Browsing by Author "Jinks-Robertson, Sue"
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Item Open Access Genetic Analysis of Mitotic Recombination in Saccharomyces cerevisiae(2016) O'Connell, Karen EileenMitotic genome instability can occur during the repair of double-strand breaks (DSBs) in DNA, which arise from endogenous and exogenous sources. Studying the mechanisms of DNA repair in the budding yeast, Saccharomyces cerevisiae has shown that Homologous Recombination (HR) is a vital repair mechanism for DSBs. HR can result in a crossover event, in which the broken molecule reciprocally exchanges information with a homologous repair template. The current model of double-strand break repair (DSBR) also allows for a tract of information to non-reciprocally transfer from the template molecule to the broken molecule. These “gene conversion” events can vary in size and can occur in conjunction with a crossover event or in isolation. The frequency and size of gene conversions in isolation and gene conversions associated with crossing over has been a source of debate due to the variation in systems used to detect gene conversions and the context in which the gene conversions are measured.
In Chapter 2, I use an unbiased system that measures the frequency and size of gene conversion events, as well as the association of gene conversion events with crossing over between homologs in diploid yeast. We show mitotic gene conversions occur at a rate of 1.3x10-6 per cell division, are either large (median 54.0kb) or small (median 6.4kb), and are associated with crossing over 43% of the time.
DSBs can arise from endogenous cellular processes such as replication and transcription. Two important RNA/DNA hybrids are involved in replication and transcription: R-loops, which form when an RNA transcript base pairs with the DNA template and displaces the non-template DNA strand, and ribonucleotides embedded into DNA (rNMPs), which arise when replicative polymerase errors insert ribonucleotide instead of deoxyribonucleotide triphosphates. RNaseH1 (encoded by RNH1) and RNaseH2 (whose catalytic subunit is encoded by RNH201) both recognize and degrade the RNA in within R-loops while RNaseH2 alone recognizes, nicks, and initiates removal of rNMPs embedded into DNA. Due to their redundant abilities to act on RNA:DNA hybrids, aberrant removal of rNMPs from DNA has been thought to lead to genome instability in an rnh201Δ background.
In Chapter 3, I characterize (1) non-selective genome-wide homologous recombination events and (2) crossing over on chromosome IV in mutants defective in RNaseH1, RNaseH2, or RNaseH1 and RNaseH2. Using a mutant DNA polymerase that incorporates 4-fold fewer rNMPs than wild type, I demonstrate that the primary recombinogenic lesion in the RNaseH2-defective genome is not rNMPs, but rather R-loops. This work suggests different in-vivo roles for RNaseH1 and RNaseH2 in resolving R-loops in yeast and is consistent with R-loops, not rNMPs, being the the likely source of pathology in Aicardi-Goutières Syndrome patients defective in RNaseH2.
Item Open Access Guidelines for DNA recombination and repair studies: Cellular assays of DNA repair pathways.(Microbial cell (Graz, Austria), 2019-01-07) Klein, Hannah L; Bačinskaja, Giedrė; Che, Jun; Cheblal, Anais; Elango, Rajula; Epshtein, Anastasiya; Fitzgerald, Devon M; Gómez-González, Belén; Khan, Sharik R; Kumar, Sandeep; Leland, Bryan A; Marie, Léa; Mei, Qian; Miné-Hattab, Judith; Piotrowska, Alicja; Polleys, Erica J; Putnam, Christopher D; Radchenko, Elina A; Saada, Anissia Ait; Sakofsky, Cynthia J; Shim, Eun Yong; Stracy, Mathew; Xia, Jun; Yan, Zhenxin; Yin, Yi; Aguilera, Andrés; Argueso, Juan Lucas; Freudenreich, Catherine H; Gasser, Susan M; Gordenin, Dmitry A; Haber, James E; Ira, Grzegorz; Jinks-Robertson, Sue; King, Megan C; Kolodner, Richard D; Kuzminov, Andrei; Lambert, Sarah Ae; Lee, Sang Eun; Miller, Kyle M; Mirkin, Sergei M; Petes, Thomas D; Rosenberg, Susan M; Rothstein, Rodney; Symington, Lorraine S; Zawadzki, Pawel; Kim, Nayun; Lisby, Michael; Malkova, AnnaUnderstanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.Item Open Access Homologous Recombination Outcomes of Double-Strand Break- initiated Events in Saccharomyces cerevisiae(2020) Gamble, Dionna MoniqueHomologous recombination (HR) is responsible for repairing otherwise lethal DNA double-strand breaks (DSBs) using a homologous donor template. The work in this dissertation, using the budding yeast Saccharomyces cerevisiae as a model system, focuses on the effect of the initiating DSB and sequence homology on the outcomes of repair. First, in Chapter 2 we investigated how the initiating DSB affects HR outcomes. Recombination studies often utilize site-specific enzymes that create a single DSB at a known location. Unlike chemical or environmental DNA damaging agents that cause multiple DSBs genome-wide, these nucleases allow precise analysis of repair intermediates and outcomes of a specific DSB. To date the most common enzymes used are I-SceI and HO which both make DSBs with 3’ overhangs. Although the current HR model is largely based on this one type of DSB, we know DSBs can vary by end structure and initiating cause in vivo. Using our lys2 ectopic assay we explored how the outcomes of recombination vary between a DSB with a 3’ overhang made by I-SceI versus a Zinc Finger Nuclease (ZFN)-generated break with a 5’ overhang. Results of this study indicate the method of DSB induction does not drastically affect HR outcomes. Although these different initiating-breaks produced a slight difference in the distributions of HR products, crossovers (COs) versus noncrossovers (NCOs), there were no detectable differences in the position/length of exchange sequence. Further physical analysis found slight differences in the rate of repair between the different breaks. These findings were significant as they suggest HR is able to handle different types of initiating DSBs and generate similar repair outcomes thus maintaining genome stability. Chapter 3 examines how additional structural challenges between our divergent ectopic substrates affect the outcomes of recombination. By manipulating the donor sequence, we introduced nonhomologous regions between the substrates that generated a gap and/or nonhomologous tails at the DSB. The HR outcomes, including repair frequencies and CO-NCO distributions/frequencies, were observed in the presence and absence of mismatch repair (MMR), as this system is responsible for limiting ectopic recombination (anti-recombination effect). Thus far data suggests these additional structural challenges impede recombination particularly in the presence of MMR activity. The addition of these structures increased the anti-recombination effect of MMR, with gap- and tail- specific effects on HR products. Sequencing of NCO products will be crucial to understanding how the gap and/or tails affect patterns of exchanged sequence, which can give insight into differences in intermediates and mechanisms of recombination. While this study is still ongoing, the findings are significant as they highlight the importance of donor homology and the regulation of ectopic recombination. Overall the work in this dissertation adds to our knowledge of the HR mechanism as defects in this process are linked to human diseases including many types of cancer and genomic disorders.
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 Recombinational Repair of Nuclease-Generated Mitotic Double-Strand Breaks with Different End Structures in Yeast.(G3 (Bethesda, Md.), 2020-10) Gamble, Dionna; Shaltz, Samantha; Jinks-Robertson, SueMitotic recombination is the predominant mechanism for repairing double-strand breaks in Saccharomyces cerevisiae Current recombination models are largely based on studies utilizing the enzyme I-SceI or HO to create a site-specific break, each of which generates broken ends with 3' overhangs. In this study sequence-diverged ectopic substrates were used to assess whether the frequent Pol δ-mediated removal of a mismatch 8 nucleotides from a 3' end affects recombination outcomes and whether the presence of a 3' vs. 5' overhang at the break site alters outcomes. Recombination outcomes monitored were the distributions of recombination products into crossovers vs. noncrossovers, and the position/length of transferred sequence (heteroduplex DNA) in noncrossover products. A terminal mismatch that was 22 nucleotides from the 3' end was rarely removed and the greater distance from the end did not affect recombination outcomes. To determine whether the recombinational repair of breaks with 3' vs. 5' overhangs differs, we compared the well-studied 3' overhang created by I-SceI to a 5' overhang created by a ZFN (Zinc Finger Nuclease). Initiation with the ZFN yielded more recombinants, consistent with more efficient cleavage and potentially faster repair rate relative to I-SceI. While there were proportionally more COs among ZFN- than I-SceI-initiated events, NCOs in the two systems were indistinguishable in terms of the extent of strand transfer. These data demonstrate that the method of DSB induction and the resulting differences in end polarity have little effect on mitotic recombination outcomes despite potential differences in repair rate.Item Open Access Spontaneous deamination of cytosine to uracil is biased to the non-transcribed DNA strand in yeast.(DNA repair, 2023-06) Williams, Jonathan D; Zhu, Demi; García-Rubio, María; Shaltz, Samantha; Aguilera, Andrés; Jinks-Robertson, SueTranscription in Saccharomyces cerevisiae is associated with elevated mutation and this partially reflects enhanced damage of the corresponding DNA. Spontaneous deamination of cytosine to uracil leads to CG>TA mutations that provide a strand-specific read-out of damage in strains that lack the ability to remove uracil from DNA. Using the CAN1 forward mutation reporter, we found that C>T and G>A mutations, which reflect deamination of the non-transcribed and transcribed DNA strands, respectively, occurred at similar rates under low-transcription conditions. By contrast, the rate of C>T mutations was 3-fold higher than G>A mutations under high-transcription conditions, demonstrating biased deamination of the non-transcribed strand (NTS). The NTS is transiently single-stranded within the ∼15 bp transcription bubble, or a more extensive region of the NTS can be exposed as part of an R-loop that can form behind RNA polymerase. Neither the deletion of genes whose products restrain R-loop formation nor the over-expression of RNase H1, which degrades R-loops, reduced the biased deamination of the NTS, and no transcription-associated R-loop formation at CAN1 was detected. These results suggest that the NTS within the transcription bubble is a target for spontaneous deamination and likely other types of DNA damage.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.
Item Open Access Studies of Spontaneous Oxidative and Frameshift Mutagenesis in Saccharomyces cerevisiae(2010) Mudrak, Sarah VictoriaPreserving genome stability is critical to ensure the faithful transmission of intact genetic material through each cell division. One of the key components of this preservation is maintaining low levels of mutagenesis. Most mutations arise during replication of the genome, either as polymerase errors made when copying an undamaged DNA template or during the bypass of DNA lesions. Many different DNA repair proteins act both prior to and during replication to prevent the occurrence of these mutations. Although the mechanisms by which mutations occur and the various repair proteins that act to suppress mutagenesis are conserved throughout all species, they are best characterized in the yeast Saccharomyces cerevisiae. In this work, we have used this model system to study two types of spontaneous mutagenesis: oxidative mutagenesis and frameshift mutagenesis. In the first part of this work, we have examined mutagenesis that arises due to one of the most common oxidative lesions in the cell, 7,8-dihydro-8-oxoguanine or GO. When present during replication, these GO lesions generate characteristic transversion events that are accurately repaired by the mismatch repair pathway. We provide the first evidence that a second pathway involving the translesion synthesis polymerase Pol&eta acts independently of the mismatch repair pathway to suppress GO-associated mutagenesis. We have also examined how differences in replication timing during S phase contribute to variations in the rate of these mutations across the genome. In the second part of this work, we have examined how spontaneous frameshift mutations are generated during replication. While most frameshift mutations occur in regions of repetitive DNA, we have designed a system to examine frameshifts that occur in very short repeats (< 4 nucleotides) and noniterated sequences. We have examined the patterns of frameshifts at these sites and how the mismatch repair pathway acts to suppress these mutations. Together, the experiments presented here provide further insight into the different mechanisms that suppress and/or influence rates of oxidative mutagenesis and describe a system in which we have begun to characterize how frameshift mutations are generated at very short repeats and non-repetitive DNA.
Item Open Access Topoisomerase 1 (Top1)-associated Genome Instability in Yeast: Effects of Persistent Cleavage Complexes or Increased Top1 Levels(2016) Sloan, Roketa ShanellTopoisomerase 1 (Top1), a Type IB topoisomerase, functions to relieve transcription- and replication-associated torsional stress in DNA. Top1 cleaves one strand of DNA, covalently associates with the 3’ end of the nick to form a Top1-cleavage complex (Top1cc), passes the intact strand through the nick and finally re-ligates the broken strand. The chemotherapeutic drug, Camptothecin, intercalates at a Top1cc and prevents the crucial re-ligation reaction that is mediated by Top1, resulting in the conversion of a nick to a toxic double-strand break during DNA replication or the accumulation of Top1cc. This mechanism of action preferentially targets rapidly dividing tumor cells, but can also affect non-tumor cells when patients undergo treatment. Additionally, Top1 is found to be elevated in numerous tumor tissues making it an attractive target for anticancer therapies. We investigated the effects of Top1 on genome stability, effects of persistent Top1-cleavage complexes and elevated Top1 levels, in Saccharomyces cerevisiae. We found that increased levels of the Top1cc resulted in a five- to ten-fold increase in reciprocal crossovers, three- to fifteen fold increase in mutagenesis and greatly increased instability within the rDNA and CUP1 tandem arrays. Increased Top1 levels resulted in a fifteen- to twenty-two fold increase in mutagenesis and increased instability in rDNA locus. These results have important implications for understanding the effects of CPT and elevated Top1 levels as a chemotherapeutic agent.
Item Open Access Trapped topoisomerase II initiates formation of de novo duplications via the nonhomologous end-joining pathway in yeast.(Proceedings of the National Academy of Sciences of the United States of America, 2020-10) Stantial, Nicole; Rogojina, Anna; Gilbertson, Matthew; Sun, Yilun; Miles, Hannah; Shaltz, Samantha; Berger, James; Nitiss, Karin C; Jinks-Robertson, Sue; Nitiss, John LTopoisomerase II (Top2) is an essential enzyme that resolves catenanes between sister chromatids as well as supercoils associated with the over- or under-winding of duplex DNA. Top2 alters DNA topology by making a double-strand break (DSB) in DNA and passing an intact duplex through the break. Each component monomer of the Top2 homodimer nicks one of the DNA strands and forms a covalent phosphotyrosyl bond with the 5' end. Stabilization of this intermediate by chemotherapeutic drugs such as etoposide leads to persistent and potentially toxic DSBs. We describe the isolation of a yeast top2 mutant (top2-F1025Y,R1128G) the product of which generates a stabilized cleavage intermediate in vitro. In yeast cells, overexpression of the top2-F1025Y,R1128G allele is associated with a mutation signature that is characterized by de novo duplications of DNA sequence that depend on the nonhomologous end-joining pathway of DSB repair. Top2-associated duplications are promoted by the clean removal of the enzyme from DNA ends and are suppressed when the protein is removed as part of an oligonucleotide. TOP2 cells treated with etoposide exhibit the same mutation signature, as do cells that overexpress the wild-type protein. These results have implications for genome evolution and are relevant to the clinical use of chemotherapeutic drugs that target Top2.