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1
Traver, Brenna E. "Exogenously-introduced Homing Endonucleases Catalyze Double-stranded DNA Breaks in Aedes aegypti." Thesis, Virginia Tech, 2009. http://hdl.handle.net/10919/40967.
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Aedes aegypti transmits the viruses which cause yellow fever, dengue fever, and dengue hemorrhagic fever. Homing endonucleases are selfish genetic elements which introduce double-stranded DNA (dsDNA) breaks in a sequence-specific manner. In this study, we aimed to validate a somatic assay to detect recombinant homing endonuclease (rHE)-induced dsDNA breaks in both cultured cells and adult female Ae. aegypti. While the cell culture-based two plasmid assay used to test rHE ability to induce dsDNA breaks was inconclusive, assays used to test rHEs in Ae. aegypti were successful. Recognition sequences for various rHEs were introduced into Ae. aegypti through germline transformation, and imperfect repair at each of these exogenous sites was evaluated. In mosquitoes containing a single exogenous HE site, imperfect gap repair was detected in 40% and 21% of clones sequenced from mosquitoes exposed to I-PpoI and Iâ SceI, respectively. In mosquitoes containing two exogenous HE sites flanking a marker gene (EGFP), 100% of clones sequenced from mosquitoes exposed to I-PpoI, I-CreI, and I-AniI demonstrated excision of EGFP. No evidence of EGFP excision or imperfect repair at any HE recognition site was detected in mosquitoes not exposed to a rHE. In summary, a somatic genomic footprint assay was developed and validated to detect rHE or other meganuclease-induced site-specific dsDNA breaks in chromosomal DNA in Ae. aegypti.Master of Science in Life Sciences
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Stephanou, Nicolas Constantinos. "Mycobacterial non-homologous end-joining : molecular mechanisms and components of a novel DNA double strand break repair pathway /." Access full-text from WCMC, 2008. http://proquest.umi.com/pqdweb?did=1528973431&sid=21&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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Aniukwu, Jideofor Flint. "The pathways and outcomes of mycobacterial NHEJ depend on the structure of the broken DNA ends /." Access full-text from WCMC :, 2008. http://proquest.umi.com/pqdweb?did=1555143361&sid=2&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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Moore, Anne Margaret. "Identification and characterisation of novel plant specific regulators of cellular responses to double stranded DNA breaks." Thesis, University of Edinburgh, 2012. http://hdl.handle.net/1842/9504.
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The ability of organisms to sense and respond to challenges to their genome integrity is key to survival. In particular, the ability to detect and respond to double-stranded DNA breaks (DSBs) is of fundamental importance as not only are DSBs potentially lethal as they can trigger apoptosis, but there is also the potential for the loss of genetic information. The response to DSBs is well conserved across Eukaryotes and comprises two stages: detection of the break and subsequent remedial action. The remedial action involves cell cycle arrest, DNA repair, and, if repair cannot be effected, possible apoptosis. Whilst many of the key components, especially in the initial detection of the break, are conserved there are also differences between plants and animals in some of the main components and their roles. In this thesis I have proposed an overall framework for the cellular response to DSBs in plants and have proposed two candidate genes, TCP20 and SOG1, as novel plant specific activators in this response. Their suitability has been addressed by considering their activation and their downstream targets. I have shown that TCP20 is necessary for growth arrest observed in shoot apical meristems after exposure to genotoxic stress. I have also shown that activation of one of the key targets of TCP20, CYCB1;1 requires TCP20 and that a key TCP20 binding motif in the promoter of CYCB1;1 is necessary for the up-regulation of CYCB1;1 in response to genotoxic stress. This motif is over-represented in the promoters of many of the genes involved in DNA damage repair, suggesting that TCP20 plays a role in the co-ordination of the cellular response to DSBs.
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Yuan, Ying. "Modulation of DNA double strand breaks end-joining pathway choice by single stranded oligonucleotides in mammalian cells." Thesis, Toulouse 3, 2015. http://www.theses.fr/2015TOU30091.
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En réponse aux dommages de son génome, le choix par la cellule de la voie de réparation de l'ADN est un crucial par ses conséquences en termes de mutagénèse et de survie. Pour faire face aux cassures double-brin de l'ADN (CDB), les cellules humaines possèdent deux voies principales qui consistent soit à rejoindre les extrémités de la cassure par jonction d'extrémités non-homologues (voie conventionnelle C-NHEJ), soit à reconstituer par recombinaison homologue la séquence clivée en copiant son double non endommagé présent après la réplication (voie RH). La RH nécessite de dégrader l'un des brins d'ADN de part et d'autre de la cassure. Cette dégradation produit de courts fragments d'ADN simple-brin, connus pour aider à signaler le dommage à la cellule. Dans ce travail, nous avons évalué directement l'effet de ces fragments d'ADN simple brin sur la réparation des CDB dans des expériences biochimiques et cellulaires. Nous montrons que de courts fragments d'ADN simple-brin inhibent la C-NHEJ en inactivant sa protéine clef Ku, tout en stimulant une forme minoritaire de jonction des cassures dite NHEJ alternative (A-EJ). Ces travaux permettent de mieux comprendre comment la réparation par la voie peu connue A-EJ peut s'exprimer dans les cellules mais aussi d'envisager des stratégies pour piloter la réponse des cellules cancéreuses aux thérapies induisant des CDBIn response to DNA damage, the choice made by the cells between DNA repair mechanisms is crucial for mutagenic and survival outcomes. In humans, DNA double-strand breaks are repaired by two mutually-exclusive mechanisms, homologous recombination or end-joining. Among end-joining mechanisms, the main process is classical non-homologous end-joining (C-NHEJ) which relies on Ku binding to DNA ends and DNA Ligase IV (Lig4)-mediated ligation. Mostly under Ku- or Lig4-defective conditions, an alternative end-joining process (A-EJ) can operate and exhibits a trend toward microhomology usage at the break junction. Homologous recombination relies on an initial MRN-dependent nucleolytic degradation of one strand at DNA ends. This process, named DNA resection generates 3' single-stranded tails necessary for homologous pairing with the sister chromatid. While it is believed from the current literature that the balance between joining and recombination processes at DSBs ends is mainly dependent on the initiation of resection, it has also been shown that MRN activity can generate short single-stranded DNA oligonucleotides (ssO) that may also be implicated in repair regulation. In this work, we evaluate the effect of ssO on end-joining at DSB sites both in vitro and in cells. Under both conditions, we report that ssO inhibit C-NHEJ through binding to Ku and favor repair by the Lig4-independent microhomology-mediated A-EJ process. Our data bring new clues in the understanding of the cellular response to DNA double-strand breaks
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Choudhury, Sibgat Ahmed. "Role of TRM2RNC1 endo-exonuclease in DNA double strand break repair." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=103373.
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DNA double strand breaks (DSB) are the most toxic of all types of DNA lesions. In Saccharomyces cerevisiae, DNA DSBs are predominantly repaired by the homologous recombination repair (HRR) pathway. The initial step of HRR requires extensive processing of DNA ends from the 5' to 3' direction by specific endo-exonuclease(s) (EE) at the DSB sites, but no endo-exonuclease(s) has yet been conclusively determined for such processing of DSBs. S. cerevisiae TRM2/RNC1 is a candidate endo-exonuclease that was previously implicated for its role in the HRR pathway and was also shown to have methyl transferase activity primarily located at its c-terminus.In this dissertation, we provided compelling biochemical and genetic evidence that linked TRM2/RNC1 to the DNA end processing role in HRR. Trm2/Rnc1p purified with a small calmodulin binding peptide (CBP) tag displayed single strand (ss) specific endonuclease and double strand (ds) specific 5' to 3' exonuclease activity characteristic of endo-exonucleases involved in HRR. Intriguingly, purified Trm2/Rnc1p deleted for its C-terminal methyl transferase domain retained its nuclease activity but not the methyl transferase activity indicating that the C-terminal part responsible for its methyl transferase function is not required for its nuclease activity.
Our genetic and functional studies with S. cerevisiae trm2/rnc1 single mutants alone or in combination with other DNA DSB repair mutants after treatment with the DNA damaging drug methyl methane sulfonate (MMS) or IR that is believed to produce DSBs, or with specific induction of DNA DSBs at the MAT locus by HO-endonuclease demonstrated an epistatic relationship of TRM2/RNC1 with the major recombination factor RAD52. These studies suggested that TRM2/RNC1 probably acts at an earlier step than RAD52 in the HRR pathway. The genetic evidence also indicated a possible functional redundancy with the bona fide endo-exonuclease EXO1 in the processing of DNA ends at the DSB sites.
In a recent report, the immuno-purified mouse homologue of TRM2/RNC1 exhibited similar enzymatic properties as the endo-exonucleases involved in HRR. A small molecular inhibitor pentamidine specifically inhibited the nuclease activity of the mouse EE and sensitized various cancer cells to DNA damaging agents commonly used in cancer chemotherapy. We specifically suppressed expression of the mouse EE using small interfering RNA (siRNA) that conferred sensitivity of B16F10 melanoma cells to a variety of DNA damaging drugs often used in cancer treatment. This further validated our earlier claim of the endo-exonuclease as a potential therapeutic target in treating cancer.
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Ku, Chuan-Chih. "TCP6, a regulator in Arabidopsis gametophyte development and DNA damage response." Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/17892.
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Plants have developed intricate mechanisms to control growth in response to a variety of environmental cues, to compensate its immobility and to survive in both normal and adverse conditions. The TCP proteins are a family of plant-specific, basic helix-loop-helix (bHLH) transcription factors that involve in different aspects in plant growth and developmental control. The Arabidopsis TCP20 has been shown to involve in coordinating cell growth and proliferation, and in growth arrest in response to DNA double-stranded breaks (DSB). In this thesis, the main interest is to examine the function of Arabidopsis TCP6, which shares the highest homology with TCP20, and like TCP20, contains a putative ATM phosphorylation motif that suggests potential involvement in the ATM/ATR-mediated DSB responses. Expressional analysis including transcript measurement and reporter gene tagging demonstrated that TCP6 is expressed in flowers, in particular in the first mitotic event of pollen and ovule/embryo sac development, indicating that TCP6 potentially involves in regulating the mitotic cell cycle during gametophyte development. Yet no gametophytic or fertility-affecting mutant phenotype was observed in the tcp6 single and tcp6/tcp20 double mutants, which may be due to high functional redundancy. The tcp6/tcp20 double mutant seedlings exhibited significantly higher growth performances in true leaf growth compared to wild type when treated with gamma radiation, implying that both functional TCP6 and TCP20 are involved in response to gamma radiation-generated DSBs. The work of this thesis provides the first expressional and functional characterizations of TCP6, with the results suggesting that TCP6 and other class I TCPs play a role in regulating growth under both normal and stress conditions.
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Vasianovich, Yuliya. "Investigating the roles of the Srs2 and Pif1 helicases in DNA double-strand break repair in Saccharomyces cerevisiae." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/17984.
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DNA double strand breaks (DSBs), which may occur during DNA replication or due to the action of genotoxic agents, are extremely dangerous DNA lesions as they can cause chromosomal rearrangements and cell death. Therefore, accurate DSB repair is vital for genome stability and cell survival. Two main mechanisms serve to repair DNA DSBs: non-homologous end joining, which re-ligates DNA ends together, and homologous recombination (HR), which restores broken DNA using homologous sequence as a template for repair. One-ended DSBs are a subject for the specialised HR-dependent repair pathway known as break-induced replication (BIR). At low frequency, DNA breaks can also be healed by telomerase, which normally extends telomeres at natural chromosome ends, but may also add de novo telomeres to DSBs due to their similarity to chromosome ends. De novo telomere addition is a deleterious event, which is effectively inhibited by the nuclear Pif1 (nPif1) helicase phosphorylated at the TLSSAES motif in response to DNA damage. In this study, it is reported that the same regulatory motif of nPif1 is also required for DSB repair via BIR. The requirement of the nPif1 TLSSAES sequence in BIR is dependent on the functional DNA damage response (DDR). Thus, nPif1 phosphorylation by the DDR machinery might mediate the role of nPif1 in BIR. In contrast, the nPif1 regulatory motif is not essential for BIR at telomeres in cells lacking telomerase. These observations indicate that the mechanism of nPif1 function in DSB repair via BIR and in BIR at telomeres might be different. In this work, a protocol for nPif1 pull-down was optimized to reveal the mechanism of the phosphorylation-dependent nPif1 functions in cells undergoing DNA repair, i. e. the mechanism of nPif1-mediated inhibition of de novo telomere addition and promoting DSB repair via BIR. In future, this protocol can be used to dissect the role of nPif1 in DNA repair through the identification of its potential interacting partners. The Srs2 helicase negatively regulates HR via dismantling Rad51 filaments. According to preliminary data from the laboratory of Sveta Makovets, Srs2 also promotes de novo telomere addition at DSBs in a Rad51-dependent manner. The work presented here establishes that Srs2 is dispensable for telomerase-mediated addition of TG1-3 repeats to DSBs. Instead, Srs2 is required for the reconstitution of the complementary DNA strand after telomerase action, thus ensuring the completion of de novo telomere addition. Overall, this study demonstrates that recombination-dependent DSB repair and de novo telomere addition share common regulatory components, i. e. the nPif1 helicase phosphorylated in response to DNA damage and the Srs2 helicase. Phosphorylated nPif1 promotes DSB repair via BIR in addition to its known role in inhibition of telomerase at DSBs, whereas Srs2 uses its well established ability to remove Rad51 from ssDNA to promote the restoration of dsDNA and thus to complete de novo telomere addition.
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Weatherbee, Jessica L. "Exploiting DNA Repair and ER Stress Response Pathways to Induce Apoptosis in Glioblastoma Multiforme: A Dissertation." eScholarship@UMMS, 2008. http://escholarship.umassmed.edu/gsbs_diss/865.
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Glioblastoma multiforme (GBM) is a deadly grade IV brain tumor characterized by a heterogeneous population of cells that are drug resistant, aggressive, and infiltrative. The current standard of care, which has not changed in over a decade, only provides GBM patients with 12-14 months survival post diagnosis. We asked if the addition of a novel endoplasmic reticulum (ER) stress inducing agent, JLK1486, to the standard chemotherapy, temozolomide (TMZ), which induces DNA double strand breaks (DSBs), would enhance TMZ’s efficacy. Because GBMs rely on the ER to mitigate their hypoxic environment and DNA repair to fix TMZ induced DSBs, we reasoned that DSBs occurring during heightened ER stress would be deleterious.
Treatment of GBM cells with TMZ+JLK1486 decreased cell viability and increased cell death due to apoptosis. We found that TMZ+JLK1486 prolonged ER stress induction, as indicated by elevated ER stress marker BiP, ATF4, and CHOP, while sustaining activation of the DNA damage response pathway. This combination produced unresolved DNA DSBs due to RAD51 reduction, a key DNA repair factor. The combination of TMZ+JLK1486 is a potential novel therapeutic combination and suggests an inverse relationship between ER stress and DNA repair pathways.
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Weatherbee, Jessica L. "Exploiting DNA Repair and ER Stress Response Pathways to Induce Apoptosis in Glioblastoma Multiforme: A Dissertation." eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/865.
Full textAbstract:
Glioblastoma multiforme (GBM) is a deadly grade IV brain tumor characterized by a heterogeneous population of cells that are drug resistant, aggressive, and infiltrative. The current standard of care, which has not changed in over a decade, only provides GBM patients with 12-14 months survival post diagnosis. We asked if the addition of a novel endoplasmic reticulum (ER) stress inducing agent, JLK1486, to the standard chemotherapy, temozolomide (TMZ), which induces DNA double strand breaks (DSBs), would enhance TMZ’s efficacy. Because GBMs rely on the ER to mitigate their hypoxic environment and DNA repair to fix TMZ induced DSBs, we reasoned that DSBs occurring during heightened ER stress would be deleterious.
Treatment of GBM cells with TMZ+JLK1486 decreased cell viability and increased cell death due to apoptosis. We found that TMZ+JLK1486 prolonged ER stress induction, as indicated by elevated ER stress marker BiP, ATF4, and CHOP, while sustaining activation of the DNA damage response pathway. This combination produced unresolved DNA DSBs due to RAD51 reduction, a key DNA repair factor. The combination of TMZ+JLK1486 is a potential novel therapeutic combination and suggests an inverse relationship between ER stress and DNA repair pathways.
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Bennett, Gwendolyn M. "Chromatin Regulators and DNA Repair: A Dissertation." eScholarship@UMMS, 2012. http://escholarship.umassmed.edu/gsbs_diss/742.
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DNA double-strand break (DSB) repair is essential for maintenance of genome stability. However, the compaction of the eukaryotic genome into chromatin creates an inherent barrier to any DNA-mediated event, such as during DNA repair. This demands that there be mechanisms to modify the chromatin structure and thus access DNA. Recent work has implicated a host of chromatin regulators in the DNA damage response and several functional roles have been defined. Yet the mechanisms that control their recruitment to DNA lesions, and their relationship with concurrent histone modifications, remain unclear. We find that efficient DSB recruitment of many yeast chromatin regulators is cell-cycle dependent. Furthering this, we find recruitment of the INO80, SWR-C, NuA4, SWI/SNF, and RSC enzymes is inhibited by the non-homologous end joining machinery, and that their recruitment is controlled by early steps of homologous recombination. Strikingly, we find no significant role for H2A.X phosphorylation (γH2AX) in the recruitment of chromatin regulators, but rather that their recruitment coincides with reduced levels of γH2AX. We go on to determine the chromatin remodeling enzyme Fun30 functions in histone dynamics surround a DSB, but does not significantly affect γH2AX dynamics. Additionally, we describe a conserved functional interaction among the chromatin remodeling enzyme, SWI/SNF, the NuA4 and Gcn5 histone acetyltransferases, and phosphorylation of histone H2A.X. Specifically, we find that the NuA4 and Gcn5 enzymes are both required for the robust recruitment of SWI/SNF to a DSB, which in turn promotes the phosphorylation of H2A.X.
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Bennett, Gwendolyn M. "Chromatin Regulators and DNA Repair: A Dissertation." eScholarship@UMMS, 2014. https://escholarship.umassmed.edu/gsbs_diss/742.
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DNA double-strand break (DSB) repair is essential for maintenance of genome stability. However, the compaction of the eukaryotic genome into chromatin creates an inherent barrier to any DNA-mediated event, such as during DNA repair. This demands that there be mechanisms to modify the chromatin structure and thus access DNA. Recent work has implicated a host of chromatin regulators in the DNA damage response and several functional roles have been defined. Yet the mechanisms that control their recruitment to DNA lesions, and their relationship with concurrent histone modifications, remain unclear. We find that efficient DSB recruitment of many yeast chromatin regulators is cell-cycle dependent. Furthering this, we find recruitment of the INO80, SWR-C, NuA4, SWI/SNF, and RSC enzymes is inhibited by the non-homologous end joining machinery, and that their recruitment is controlled by early steps of homologous recombination. Strikingly, we find no significant role for H2A.X phosphorylation (γH2AX) in the recruitment of chromatin regulators, but rather that their recruitment coincides with reduced levels of γH2AX. We go on to determine the chromatin remodeling enzyme Fun30 functions in histone dynamics surround a DSB, but does not significantly affect γH2AX dynamics. Additionally, we describe a conserved functional interaction among the chromatin remodeling enzyme, SWI/SNF, the NuA4 and Gcn5 histone acetyltransferases, and phosphorylation of histone H2A.X. Specifically, we find that the NuA4 and Gcn5 enzymes are both required for the robust recruitment of SWI/SNF to a DSB, which in turn promotes the phosphorylation of H2A.X.
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Oza, Pranav O. "Nuclear Dynamics of a Broken Chromosome: A Dissertation." eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/422.
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In order to preserve its genomic integrity, an organism needs to detect and repair DNA double-strand breaks (DSBs) in a prompt and accurate fashion. This goal is accomplished by enabling an exquisitely sensitive DSB sensing apparatus as well as multiple and often overlapping pathways for repair. All of these processes are carried out on a highly organized and compacted chromatin substrate in the nucleus. An important question is whether chromatin plays an active role in the process and whether it helps in the signaling or repair of this damage. We have used Chromosome Conformation Capture (3C) to show that there are no large scale changes in chromosome structure at a single site-specific DNA double-strand break, although looping interactions between DSBs and donors can be detected.
In a surprising result, we found that 3C detected a nucleus-wide decrease in interactions with the DSB. We have used a combination of 3C, fluorescence microscopy and chromatin immunoprecipitation to show that the decrease in interactions is a result of the relocalization of persistent DSB to the nuclear periphery. We also show that this is dependent on the recruitment of telomerase complex to the DSB, which then interacts with its natural partner in the Inner nuclear membrane, Mps3, and relocalizes the DSB to the periphery. Thus, a DSB that cannot be repaired is shunted into a pathway where the cell attempts to survive by putting a de novotelomere on the broken chromosome.
Remarkably, this is not an irreversible phenomenon despite the recruitment of telomerase and the relocalization to the periphery. DSBs which are repaired slowly due to the presence of homology on a different chromosome, or merely usage of a kinetically slower form of repair, undergo this pathway switch, but can still recover and repair the DSB if homology is present. We also show that the role of the periphery is to ensure repair through de novotelomere formation or other non-canonical repair pathways. Indeed, loss of peripheral localization results in a dramatic suppression of the genomic instability of the Slx5/8 mutants, which have been implicated in the persistent DSB response at the Nuclear pores.
Thus, the nuclear periphery is a special compartment where DSBs go after they cannot be repaired by canonical pathways. Specialized components such as telomerase, silencing proteins and components of the SUMO pathway, all seem to play roles in the healing of these chromosomes. Importantly, the SUN domain homologues of Mps3 have been shown to play roles similar to their yeast homologues in meiotic bouquet formation through their interactions with telomeres. Thus, they may represent a conserved mechanism for chromosome healing and telomere anchoring, despite the fact that mammalian telomeres are rarely found at the nuclear periphery. Such survival mechanisms may be expected to operate in cancer cells which may or may not have upregulated telomerase expression.
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Ranjit, Sanjay. "The C Terminus of Activation Induced Cytidine Deaminase (AID) Recruits Proteins Important for Class Switch Recombination to the IG Locus: A Dissertation." eScholarship@UMMS, 2012. http://escholarship.umassmed.edu/gsbs_diss/516.
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Activation-induced cytidine deaminase (AID) is a key protein required for both class switch recombination (CSR) and somatic hypermutation (SHM) of antibody genes. AID is induced in B cells during an immune response. Lack of AID or mutant form of AID causes immunodeficiency; e.g., various mutations in the C terminus of AID causes hyper IgM (HIGM2) syndrome in humans. The C terminal 10 amino acids of AID are required for CSR but not for SHM. During both CSR and SHM, AID deaminates dCs within Ig genes, converting them to dUs, which are then either replicated over, creating mutations, or excised by uracil DNA glycosylase (UNG), leading to DNA breaks in Ig switch regions. Also, the mismatch repair (MMR) heterodimer Msh2-Msh6 recognizes U:G mismatches resulting from AID activity and initiates MMR, which leads to increased switch region double strand breaks (DSBs). DSBs are essential intermediates of CSR; lack of UNG or MMR results in a reduction of DSBs and CSR. The DSBs created in the Sμ and one of the downstream S-regions during CSR are recombined by non-homologous end joining (NHEJ) to complete CSR. Available data suggest that AID is required not only for the deamination step of CSR, but also for one or more of the steps of CSR that are downstream of deamination step. This study investigates the role of C terminus of AID in CSR steps downstream of deamination.
Using retroviral transduction into mouse splenic B cells, I show that AID binds cooperatively with UNG and Msh2-Msh6 to the Ig Sμ region, and this depends on the AID C terminus. I also show that the function of MMR during CSR depends on the AID C terminus. Surprisingly, the C terminus of AID is not required for Sμ or Sγ3 DSBs, suggesting its role in CSR occurs during repair and/or recombination of DSBs.
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Ranjit, Sanjay. "The C Terminus of Activation Induced Cytidine Deaminase (AID) Recruits Proteins Important for Class Switch Recombination to the IG Locus: A Dissertation." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/516.
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Activation-induced cytidine deaminase (AID) is a key protein required for both class switch recombination (CSR) and somatic hypermutation (SHM) of antibody genes. AID is induced in B cells during an immune response. Lack of AID or mutant form of AID causes immunodeficiency; e.g., various mutations in the C terminus of AID causes hyper IgM (HIGM2) syndrome in humans. The C terminal 10 amino acids of AID are required for CSR but not for SHM. During both CSR and SHM, AID deaminates dCs within Ig genes, converting them to dUs, which are then either replicated over, creating mutations, or excised by uracil DNA glycosylase (UNG), leading to DNA breaks in Ig switch regions. Also, the mismatch repair (MMR) heterodimer Msh2-Msh6 recognizes U:G mismatches resulting from AID activity and initiates MMR, which leads to increased switch region double strand breaks (DSBs). DSBs are essential intermediates of CSR; lack of UNG or MMR results in a reduction of DSBs and CSR. The DSBs created in the Sμ and one of the downstream S-regions during CSR are recombined by non-homologous end joining (NHEJ) to complete CSR. Available data suggest that AID is required not only for the deamination step of CSR, but also for one or more of the steps of CSR that are downstream of deamination step. This study investigates the role of C terminus of AID in CSR steps downstream of deamination.
Using retroviral transduction into mouse splenic B cells, I show that AID binds cooperatively with UNG and Msh2-Msh6 to the Ig Sμ region, and this depends on the AID C terminus. I also show that the function of MMR during CSR depends on the AID C terminus. Surprisingly, the C terminus of AID is not required for Sμ or Sγ3 DSBs, suggesting its role in CSR occurs during repair and/or recombination of DSBs.
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Marafona, Ana Marlene Neto. "The novel LAP1: TRF2 complex is associated to DNA damage." Master's thesis, Universidade de Aveiro, 2016. http://hdl.handle.net/10773/22000.
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Mestrado em Biomedicina MolecularLamin associated protein 1 (LAP1) is a type II integral membrane protein located at the inner nuclear membrane (INM). The role of LAP1 remains poorly understand, however, this protein has been associated with several cellular functions due to its interactions with lamins, phosphatase protein 1 (PP1), emerin and torsinA. Moreover, novel putative LAP1 interactors are emerging. A recent study from our group allowed the identification of several novel putative LAP1 interactors involved in telomere signaling and DNA damage responses, namely Ataxia-telangiectasia mutated (ATM), Telomeric repeat binding factor 2 (TRF2), Repressor Activator Protein 1 (RAP1), RAP1 interacting factor 1 homologue (RIF1), Mitotic arrest deficient-like1 (MAD2L1) and Mitotic arrest deficient-like1 binding protein (MAD2L1BP). Protein-protein interactions are crucial in the study of signaling pathways. In this study, TRF2 was identified as a novel LAP1 binding protein using both co-immunoprecipitations and mass spectrometry based methodologies. To determine the functional relevance of the novel complex LAP1:TRF2, HeLa cells were subjected to DNA damage using hydrogen peroxide (H2O2), namely double-stranded breaks (DSBs). In response to DSBs, the expression levels of LAP1 and TRF2 were significantly reduced. The phosphorylation of Histone 2A family member (γ-H2AX) that is considered the hallmark of DSBs was also evaluated. Upon DNA damage, LAP1 not only co-localizes with γ-H2AX in some specific points near nuclear envelope (NE) and nucleus, but also with TRF2 in the nuclear periphery. Moreover, LAP1 and TRF2 have been reported to be crucial for cell cycle progression. Therefore, we decided to pursued this issue. When the NE is reassembled, the complex is located mainly in specific regions of the NE, evidencing that TRF2 allows the attachment of chromosomes to NE membrane in somatic cells. In conclusion, our results are of paramount importance since novel functional insights regarding the novel LAP1:TRF2 complex were achieved particularly related with DNA damage response and cell cycle progression.
Proteína 1 associada com a lâmina (LAP1) é uma proteína integral da membrana do tipo II localizada na membrana nuclear interna (INM). O papel da LAP1 não é inteiramente sabido, no entanto esta proteína tem sido associada a algumas funções celulares devido às suas interações com as lâminas, proteína fosfatase 1 (PP1), emerina e torsinA. Além disso, novos putativos interactores da LAP1 estão a surgir. Um recente estudo do nosso grupo permitiu a identificação de vários novos putativos interactores da LAP1 envolvidos na sinalização dos telómeros e em respostas a danos no DNA, nomeadamente a mutação da ataxia telangiectasia (ATM), fator 2 de ligação às repetições teloméricas (TRF2), proteína 1 ativadora repressora (RAP1), fator homólogo 1 de interação com a RAP1 (RIF1), proteína 1 do checkpoint do fuso mitótico (MAD2L1) e a proteína de ligação à proteína 1 do checkpoint do fuso mitótico (MAD2L1BP). As interações proteína-proteína são cruciais no estudo das vias de sinalização. Neste estudo, a TRF2 foi identificada como uma nova proteína interatora da LAP1 utilizando tanto co-immunoprecipitação como metodologias baseadas em espectrometria de massa. Para determinar a relevância funcional do novo complexo LAP1:TRF2, células HeLa foram submetidas a danos no DNA através do peróxido de hidrogénio (H202), nomeadamente a quebras de DNA de cadeia dupla (DSBs). Em resposta a DSBs, os níveis de expressão da LAP1 e da TRF2 estavam significativamente reduzidos. A fosforilação do membro da família da histona 2A (γ-H2AX) que é considerado um biomarcador de DSBs foi também avaliada. Em resposta a danos no DNA, a LAP1 não só co-localiza com a γ-H2AX em alguns pontos específicos perto do invólucro nuclear (EN) e núcleo, mas também com TRF2 na periferia nuclear. Além disso, a LAP1 e a TRF2 têm sido reportadas como proteínas cruciais na progressão do ciclo celular. Por isso, decidimos prosseguir com esta questão. Quando o EN é remontado, o complexo está localizado principalmente em regiões especificas do EN, evidenciado que a TRF2 permite a ligação dos cromossomas à membrana do NE em células somáticas. Como conclusão, os nossos resultados são de uma importância suprema, uma vez que novas descobertas funcionais relativas ao novo complexo LAP1:TRF2 foram alcançadas, particularmente relacionadas com respostas a danos no DNA e progressão do ciclo celular.
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Prudden, Giulia. "Responses to a site-specific DNA double-stranded break in Schizosaccharomyces pombe." Thesis, University of Oxford, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.249323.
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Bryntesson, Fredrik Anders. "An investigation of genes involved in double stranded break repair of DNA." Thesis, University College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.268453.
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Seo, Jooheon. "Modulation of DNA repair pathway after CRISPR/Cas9 mediated Double Stranded Break." Thesis, Virginia Tech, 2017. http://hdl.handle.net/10919/74884.
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The CRISPR/Cas9 system has become the predominant tool for genome editing. Targeted modifications can be introduced while repairing double strand breaks (DSBs), induced by the CRISPR/Cas9 system. The DSB is repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR), and the repair is commonly processed through NHEJ because it is the dominant repair pathway in most cell types. The goal of this study is to modulate DNA repair system of somatic cells to increase the frequency of homology-directed repair (HDR) through HR by chemical treatment and the frequency of NHEJ by serum starvation. CRISPR/Cas9 systems targeting RAG2 gene and donor DNA to replace endogenous RAG2 were transfected into porcine fetal fibroblast (PFF) cells and the cells were treated with various chemicals that were known to inhibit NHEJ or stimulate HR. Among the chemical treated groups, cells treated with thymidine showed an average of 5.85-fold increase in HDR compared to the control group; the difference ranged from 1.37 to 9.59. There was no positive effect on the frequency of HDR after treating transfected cells with other chemicals. Placing PFFs under low amount of serum (serum deprivation) could enrich the cells in G0/G1 phase, but there was little difference in the frequency of NHEJ. Our results indicate that modulating DNA repair pathways during CRISPR/Cas9-mediated gene targeting could change the outcome of the targeted events.Master of Science
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Yeeles, Joseph. "The initiation of double-stranded DNA break repair by an AddAB-type helicase-nuclease." Thesis, University of Bristol, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.503857.
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The DNA of all living organisms is constantly damaged by a wide variety of endogenous and exogenous agents. Double-stranded DNA breaks are particularly toxic and under certain conditions a single break will result in cell death. To preserve genome integrity, double-stranded breaks are faithfully repaired via homologous recombination. Recombinational repair in prokaryotes is generally initiated by a class of enzymes known as helicase-nucleases. These enzymes bind tightly to broken duplex DNA ends and use ATP driven motors to unwind the DNA.
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Odeh, Mona. "Establishment of Recombinant Adeno-Associated Virus Vector Integration Frequency In Vitro and In Vivo." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1338435720.
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Zierhut, C. "The involvement of single-stranded DNA, replication protein A, and the DNA double-strand break dose in the damage checkpoint of Saccharomyces cerevisiae." Thesis, University College London (University of London), 2007. http://discovery.ucl.ac.uk/1445169/.
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In response to DNA damage, eukaryotic cells activate a checkpoint signalling cascade, resulting in cell cycle arrest, stabilisation of replication forks and activation of repair. While many players in these pathways have been identified, little is known about the original sensors, or of the DNA structures involved. Because it is present in all checkpoint-inducing lesions, single-stranded DNA (ssDNA) is a good candidate for a common structure recognised by the DNA damage response. The role of ssDNA in checkpoint activation in the yeast Saccharomyces cerevisiae was investigated using three different approaches. Firstly, an attempt was made to produce ssDNA independently of strand breaks by inducing replication-independent plasmid unwinding. Secondly, the effects of depleting the major ssDNA-binding complex, replication protein A (RPA) were analysed. Lastly, an assay to quantify ssDNA generated at a defined DNA double-strand break (DSB) was developed. Despite extensive efforts, the first approach proved unsuccessful, as the method used did not generate unwound plasmid. Using the second approach, it was found that depletion of RPA did not inhibit checkpoint activation during replication stress. Furthermore, replication with limiting amounts of RPA led to rapid cell death and checkpoint activation that was mediated independently of the response to stalled replication forks. Lastly, at a defined DSB it was found that less ssDNA was being generated than had previously been estimated from results based on non-quantitative methods. Additionally, an element of dose dependency was observed in the checkpoint response to DSBs, with stronger and more rapid responses being generated by higher numbers of breaks. Formation of four DSBs resulted in checkpoint activation even in G1 arrested cells. Together, these results raise the possibility of a DNA damage checkpoint pathway largely independent of long tracts of RPA-coated ssDNA and show that checkpoint activation to DSB-damage is possible in G1.
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Rajan, Rakhi. "Structural and functional studies of the bacterial RECA protein." The Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=osu1186676763.
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Ramírez-Lugo, Juan S. "The Activation of ATR in Response to Double-Stranded DNA Breaks." Thesis, 2010. https://thesis.library.caltech.edu/5847/2/JSRL-Thesis.pdf.
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The cellular response to the presence of double-stranded DNA breaks (DSBs) is primarily mediated by the ATM protein kinase. A related kinase, ATR, regulates the responses to dysfunctional DNA replication and is also activated, in an ATM-dependent manner, when breaks occur during S-phase. The latter is achieved by the ability of ATM to interact with TopBP1, an inducer of ATR activity. Additionally, in Xenopus egg extracts, the Mre11-Rad50-Nbs1 (MRN) complex is required to bridge ATM and TopBP1 together. With our current work, we show that CtIP, a known MRN-interacting protein, is recruited to DSB-containing chromatin and interacts with TopBP1 in a damage-dependent manner. A region containing the first two BRCT repeats of TopBP1 is essential for this interaction. Furthermore, two distinct regions of CtIP participate in mediating the association between CtIP and TopBP1. The first region includes two putative ATM/ATR phosphorylation sites. Secondly, an MRN-binding region in the N-terminal region of CtIP is involved. In addition, the binding between CtIP and TopBP1 is diminished in Nbs1-depleted extracts and, reciprocally, the binding of Nbs1 to TopBP1 decreases in the absence of CtIP. This suggests the formation of a complex containing CtIP, TopBP1 and the MRN complex. When CtIP is removed from egg extracts, the levels of TopBP1 and Nbs1 in damaged nuclei are reduced, thereby compromising the activation of the damage response. Thus, CtIP interacts with TopBP1 in a damage-stimulated, MRN-dependent manner to mediate the activation of ATR in response to DSBs.
We additionally explore the involvement of the chromatin remodeling ATPase ISWI in the responses to DNA damage. We find that ISWI associates with ATR, ATRIP, and TopBP1 on DNA in the presence of damage. In addition, ISWI is a substrate of both ATM and ATR in vitro. Furthermore, the activities of ATM and ATR stimulate an increase in the levels of ISWI on chromatin that contains DSBs. Finally, we assessed the role of ISWI in the activation of multiple damage responses in Xenopus egg extracts. Taken together, our work describes several previously uncharacterized features of ISWI with implications in the response to damaged and incompletely replicated DNA.
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Ghodke, Indrajeet Laxman. "The Role of Saccharomyces Cerevisiae MRX Complex and Sae2 in Maintenance of Genome Stability." Thesis, 2015. http://etd.iisc.ernet.in/2005/3679.
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In eukaryotes, the repair of DSBs is accomplished through two broadly defined processes: Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). The central step of HR is pairing and exchange of strands between two homologous DNA molecules, which is catalyzed by the conserved Rad51/RecA family of proteins. Prior to this step, an essential step in all HR pathways i.e. 5'→3' resection of broken DNA ends to generate 3' single stranded DNA tails. At the molecular level, initiation of DNA end resection is accomplished through the concerted action of MRX complex (Mre11, Rad50 and Xrs2) and Sae2 protein.
To elucidate the molecular basis underlying DSB end resection in S. cerevisiae mre11 nuclease deficient mutants, we have performed a comprehensive analysis of the role of S. cerevisiae Mre11 (henceforth called as ScMre11) in the processing of DSB ends using a variety of DNA substrates. We observed that S. cerevisiae Mre11(ScMre11) exhibits higher binding affinity for single- over double-stranded DNA and intermediates of recombination and repair and catalyzes robust unwinding of substrates possessing a3' single-stranded DNA overhang but not of 5' overhangs or blunt-ended DNA fragments. Furthermore, reconstitution of DSB end resection network in-vitro revealed that Rad50, Xrs2, and Sae2 potentiated the DNA unwinding activity of Mre11. Since the exonuclease activity of Mre11 is of the opposite polarity to that expected for resection of DSBs, unwinding activity of Mre11 in conjunction with Rad50, Xrs2, and Sae2 might provide an alternate mechanism for the generation of ssDNA intermediates for DSB end repair and HR. Additionally, ScMre11 displays strong homotypic as well as heterotypic interaction with Sae2. In summary, our results revealed important insights into the mechanism of DSB end processing and support a model in which Sae2, Rad50, and Xrs2 positively regulate the ScMre11-mediated DNA unwinding activity via their direct interactions or through allosteric effects on the DNA or cofactors.
Prompted by the closer association of MRX and Sae2 during DSB end processing, we asked whether Sae2 and its endonuclease activity is required for cellular response to replication stress caused by DNA damage. Toward this end, we examined the sensitivity of S. cerevisiae wild type, sae2Δ and various SAE2 mutant strains defective in phosphorylation and nuclease activity in the presence of different genotoxic agents, which directly or indirectly generate DSBs during replication. We found that S. cerevisiae lacking SAE2 show decreased cell viability, altered cell cycle dynamics after DNA damage, and more specifically, that Sae2 endonuclease activity is essential for these biological functions. To corroborate the genetic evidences for role of SAE2 during replicative stress, we investigated SAE2 functions in-vitro. For this, we purified native Sae2 protein and nuclease dead mutant of Sae2 i.e. sae2G270D. Our studies revealed dimeric forms of both the wild type and mutant forms of Sae2. Furthermore, Sae2 displays higher binding affinity and catalytic activity with branched DNA structures, such as Holliday junction and replication forks. By using nuclease dead Sae2 protein i.e. sae2G270D, we confirmed that the endonuclease activity is not fortuitous and is intrinsic to Sae2 polypeptide. Furthermore, nuclease-defective Mre11 stimulates Sae2endonuclease activity. Mapping of the cleavage sites of Sae2 revealed a distinct preference for cleavage on the 5' end of the Holliday junction, suggesting the importance of Sae2 nuclease during recombination mediated restart of the reversed replication fork. In summary, our data clearly demonstrate a previously uncharacterized role for Sae2 nuclease activity in resection of DSB ends, processing of intermediates of DNA replication/repair and attenuation of DNA replication stress-related defects in S. cerevisiae.
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Lepage, Étienne. "Le maintien de la stabilité génomique du plastide : un petit génome d’une grande importance." Thèse, 2015. http://hdl.handle.net/1866/13030.
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Chez les plantes, le génome plastidique est continuellement exposé à divers stress mutagènes,
tels l’oxydation des bases et le blocage des fourches de réplication. Étonnamment, malgré ces
menaces, le génome du plastide est reconnu pour être très stable, sa stabilité dépassant même celle
du génome nucléaire. Néanmoins, les mécanismes de réparation de l’ADN et du maintien de la
stabilité du génome plastidique sont encore peu connus.
Afin de mieux comprendre ces processus, nous avons développé une approche, basée sur
l’emploi de la ciprofloxacine, qui nous permet d’induire des bris d’ADN double-brins (DSBs)
spécifiquement dans le génome des organelles. En criblant, à l’aide de ce composé, une collection de
mutants d’Arabidopsis thaliana déficients pour des protéines du nucléoïde du plastide, nous avons
identifié 16 gènes vraisemblablement impliqués dans le maintien de la stabilité génomique de cette
organelle. Parmi ces gènes, ceux de la famille Whirly jouent un rôle primordial dans la protection du
génome plastidique face aux réarrangements dépendants de séquences de microhomologie. Deux
autres familles de gènes codant pour des protéines plastidiques, soit celle des polymérases de types-I
et celle des recombinases, semblent davantage impliquées dans les mécanismes conservateurs de
réparation des DSBs. Les relations épistatiques entre ces gènes et ceux des Whirly ont permis de
définir les bases moléculaires des mécanismes de la réparation dépendante de microhomologies
(MHMR) dans le plastide. Nous proposons également que ce type de mécanismes servirait en quelque
sorte de roue de secours pour les mécanismes conservateurs de réparation.
Finalement, un criblage non-biaisé, utilisant une collection de plus de 50,000 lignées mutantes
d’Arabidopsis, a été réalisé. Ce criblage a permis d’établir un lien entre la stabilité génomique et le
métabolisme des espèces réactives oxygénées (ROS). En effet, la plupart des gènes identifiés lors de ce
criblage sont impliqués dans la photosynthèse et la détoxification des ROS. Globalement, notre étude
a permis d’élargir notre compréhension des mécanismes du maintien de la stabilité génomique dans
le plastide et de mieux comprendre l’importance de ces processus.The plant plastidial genome is constantly threatened by many mutagenic stresses, such as base oxidation and replication fork stalling. Despite these threats, the plastid genome has long been known to be more stable than the nuclear genome, suggesting that alterations of its structure would have dramatic consequences on plant fitness. At the moment, little is known about the genes and the pathways allowing such conservation of the organelle genome sequences. To gain insight into these mechanisms, we developed an assay which uses ciprofloxacin, a gyrase inhibitor, to generate DNA double-strand breaks (DSBs) exclusively in plant organelles. By screening mutants deficient for proteins composing the plastid nucleoid on ciprofloxacin, we were able to identify 16 candidate genes, most likely involved in the repair of DSBs in plastid. Among these genes, those of the Whirly family of single-stranded DNA binding proteins are shown to be key factors in protecting the genome from error-prone microhomology mediated repair (MHMR). Two other family of proteins, the plastid type-I polymerases and the plastid recombinases, seem to be involved in the conservative repair pathways. The evaluation of the epistatic relationship between those two genes and the Whirly genes led us to define the molecular basis of MHMR and to propose that they might act as a backup system for conservative repair pathways. Finally, a non-biased screen, using 50,000 different insertion lines, allowed the identification of numerous genes that were already associated with ROS homeostasis, suggesting a link between DNA repair and ROS imbalance. Globally, our study shed light on the mechanisms that allow the maintenance of plastid genome, while explaining the importance of such conservation of the plastid genome.