Browsing by Author "Wiese, Claudia, committee member"
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Item Open Access Characterization of biased partner choice in mitotic non-allelic homologous recombination of Saccharomyces cerevisiae(Colorado State University. Libraries, 2023) Merriman, Sean, author; Argueso, Juan Lucas, advisor; Markus, Steven, committee member; Nishimura, Erin, committee member; Wiese, Claudia, committee memberUsing yeast as a model in which to study copy number variation (CNV)-generating mutations, the J.L. Argueso lab has discovered that a specific region of S. cerevisiae genome (the right arm of chromosome 7; Chr7R) is much more susceptible to sustaining deletions as a translocation recipient than other apparently similar segments of the genome. Further, Chr7R acquires amplifications as a translocation donor less frequently than other chromosomes. To begin unraveling the cause of this unusual behavior, we evaluated the effect of several candidate genes involved in chromatin mobility and sister chromatid cohesion on the mutational spectra involving Chr7R. Our results suggest that regulatory factors of chromatin mobility or sister chromatid cohesion affect the outcomes of HR-mediated repair events at Ch7R. We are hopeful that our findings will open a window into the fundamental cellular processes that are responsible for CNVs found in eukaryotic genomes, and inform translational implications for modeling this class of mutation in cancer.Item Embargo Characterization of modes and kinetics of mutation accumulation in Saccharomyces cerevisiae through the analysis of defined cellular lineages(Colorado State University. Libraries, 2024) Stewart, Joseph, author; Argueso, Juan Lucas, advisor; Moreno, Julie, committee member; Regan, Daniel, committee member; Wiese, Claudia, committee memberIn the field of evolution, gradualism is the process of incremental adaptation supported by a slow and random accumulation of mutations that, over time, lead to genetic diversification and fitness gains. Although this Darwinian model is well supported and widely accepted, it cannot always explain the rapid changes seen in some instances such as tumors with extremely high and complex mutation loads. Recent reports in various organisms, including from our group using Saccharomyces cerevisiae, provide evidence for an additional mode of rapid and non-independent accumulation of chromosomal rearrangements. We have used a yeast model to follow the accumulation of structural genomic rearrangements such as loss of heterozygosity (LOH). We found that while chances of a single LOH event happening are very low, two or more LOH tracts co-occurred at rates 25- to 200-fold higher than expected if these events were independent of each other; therefore, the conventional process of slow and independent accumulation of mutations are not sufficient to account for every change in the genome. In the present study, we focused on temporal kinetics of bursts of LOH accumulation in yeast. We developed a hybrid diploid yeast experimental strain that enables identification of LOH event both through counter-selection and visual screening for colony color. This hybrid strain, made from the S288c and SK1 genetic backgrounds, possesses ~55,000 heterozygous SNPs distributed throughout the genome and allows for ease of tracking LOH events through sequencing. The screening approach was used in combination with microcultures (one cell grown for 5 or 6 divisions) in phylogenetic analyses that unambiguously revealed 18 cases where multiple LOH events co-occurred in the same cell division cycle. Collectively, these studies offer support for punctuated bursts of mutation accumulation caused by systemic genomic instability (SGI). Additionally, we have investigated a potential mechanism that influences SGI, namely global noise in gene expression.Item Open Access Characterization the role of telomeric RNA (TERRA) in telomeric DNA double-strand break (DSB) repair in human ALT (telomerase independent) cells(Colorado State University. Libraries, 2019) Alturki, Taghreed Mohammed, author; Bailey, Susan, advisor; Argueso, Lucas, committee member; Wiese, Claudia, committee member; Yao, Tingting, committee memberTelomeres are specialized nucleoprotein complexes that protect natural chromosomal termini from degradation and prevent their detection as of DNA damage. Therefore, telomeres play critical roles in maintaining genomic stability. Telomeres are composed of tandem arrays of conserved repetitive sequences (TTAGGG in vertebrates), bound by a suite of proteins collectively termed "shelterin". Shelterin proteins are essential for telomere length regulation and end-capping structure/function. Due to their repetitive nature and together with telomeres possessing an abundance of heterochromatic marks, telomeres have long been regarded as silenced, non-transcribed features of the genome. The relatively recent discovery of telomeric RNA (TElomere Repeat- containing RNA; TERRA) opened many new avenues of investigation. TERRA is a long, noncoding RNA (lncRNA) that serves a structural role at telomeres, as well as function in regulation of telomere length and telomerase activity, the specialized reverse transcriptase capable of elongating telomeres de novo. Further, TERRA participates in telomeric recombination in tumors that maintain telomere length in a telomerase independent fashion via the Alternative Lengthening of Telomeres (ALT) pathway. Emerging evidence also supports telomeric "DNA- TERRA hybrids" as indispensable for end protection and capping function; e.g., RNA interference mediated depletion of TERRA induced telomeric DNA damage responses (DDRs) and aberrations. Thus, TERRA participates in facilitating telomeric recombination and in preventing inappropriate telomeric DNA damage responses. We hypothesized that TERRA plays a critical role in the repair of telomeric DNA damage. To address the intriguing possibility that TERRA plays a role in the telomeric DNA damage response, we evaluated the colocalization of TERRA and γ-H2AX, a well-accepted marker of double-strand breaks (DSBs), at broken telomeres in human Osteosarcoma U2OS-ALT cells in different phases of the cell cycle. To test our hypothesis, we generated U2OS-ALT cells that stably expressed FUCCI green fluorescent signals to label cells in G2 phase. Telomeric DSBs were then induced in FUCCI-U2OS cells utilizing the ENT endonuclease fused to Telomere Repeat Factor 1 TRF1 (ENT-TRF1), and validated via colocalization of telomeres with γ-H2AX-FLAG. Forty-eight hours following transfection, FUCCI-U2OS cells were also treated with EdU to label cells in S phase. Cells negative for both FUCCI and EdU identified cells in G1 phase. Using this powerful strategy to distinguish cells in G1, S and G2 phases of the cell cycle, we showed that FUCCI-U2OS cells accumulate in G2 phase following transient transfection with ENT-TRF1. We validated that expression of ENT-TRF1 generates telomeric DSBs in U2OS-ALT cells through detection of telomere- γ-H2AX-FLAG colocalization events. Importantly, our data revealed that telomeric DSB induction triggers enrichment of TERRA in G2 phase. Taken together, these observations suggested that TERRA is increased in cells transfected with ENT-TRF1; i.e., in U2OS cells harboring telomere-specific DSBs. TERRA recruitment to telomeric DSB damage sites in G2 was validated by assessing co-localization between TERRA and ENT (FLAG). Similarly, TERRA recruitment to telomeric DSBs in G1/S was also evaluated. Futhermore, non-denaturing Telomere DNA FISH was employed to visualize G-rich and C-rich single-stranded (ss)telomeric DNA. Treatment of U2OS ENT transfected cells with Rnase A and Rnase H to remove TERRA, uncovered elevated levels of resected 5' C-rich (ss)telomeric DNA (complementary TERRA sequence), suggesting a potential role for TERRA in protecting resected telomeric DNA prior to cells entering G2 phase where Homologous Recombination (HR)-mediated elongation/repair would be possible. Consistent with published reports, telomeric DSBs were also significantly induced in cells transfected with TRF1-only (positive control). However, although TERRA co-localized with FLAG/broken telomeres, resected (ss)telomeric DNA was not detected upon removal of TERRA. Therefore, our results further support induction of telomeric DSBs with overexpression of TRF1, and additionally indicate that they are repaired via a different pathway than those induced by ENT, potentially alternative End Joining (alt-EJ), as previously proposed. In conclusion, our work revealed for the first time that the telomeric RNA TERRA, is enriched specifically at telomeric DNA DSB sites in U2OS (ALT) cells.Item Open Access Genome instability: a pre-existing condition(Colorado State University. Libraries, 2018) Sedam, Hailey Nicole Conover, author; Argueso, Lucas, advisor; DeLuca, Jennifer, committee member; Nickoloff, Jac, committee member; Wiese, Claudia, committee memberCopy number variations (CNV), or large amplifications or deletions in the genome, account for about 50% of human genetic diversity. CNVs across genomic regions essential for development and function can lead to disease. The underlying mechanisms of CNV formation are typically traced to a combination of endogenous or environmental sources of DNA damage coupled with defects in DNA repair, replication, and recombination. This dissertation describes two endogenous sources of genome instability involved in both mitotic and meiotic CNVs. Each chapter of this dissertation focuses on one endogenous contribution to genome instability, using the budding yeast Saccharomyces cerevisiae as a model system to investigate the conserved cellular processes that, when gone awry, can lead to CNVs. In the first phase of my research, I focused on the mitotic mutagenic effects of ribonucleotide incorporation into DNA. In the absence of RNase H2, RNA-DNA hybrids (R-loops) accumulate in the genome and ribonucleotides that are misincorporated into the DNA are not efficiently excised. Instead, the latter function is taken over by topoisomerase 1 which inappropriately removes ribonucleotides in a way that leads to accidental/unscheduled DNA double strand breaks (DSBs). My work showed that the accumulation of these lesions in RNase H deficient mutants was sufficient to increase the rate of genome rearrangements through both Loss of Heterozygosity (LOH) and Non Allelic Homologous Recombination (NAHR). Modulating the number of ribonucleotides incorporated into the leading DNA strand during replication through the use of DNA polymerase epsilon mutants affected the rate of LOH and NAHR. Additionally, the RNase H2-Ribonucleotide Excision Repair Deficient (RNase H2-RED) separation of function allele allowed further investigation of genomic instability when R-loops are properly processed but misincorporated ribonucleotides are not. The RNaseH2-RED study revealed that both ribonucleotide excision repair and R-loop removal contribute roughly equally to chromosomal stability under normal conditions. Together, the results of these studies indicated that the effects of ribonucleotides and R-loops on chromosomal instability may vary under different genomic contexts of variable R-loop formation and ribonucleotide density. Next, I designed, constructed, optimized and validated a new yeast assay system to study meiotic NAHR leading to de novo recurrent CNVs. The chromosomal rearrangements analyzed through this system are directly analogous to human pathogenic CNVs that are formed in germ cells through recombination between Low Copy Repeat elements (LCRs). While there are assays available to investigate factors involved in mitotic CNV formation, few assays have been developed to experimentally test factors involved in meiotic recurrent CNVs. Previous studies of human patient cohorts have shown that the size and distance between LCRs is strongly correlated with the frequency of recurrent CNV formation. We used this basic observation to validate our experimental system and ask whether it could faithfully recapitulate the phenomenon in our yeast model system. I constructed four diploid strains containing LCRs engineered to range in size from 5-35 Kb and determined the meiotic NAHR frequency in each construct. We detected a very clear linear correlation between LCR size and CNV frequency, and thus established our system as a pertinent assay for interrogation of factors involved in meiotic recurrent CNV formation. The results described within this dissertation have deepened our understanding of the endogenous causes of genome instability leading to CNVs, and provide perspective into the ability of normal cellular processes to trigger both mitotic and meiotic CNV formation. Additionally, I describe a unique method for future screens of both endogenous and exogenous stimulants of meiotic CNV.Item Open Access Modeling the evolution of SIV progenitor viruses towards HIV-1 and HIV-2 in a humanized mouse surrogate model(Colorado State University. Libraries, 2020) Curlin, James Zachary, author; Akkina, Ramesh, advisor; Aboellail, Tawfik, committee member; Stenglein, Mark, committee member; Wiese, Claudia, committee memberHuman Immunodeficiency Virus Type 1 (HIV-1) and Type 2 (HIV-2), the causative agents of Acquired Immunodeficiency Syndrome (AIDS) first emerged in humans over the past century. Despite significant advances in treatment options, the pandemics continue with millions of new infections every year. Both HIV-1 and HIV-2 likely emerged through the cross-species transmission of primate lentiviruses originating from nonhuman primates (NHPs) including chimpanzees (SIVcpz), gorillas (SIVgor), and sooty mangabeys (SIVsm). SIVsm shares a remarkable degree of homology with HIV-2, while SIVcpz and SIVgor are most closely related to HIV-1. Nonhuman primates infected with these lentiviruses frequently come into contact with humans due to the prevalence of bushmeat hunting practices in various African countries. Other lentiviruses such as SIVmac239 represent independent instances of primate lentiviruses crossing into novel host species. The repeated exposure of primate lentiviruses to a human immune environment allowed the accumulation of adaptive genetic changes uniquely suited to overcoming the evolutionary pressures of a new host. Host-restriction factors such as tetherin, SAMHD1, APOBEC3G and SERINC3/5 exert species-specific antiviral activity and must be overcome for a virus to adapt to a new host cell. These evolutionary pressures could be a guiding force in the direction that these viruses adapt. In order to recapitulate these genomic cross-species adaptations, we used humanized mice engrafted with human hematopoietic stem cells (hu-HSC mice). These mice produce a full spectrum of human immune cells such as B cells, T cells, macrophages, monocytes, and dendritic cells, and are susceptible to HIV infection. Representative progenitor viruses of both HIV-1 (SIVcpzEK505, SIVcpzMB897, and SIVcpzLB715) and HIV-2 (SIVsmE041) as well as other viruses of interest, namely, SIVmac239, SIVhu and SIVB670 lineages were intraperitoneally injected into hu-HSC mice. Following successful infections, the derivative viruses were subsequently passaged serially through multiple generations to simulate the repeated exposures that originally produced HIV-1 and HIV-2. Viral adaptation was assessed primarily through three different criteria. Plasma viral RNA levels were measured on a weekly basis using qRT-PCR to determine changes in viral replication kinetics over time. We found that the plasma viral loads of the viruses tested varied during serial passages, and mostly increased over time in many cases. Human CD4+ T cell engraftment decline as assessed by flow cytometry biweekly acts as a measure of AIDS progression in cases of human infection. CD4+ T cell levels declined over time with increasing rapidity upon further passaging in many cases. Additionally, viral RNA collected from the infected mice at multiple timepoints in each generation was used to generate overlapping amplicons spanning the length of the viral genome in order to be used with Illumina-based deep sequencing. Numerous nonsynonymous mutations arose in the first generation of passaging and were maintained across multiple sequential passages. While the mutations occurred throughout the viral genome, the bulk of the mutations were found in env and nef. Many of these mutations were present in known CD4+ binding sites, motifs involved in protein interactions, and other areas involved in host-restriction factor antagonism. While these results are revealing, further inquiry is needed to determine the true functionality of these genetic changes. These data showcase the value of using humanized mice to model lentiviral evolution and provide important insights into understanding the origin of HIVs.Item Open Access Telomeric double strand breaks undergo resection - but not repair - in G1 human cells(Colorado State University. Libraries, 2017) Nelson, Christopher Boulanger, author; Bailey, Susan, advisor; Argueso, Lucas, committee member; Kato, Takamitsu, committee member; Wiese, Claudia, committee member; Miller, Benjamin, committee member; Chicco, Adam, committee memberTelomeres are specialized G-rich repetitive regions at the ends of eukaryotic chromosomes (TTAGGGn in mammalian cells). Telomeres function to prevent double strand break (DSB) repair activities at chromosome ends, in order to avoid fusion events which result in lethal dicentric chromosomes. Telomeric repeats make up an appreciable amount of genomic DNA (1-15kb per chromosome end). Therefore, an interesting question becomes, how is the inevitable DSB occurring within a telomere dealt with by the cell? It has been suggested that DSBs within telomeric DNA may not be repaired at all, as DSB DNA damage response (DDR) foci at telomeres do not resolve following large amounts of global DNA damage (e.g. ionizing radiation). Such studies also suggest that telomere repair may be inhibited specifically in G1, as the majority of surviving cells with unresolved telomere damage responses were senescent (a G1 phenotype). On the other hand, studies on the fragmentation of telomeric DNA following cutting with a telomere-targeted endonucleases indicate that repair of telomere-specific DSBs involves Homologous Recombination (HR) and Break-Induced Replication (BIR). However, a marker of telomeric DSB DDRs was only observed in cells with BrdU incorporation, in support of the view that repair of telomeric DSBs is an S/G2-related process, which does not occur in G1. To follow up on these studies, we investigated telomeric DDRs and DSB repair in individual G1 cells using ionizing radiation (IR) and a targeted telomere-cutting endonuclease. IR exposure could potentially induce loss of telomere function, such that persistent DDRs may not represent actual DSBs. To rule out this possibility, we evaluated whether persistent telomeric DDRs following IR occurred at telomeres that were critically short or lacking TRF2. We found that persistent telomeric DDRs occurred at telomeres of normal length and TRF2 status, in support of the conclusion that G1 telomeric DSBs are irreparable. Additionally, using the telomere-targeted endonuclease we observed that telomeric DSBs in G1 cells elicited a relatively conventional DSB DDR – with one important exception – G1 telomeric DDRs failed to recruit 53BP1, an event implicated in the completion of DSB repair by most pathways, but especially, canonical non-homologous end joining (cNHEJ). Further, shRNA knockdown and kinase inhibition of the cNHEJ factor DNA-PKcs, provided evidence that cNHEJ is not responsible for repair of telomeric DSBs, and that DNA-PKcs does not influence recruitment of 53BP1 to telomeric DSBs in G1. Partial deprotection of telomeres, achieved by siRNA depletion of TRF2, also failed to alleviate inhibition of 53BP1 recruitment to G1 telomeric DSBs, suggesting that 53BP1 recruitment to telomeric DSBs may require full deprotection of telomeres. However, as 53BP1 recruitment occurs at de-protected telomeres, this idea would be difficult to test. Most likely related to the lack of 53BP1 recruitment, an abundance of bidirectionally occurring single-stranded DNA was observed at G1 telomeric DSBs, a characteristic of long-range repair-associated resection. In support of long-range resection, RPA70 and phospho-RPA32 were observed at G1 telomeric DSBs. Additionally, conventional DSB repair-associated resection machinery, including MRE11 and EXO1, but not the telomere processing exonuclease Apollo, promoted resection at telomeric DSBs. We then investigated whether long-range resection-dependent repair was occurring at G1 telomeric DSBs via RAD51 or RAD52 foci, and DNA synthesis (S/G2 related processes). Despite activity resembling long-range repair-associated resection at G1 telomeric DSBs, no evidence for repair by these pathways was found. Taken together, the results presented here provide strong evidence in support of the view that telomeric DSBs in G1 are unrepairable. Therefore, the extensive resection observed at telomeric DSBs must be reflective of an alternative, non-repair related function, perhaps related to structural end-protection. We speculate that resection at G1 telomeric DSBs may serve to prevent 53BP1 recruitment, thereby circumventing a full DDR and activation of cNHEJ, a scenario that would create a serious threat to genome stability.