Browsing by Subject "Double-strand breaks"
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Item Open Access Interrogating Chromatin Dynamics Surrounding a DNA Double-Strand Break and Ensuing Non-Homologous End-Joining Mediated Repair(2020) Tripuraneni, VinayThe DNA double-strand break (DSB) is one of the most toxic genomic lesions that can occur in any living cell. Failure to repair DSBs results in cell cycle arrest and ultimately programmed cell death, while improper repair can lead to profound alterations or loss of genomic information through translocations, inversions, deletions and other genomic aberrations. Although the molecular events required for the repair of double-strand breaks (DSB) have been well characterized, the role of epigenetic processes in the recognition and repair of DSBs has only been investigated at low resolution. I tested several site-specific DSB induction systems and found that that the HO endonuclease was able to rapidly and synchronously induce a site-specific DSB in Saccharomyces cerevisiae upstream of the PHO5 locus. This region of the genome is recognized for its chromatin organization, which is comprised of well-positioned nucleosomes. Utilizing MNase digestion of chromatin followed by paired-end fragment sequencing I was able to interrogate the order of chromatin changes that occur immediately following a DSB by generating a base-pair resolution map of the chromatin landscape. In wild-type cells, the first nucleosome left of the break was rapidly evicted. The eviction of this flanking nucleosome was dynamic and proceeded through an early intermediate chromatin structure where the nucleosome was repositioned in the adjacent linker DNA. Other nucleosomes bordering the break were also shifted away from the break; however, their loss was more gradual. These local changes preceded a broader loss of chromatin organization and nucleosome eviction that was marked by increased MNase sensitivity in the regions ~8 kb on each side of the break. While the broad loss of chromatin organization was dependent on the end-processing complex, Mre11-Rad50-Xrs2 (MRX), the early remodeling and repositioning of the nucleosome adjacent to the break was independent of the MRX and yKU70/80 complexes. I also examined the temporal dynamics of non-homologous end joining (NHEJ) mediated repair in a G1-arrested population. Concomitant with DSB repair, I observed the re-deposition and precise re-positioning of nucleosomes at the originally occupied positions. This re-establishment of the pre-lesion chromatin landscape suggests that a DNA replication-independent mechanism exists in G1 cells to preserve epigenome organization following DSB repair.
Item Open Access Molecular Characterization of Mitotic Homologous Recombination Outcomes in Saccharomyces cerevisiae(2017) Hum, Yee FangMitotic homologous recombination (HR) is vital for accurate repair of DNA strand breaks caused by endogenous and exogenous sources. However, this high-fidelity repair pathway also can lead to genome rearrangements when dispersed sequences are used for repair. During normal growth, spontaneous DNA strand breaks are presumably generated during DNA replication and transcription, and from the attack by endogenous agents such as reactive oxygen species (ROS). Though the exact nature of endogenous lesions that initiate HR is not well understood, double-strand breaks (DSBs) rather than single-strand breaks (SSBs) are thought to be the main culprit. Because spontaneous HR events can lead to development of human diseases and sporadic cancers, identifying the primary type of DNA strand breaks, either DSBs or SSBs, is central to understanding how genome instability arises. Using the yeast Saccharomyces cerevisiae as a model system, the focus of this thesis is to delineate early molecular steps (DNA end resection and synthesis) during mitotic DSB-induced HR events and to perform comparative analysis of DSB-induced and spontaneous HR repair outcomes. To this end, the first part of this thesis examined the relative contribution of DNA end resection and DNA synthesis in determining the DSB-associated repair outcomes, such as distributions of crossover and noncrossover outcomes, as well as the length of a key recombination intermediate, heteroduplex (hetDNA). The main conclusion from this work is that both end resection and DNA synthesis are required to obtain normal DNA repair outcomes and hetDNA profiles. A unifying model is that decreased end resection reduces stability of strand invasion intermediates, limiting the extent of DNA synthesis and hence shortening hetDNA. The second and third parts of this work directly compared the molecular structures of HR outcomes associated with a defined DSB and with those arising spontaneously. Two different approaches were employed to systematically characterize the molecular structures of recombination intermediates in repair events. In the second part of this thesis, mapping of gene conversion events (nonreciprocal transfer of information that results from mismatch-repair activity) following allelic repair of a DSB revealed that DSB-induced HR events shared similar repair profiles with those of previously described spontaneous recombination events, confirming that DSBs are the main contributor to spontaneous HR. In the third part of this thesis, mapping of hetDNA in DSB-induced and spontaneous HR events in cells with normal and elevated ROS levels further confirmed that DSBs are the primary initiator of spontaneous HR. Mapping of hetDNA additionally revealed complexities within hetDNA associated with a defined DSB. Collectively, this work not only advances our knowledge of the fundamental molecular mechanisms of HR, but also provides in vivo experimental support for DSBs as the major physiological lesion that initiates spontaneous HR.
Item Open Access Stabilization of Topoisomerase 2 Mutants Initiates the Formation of Duplications in DNA(2021) Stantial, NicoleTopoisomerase 2 (Top2) is an enzyme that helps maintain genome integrity by resolving topological structures that arise during cellular processes such as replication and transcription. To resolve these structures, a Top2 dimer creates a transient double-strand break (DSB) in the DNA. Each subunit forms a phosphotyrosyl bond with the 5’ ends of the break, and this DNA-protein intermediate is called a Top2 cleavage complex (Top2cc). Following the passage of an intact duplex, Top2 re-ligates the DNA and is released to restore genome integrity. Top2cc stabilization by chemotherapeutic drugs such as etoposide leads to persistent and potentially toxic DSBs. This thesis characterizes two novel top2 mutants, both of which are associated with a mutation signature characterized by de novo duplications. These duplication events are dependent on clean removal of the Top2cc from the DNA and DSB repair by nonhomologous end-joining. The first mutant (top2-FY,RG) was identified through a screen for etoposide hypersensitivity, and it generates a stabilized cleavage intermediate in vitro. The second mutant (top2-K720N) is the yeast equivalent of a somatic mutation in TOP2A identified in gastric cancers and choloangiocarcinomas that is also associated with a duplication mutation signature (ID17). Overall, the findings in this thesis are relevant for clinical use of chemotherapeutic drugs that target Top2 and have implications for genome evolution.