated that cells can compensate for damaged DNA during mitosis. However, little is known about this mitotic DNA damage response.

In this work, I have utilized a genetically tractable system to study mitotic DNA damage responses in Drosophila. During development, Drosophila rectal papillar cells undergo developmentally programmed inactivation of DNA damage responses. Following inactivation, papillar cells undergo two rounds of mitosis. We find that papillar cells fail to undergo cell death or high-fidelity DNA repair prior to mitosis and instead enter mitosis with DNA double stranded breaks (DSBs). Remarkably, papillar cells segregate acentric DNA fragments into daughter cells during mitosis resulting in viable daughter cells, normal organ development and function. Proper segregation and organ formation is dependent on the FANCONI Anemia gene FANCD2. Loss of FANCD2 results in unaligned acentric fragments and mis-segregation of broken DNA resulting acentric micronuclei formation. Mis-segregation of acentric DNA results in cell death and failure to form a developmentally normal and functional organ. Thus, we have uncovered a role for FANCD2 in mitotic DNA damage responses.

Additionally, we find that single-stranded DNA (ssDNA) is present during papillar cell mitosis following DNA DSB induction. ssDNA is present on both the edge of segregating and lagging DNA as well as spanning short regions between fragments of lagging DNA. The observation that ssDNA is present suggests that while papillar cells do not initiate complete repair, some level of DNA resection must occur following DNA DSB induction. In line with this reasoning, we find a role for the DNA damage sensor complex, the MRN complex, in papillar cell survival following I-Cre induction. The MRN complex consists of three components, Mre11, Rad50 and NBS1. Loss of Mre11 or NBS1 results in reduced papillar cell survival following I-Cre induction. Furthermore, Mre11 is a nuclease. Thus, we propose that MRE11 acts at sites of DNA DSBs in papillar cells to create ssDNA. We hypothesize that formation of ssDNA is sufficient to form a DNA/protein bridge between segregating and lagging DNA to enable proper DNA segregation. Interestingly, resistance to DNA damage is also observed in many cancers. We speculate that such DNA damage resistant cancer cells may utilize similar mechanisms to compensate for DNA breaks during mitosis.

Epstein-Barr virus (EBV) is an oncogenic gamma-herpesvirus that infects over 90% of adults worldwide. Typically, EBV establishes a benign latent infection that is controlled by a strong cytotoxic T cell immune response. However, EBV infection in immunocompromised patients has been associated with development of several lymphoid and epithelial cell malignancies, including Burkitt’s lymphoma, Hodgkin’s lymphoma, post-transplant lymphoproliferative disease, and nasopharyngeal carcinoma. In primary human B cells, EBV infection has been shown to induce a transient period of hyper-proliferation, but many of these infected cells succumb to a DNA damage response (DDR)-mediated growth arrest. We hypothesize that EBV infection triggers replicative stress early after infection and facilitates persistent activation of the DDR establishing oncogene-induced senescence. To test this hypothesis, we infected primary human B cells with EBV and examined cellular proliferation and host DNA damage response pathways at early and late stages post infection. We found that early after EBV infection, rapidly proliferating B cells exhibited signs of replication stress and reduced levels of purine dNTP nucleotide pools that are necessary for sustained proliferation. These findings suggest that purine dNTP biosynthesis plays a critical role in the early stages of EBV-mediated B cell immortalization. Furthermore, we observed the formation of persistent DDR foci in arrested B cells and identified key regulators of long-term outgrowth of EBV-infected B cells. Ultimately, this work has shown that early after EBV infection, cells that experience aberrant proliferation establish oncogene-induced senescence by chronically activating the DDR in the context of reduced dNTP nucleotide pools.

Mitochondria are responsible for producing the vast majority of cellular ATP, and are therefore critical to organismal health [1]. They contain thir own genomes (mtDNA) which encode 13 proteins that are all subunits of the mitochondrial respiratory chain (MRC) and are essential for oxidative phosphorylation [2]. mtDNA is present in multiple copies per cell, usually between 103 and 104 , though this number is reduced during certain developmental stages [3, 4]. The health of the mitochondrial genome is also important to the health of the organism, as mutations in mtDNA lead to human diseases that collectively affect approximately 1 in 4000 people [5, 6]. mtDNA is more susceptible than nuclear DNA (nucDNA) to damage by many environmental pollutants, for reasons including the absence of Nucleotide Excision Repair (NER) in the mitochondria [7]. NER is a highly functionally conserved DNA repair pathway that removes bulky, helix distorting lesions such as those caused by ultraviolet C (UVC) radiation and also many environmental toxicants, including benzo[a]pyrene (BaP) [8]. While these lesions cannot be repaired, they are slowly removed through a process that involves mitochondrial dynamics and autophagy [9, 10]. However, when present during development in C. elegans, this damage reduces mtDNA copy number and ATP levels [11]. We hypothesize that this damage, when present during development, will result in mitochondrial dysfunction and increase the potential for adverse outcomes later in life.

To test this hypothesis, 1st larval stage (L1) C. elegans are exposed to 3 doses of 7.5J/m2 ultraviolet C radiation 24 hours apart, leading to the accumulation of mtDNA damage [9, 11]. After exposure, many mitochondrial endpoints are assessed at multiple time points later in life. mtDNA and nucDNA damage levels and genome copy numbers are measured via QPCR and real-time PCR , respectively, every 2 day for 10 days. Steady state ATP levels are measured via luciferase expressing reporter strains and traditional ATP extraction methods. Oxygen consumption is measured using a Seahorse XFe24 extra cellular flux analyzer. Gene expression changes are measured via real time PCR and targeted metabolomics via LC-MS are used to investigate changes in organic acid, amino acid and acyl-carnitine levels. Lastly, nematode developmental delay is assessed as growth, and measured via imaging and COPAS biosort.

I have found that despite being removed, UVC induced mtDNA damage during development leads to persistent deficits in energy production later in life. mtDNA copy number is permanently reduced, as are ATP levels, though oxygen consumption is increased, indicating inefficient or uncoupled respiration. Metabolomic data and mutant sensitivity indicate a role for NADPH and oxidative stress in these results, and exposed nematodes are more sensitive to the mitochondrial poison rotenone later in life. These results fit with the developmental origin of health and disease hypothesis, and show the potential for environmental exposures to have lasting effects on mitochondrial function.

Lastly, we are currently working to investigate the potential for irreparable mtDNA lesions to drive mutagenesis in mtDNA. Mutations in mtDNA lead to a wide range of diseases, yet we currently do not understand the environmental component of what causes them. In vitro evidence suggests that UVC induced thymine dimers can be mutagenic [12]. We are using duplex sequencing of C. elegans mtDNA to determine mutation rates in nematodes exposed to our serial UVC protocol. Furthermore, by including mutant strains deficient in mitochondrial fission and mitophagy, we hope to determine if deficiencies in these processes will further increase mtDNA mutation rates, as they are implicated in human diseases.

Early life exposure to mitochondrial toxicants, including paraquat, rotenone, and manganese, has been hypothesized to promote early onset of genetic mitochondrial disorders as well as common degenerative diseases such as Parkinson's Disease and Alzheimer's Disease. This dissertation aimed to investigate the biochemical and physiological effects of early life exposure to mitochondrial genotoxicants during development in the whole organism modelC. elegans. In the first experiment, a panel of model mammalian neurotoxicants and heavy metal ions was screened for mitochondrial genotoxicity by measuring mitochondrial DNA (mtDNA) copy number and damage in C. elegans. Exposures to paraquat, cumene hydroperoxide, rotenone, maneb, cadmium (II) chloride, and manganese (II) chloride have no significant effect on the mtDNA : nuclear DNA (nuDNA) ratio; only exposure to paraquat resulted in higher mtDNA than nuDNA damage level.In the second experiment, a laboratory method was developed to generate persistent mtDNA damage in larval C. elegans using serial ultraviolent C (UVC) exposures. While the mitochondrial DNA damage persisted from L1 to L4 stage, there was no difference between mitochondrial copy number of the control and UVC treated worms. The UVC treatment significantly inhibited both ATP level and oxygen consumption 24 and 48 hr after the exposure, while the mitochondrial mRNA expression was inhibited 3 hr after the exposure. The pink-1 mutation, a mitochondrial serine/threonine-protein kinase involved in the mitophagy process, appeared to limit the growth inhibitory effect of UVC treatment and increase the mitochondrial DNA content of the organism. In the third experiment,larval C. elegans was exposed to UVC and paraquat and examined using differential interference contrast and fluorescence confocal microscopy. Both resulted in detectable, dose-dependent lesions in dopaminergic CEP neurons in adult C. elegans. Neither significant lesions in the GABAergic dorsal nerve cord nor any sign of pharyngeal necrosis were detected. This work demonstrated a mechanism in which early life exposure to mitochondrial genotoxicants could result in both biochemical and physiological changes in later stages of life, thereby highlighting the potential health hazard of time-delayed effects of these chemicals in the environment.

Mitosis involves the faithful segregation of two identical copies of chromosomes into two daughter cells. This process is highly regulated to maintain genome integrity, as mis-segregation of partial or whole chromosomes can lead to genomic instability. Cells are constantly exposed to both endogenous and exogenous forms of DNA damage, which if left unattended to, can contribute to mitotic errors. Cells therefore possess DNA damage responses (DDRs) which involves enacting cell cycle checkpoints, DNA damage repair, and in cases of extreme damage – cell death or senescence.While several lines of investigation have identified key mechanisms of the DDR during interphase of the cell cycle, there are several key questions that remain with regards to how cells deal with damage that persists into mitosis. Further, there is currently a gap in knowledge on the mechanisms, timing, and conditions in which different aspects of the DDR are active and coordinated. In this dissertation, I will demonstrate how I implemented genetic and imaging tools using our laboratory’s previously established model system, Drosophila rectal papillar cells [hereafter papillar cells]. Using this model, I studied (1) mechanisms of the DDR during mitosis, (2) mechanisms that act in the absence of key DDR components, and (3) novel regulators and protein-protein interactions of the mitotic DDR. This body of work contributes to the growing knowledge of how cells tolerate DNA damage that persists into mitosis.

To ensure genomic integrity, the genome must be accurately duplicated once and only once per cell division. DNA replication is tightly regulated by replication licensing mechanisms which ensure that origins only initiate replication once per cell cycle. Disruption of replication licensing mechanisms may lead to re-replication and genomic instability.

DNA licensing involves two steps including the assembly of the pre-replicative compelx at origins in G1 and the activation of pre-RC in S-phase. Cdt1, also known as Double-parked (Dup) in Drosophila Menalogaster , is a key regulator of the assembly of pre-RC and its activity is strictly limited to G1 by multiple mechanisms including Cul4Ddb1 mediated proteolysis and inhibitory binding by geminin. Previous studies have indicated that when the balance between Cdt1 and geminin is disrupted, re-replication occurs but the genome is only partially re-replicated. The exact sequences that are re-replicated and the mechanisms contributing to partial re-replication are unknown. To address these two questions, I assayed the genomic consequences of deregulating the replication licensing mechanisms by either RNAi depletion of geminin or Dup over-expression in cultured Drosophila Kc167 cells. In agreement with previously reported re-replication studies, I found that not all sequences were sensitive to geminin depletion or Dup over-expression. Microarray analysis and quantitative PCR revealed that heterochromatic sequences were preferentially re-replicated when Dup was deregulated either by geminin depletion or Dup over-expression. The preferential re-activation of heterochromatic replication origins was unexpected because these origins are typically the last sequences to be duplicated during normal S-phase.

In the case of geminin depletion, immunofluorescence studies indicated that the re-replication of heterochromatin was regulated not at the level of pre-RC activation, but rather due to the restricted formation of the pre-RC to the heterochromatin. Unlike the global assembly of the pre-RC that occurs throughout the genome in G1, in the absence of geminin, limited pre-RC assembly was restricted to the heterochromatin. Elevated cyclin A-CDK activity during S-phase could be one mechanism that prevents pre-RC reassembly at euchromatin when geminin is absent. These results suggest that there are chromatin and cell cycle specific controls that regulate the re-assembly of the pre-RC outside of G1.

In contrast to the specific re-replication of heterochromatin when geminin is absent, re-replication induced by Dup over-expression is not restricted to heterochromatin but rather includes re-activation of origins throughout the genome, although there is a slight preference for heterochromatin when re-replication is initiated. Surprisingly, Dup over-expression in G2 arrested cells result in a complete endoreduplication. In contrast to the ordered replication of euchromatin and heterochromatin during early and late S-phase respectively, endoreduplication induced by Dup over-expression does not exhibit any temporal order of replication initiation from these two types of chromatin, suggesting replication timing program may be uncoupled from local chromatin environment. Taken together, these findings suggest that the maintenance of proper levels of Dup protein is critical for genome integrity.

The ultimate goal of any living cell is to pass on a complete, unaltered copy of its DNA to its daughter cell. The DNA damage response (DDR) and spindle checkpoint are two essential signaling pathways that make it possible for a cell to achieve this goal. The DDR protects genetic integrity by sensing errors in the DNA sequence and activating signaling pathways to arrest the cell cycle and repair the DNA. The spindle checkpoint protects chromosomal integrity by preventing the separation of chromosomes during mitosis until all chromosomes are correctly attached to the mitotic spindle. Proper regulation of both the DDR and the spindle checkpoint is critical for cell survival. In this dissertation I will describe our discovery of novel regulatory mechanisms involved in each of these signaling networks.

In the first research chapter of this dissertation, we describe our findings concerning how the DDR regulates cyclin F levels. Cyclin F is an F-box protein that associates with the SCF E3 ubiquitin ligase complex to target proteins for degradation. In response to DNA damage, cyclin F levels are downregulated to facilitate increased dNTP production for efficient DNA repair, but the molecular mechanisms regulating this downregulation of cyclin F are largely unknown. We discovered that cyclin F downregulation by the DDR is the combined result of increased protein degradation and decreased mRNA expression. At the level of protein regulation, cyclin F is targeted for proteasomal degradation by the SCF complex. Interestingly, we found that the half-life of cyclin F protein is significantly increased in cells treated with the phosphatase inhibitor calyculin A, which caused cyclin F to be hyper-phosphorylated. Calyculin A also partially prevented cyclin F downregulation following DNA damage. This result suggests that cyclin F phosphorylation stabilizes the protein, and dephosphorylation of cyclin F may be required for its degradation in both unperturbed and DNA damaged cells. We also found that cyclin F downregulation is dependent on the Chk1 kinase, which is predominately activated by the ATR kinase. In examining the mechanism by which Chk1 promotes cyclin F downregulation, we determined that Chk1 represses cyclin F transcription. Lastly, we investigated the role of cyclin F in cell cycle regulation and discovered that both increased and decreased cyclin F expression delay mitotic entry, indicating that an optimal level of cyclin F expression is critical for proper cell cycle progression.

The second research chapter of this dissertation details our discovery of the requirement for phosphatase activity to inhibit the APC/C E3 ubiquitin ligase during the spindle checkpoint. Early in mitosis, the mitotic checkpoint complex (MCC) inactivates the APC/C until the chromosomes are properly aligned and attached to the mitotic spindle at metaphase. Once all the chromosomes are properly attached to the spindle, the MCC dissociates, and the APC/C targets cyclin B and securin for degradation so that the cell progresses into anaphase. While phosphorylation is known to drive many of the events during the checkpoint, the precise molecular mechanisms regulating spindle checkpoint maintenance and inactivation are still poorly understood. In our studies, we sought to determine the role of mitotic phosphatases during the spindle checkpoint. To address this question, we treated spindle checkpoint-arrested cells with various phosphatase inhibitors and examined their effect on the MCC and APC/C activation. Using this approach we found that two phosphatase inhibitors, calyculin A and okadaic acid (1 µM), caused MCC dissociation and APC/C activation in spindle checkpoint-arrested cells. Although the cells were able to degrade cyclin B, they did not exit mitosis as evidenced by high levels of Cdk1 substrate phosphorylation and chromosome condensation. Our results provide the first evidence that phosphatases are essential for maintenance of the MCC during operation of the spindle checkpoint.

The DNA of a cell operates as its blueprint, providing coded information for the production of the RNA and proteins that allow the cell to function. Cells can face a myriad of insults to their genomic integrity during their lifetimes, from simple errors during growth and division to reactive oxygen species to chemotherapeutic reagents. To deal with these mutagenic insults and avoid passing them on to progeny, cells are equipped with multiple defenses. Checkpoints can sense problems and halt a cell’s progression through the cell cycle in order to allow repairs. More drastically, cells can also prevent passing on mutations to progeny by triggering apoptosis, or programmed cell death. This work will present two separate discoveries regarding the regulation of DNA damage-induced apoptosis and the regulation of the spindle checkpoint.

The protease caspase-2 has previously been shown to be an important regulator of DNA damage-induced apoptosis. In unstressed cells caspase-2 is present as an inactive monomer, but upon sensing a stress caspase-2 dimerizes and becomes catalytically active. The mechanisms that regulate this dimerization are poorly understood. The first research chapter details our development of a novel method to study dimerized caspase-2, which in turn identified TRAF2 as a direct activator of caspase-2. Specifically, we utilized the Bimolecular Fluorescence Complementation technique, wherein complementary halves of the Venus fluorophore are fused to caspase-2: when caspase-2 dimerizes, the non-fluorescent halves fold into a functional Venus fluorophore. We combined this technique with a Venus-specific immunoprecipitation that allowed the purification of caspase-2 dimers. Characterization of the caspase-2 dimer interactome by MS/MS identified several members of the TNF Receptor Associated Factor (TRAF) family, specifically TRAF1, 2, and 3. Knockdown studies revealed that TRAF2 plays a primary role in promoting caspase-2 dimerization and downstream apoptosis in response to DNA damage. Identification of a TRAF Interacting Motif (TIM) on caspase-2 indicates that TRAF2 directly acts on caspase-2 to induce its activation. TRAF2 is known to act as an E3 ubiquitin ligase as well as a scaffold for other E3 ubiquitin ligases. Indeed, we identified three lysine residues in the caspase-2 prodomain (K15, K152, and K153) important for its ubiquitination and complex formation. Together these results revealed a novel role for TRAF2 as a direct activator of caspase-2 apoptosis triggered by DNA damage.

During mitosis, when the cell prepares to divide, great care is taken to ensure that the chromosomes are properly segregated between the two daughter cells by the mitotic spindle. This is primarily accomplished through the spindle checkpoint, which becomes activated when the mitotic spindle is not properly attached to each chromosome’s kinetochore. When activated, the primary effector of the spindle checkpoint, the mitotic checkpoint complex (MCC), inhibits the anaphase-promoting complex (APC/C) by binding to the APC/C co-activator, CDC20. This prevents the APC/C from targeting critical pro-mitotic proteins, like cyclin B and securin, to promote mitotic exit. Although the function of the MCC is well understood, its regulation is not, especially in regard to protein phosphatases To investigate this, we activated the spindle checkpoint with microtubule inhibitors and then treated with a variety of phosphatase inhibitors, examining the effect on the MCC and APC/C. We found that two separate inhibitors, calyculin A and okadaic acid (1uM), were able to promote the dissociation of the MCC. This led to the activation of the APC/C, but the cells remained in mitosis as evidenced by high levels of Cdk1 activity and chromosome condensation. This is the first time that phosphatases have been shown to be essential to maintaining the MCC and an active spindle checkpoint.