Dissertations / Theses on the topic 'Human DNA repair and recombination pathways'

The present thesis concerns of two sections. The first one focuses on the application of Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR) system as a tool for precise genome targeting and genome editing; the association between specific endonuclease and RNA guides complementary to the DNA target allows its targeting with single-nucleotide precision. CRISPR/Cas is able to perform Double-Strand Breaks (DSBs) at a target site which are soon repaired by cellular repairing mechanism, non-homologous end joining (NHEJ) or homology-directed repair (HDR). The first part of my project aims to explore and demonstrate the efficacy of a personalized therapeutic approach based on the CRISPR/Cas9 technology associated with adeno-associated viral vectors (AAVs)s, a mutation-specific gene therapy to restore mutated genes in genetic diseases to their original sequence trough the HDR-mediated correction. I developed an mCherry/EGFP reporter cassette where the reporter gene bears a mutation-specific target. It connects the mCherry and the EGFP (out of frame) coding sequences. Due to a frameshift, the reactivation of the EGFP allows the visualization of cells in which Cas9 had targeted the mutation-specific sequence leading to the production of Indels. I worked to edit mutations involved in specific genetic diseases such as mutations in FOXG1 or in MECP2, which are responsible for Rett syndrome, in the IQSEC2 gene that causes an intellectual disability clinically related to the Rett syndrome and in COL4A3 and COL4A5 causing Alport syndrome. In the second part of my study, I worked on developing a gene editing system aims to selective targeting to cancer cells while preserving the genetic integrity of normal cells. To this aim, I plan to exploit microhomology-mediated end joining (MMEJ) through Cas12a, an RNA-directed endonuclease that causes double-strand breaks with staggered ends, to insert in-frame the Herpes Simplex Virus –Thymidine Kinase suicide gene to trigger cell death. I designed and developed a construct to target a patient-specific single nucleotide variant within a coding sequence of the TP53 gene, from a patient with Chronic Lymphocytic Leukemia characterized by clonal expansion of clones bearing this TP53 mutation. I am able to detect the proper integration of the suicide gene sequence by analyzing the treated cells by fluorescence-activated cell sorting (FACS). Indeed, a green fluorescent protein (EGFP) sequence is linked to the TK by a 2A peptide system, thus green fluorescent cells are also the one expressing for the TK gene. The second section of my thesis concerns the COVID-19 pandemic global crisis and the need to understand how best to study and treat COVID-19. A key focus is sharing and analyzing data to learn about the genetic determinants of COVID-19 susceptibility, severity, and outcomes. In particular, my work has been focused on the TLR7 gene that has been involved as an important pattern recognition receptor for the ssRNA of SARS-CoV-2. We demonstrate that rare loss-of-function variants in the TLR7 gene in young men with severe COVID-19 and with no prior history of major chronic diseases were associated with impaired TLR7 signaling and type I and II IFN responses.

Maintaining genome integrity is indispensible for cells to prevent and limit accruement of deleterious mutations and to promote viable cell growth and proliferation. Cells possess a myriad of mechanisms to detect, prevent and repair incurred cellular damage. Here we discuss various proteins and their accompanying cellular pathways that promote genome stability. We first investigate the NEDD8 protein and its role in promoting homologous recombination repair via multiple Cullin E3 ubiquitin ligases. We provide specific mechanisms through which, UBE2M, an E2 conjugating enzyme, neddylates various Cullin ligases to render them catalytically active to degrade their substrates by the proteasome. We show that CUL1, CUL2 and CUL4 are important in regulating various steps in the DNA damage response. Our data indicates that UBE2M and the neddylation pathway are important for genome stability. Our second topic discusses the role of the USP1- UAF1 deubiquitinating enzyme in promoting homologous recombination. We show that USP1-UAF1 interact with and stabilize RAD51AP1 (RAD51- Associated Protein 1). RAD51AP1 has previously been reported to promote homologous recombination by facilitating recombinase activity of RAD51, an essential protein involved in homologous recombination repair. We show that USP1, UAF1 and RAD51AP1 depletion leads to genome instability. Our data demonstrates the importance of these proteins in promoting genome integrity via homologous recombination.

Homologous recombination (HR) is essential for the repair of DNA doublestrand breaks (DSBs) and damaged replication forks. However, HR can also cause gross chromosomal rearrangements (GCRs) by producing crossovers (COs), resulting in the reciprocal exchange of sequences between non-sister chromatids. Therefore, HR-mediated GCRs are suppressed via the promotion of HR pathways that favour noncrossover (NCO) formation, such as the synthesis-dependent strand annealing (SDSA) and dissolution pathways, which are modulated by Mph1 and Sgs1 helicases, respectively. The mismatch repair (MMR) pathway is intricately associated with HR via its roles in repairing mismatches on heteroduplex DNA that can arise during HR and in preventing homeologous recombination. Using a plasmid break-repair assay, we have revealed a novel, MMR-independent role of MutSα in promoting the formation of a subset of COs that is specifically supressible by Mph1, during HR between two completely homologous sequences. In contrast, the MMR-dependent function of MutSα, together with Mph1 and Sgs1, was shown to be required for the suppression of CO formation during homeologous recombination. These data indicate that Mph1 can both antagonise and promote the functions of MutSα during DSB repair, depending on the levels of homology between the two recombining sequences. COs are generated by the resolution of Holliday junction (HJ) intermediates formed at the terminal stages of HR. Several S.cerevisiae proteins such as Yen1, Mus81, Slx1 and Rad1 have been implicated in HJ resolution. However, the in vivo roles of these proteins in HJ resolution remain to be confirmed. To directly and quantitatively monitor in vivo HJ resolution in S.cerevisiae, a transformation-based HJ resolution assay using a plasmid-borne HJ substrate has been developed. Using this system, we have demonstrated an in vivo HJ resolution function of Yen1, which acts redundantly with Mus81. Moreover, these redundant activities of Yen1 and Mus81 are essential for survival during replication stress, but are dispensable for DSB repair. An Slx4 and Rad1-dependent in vivo HJ resolution activity was also observed in the absence of Yen1 and Mus81 that was suppressed by presence of Slx1. Models describing how the nucleases interact to process HJs in vivo will be discussed.

The stability of DNA is a critical factor for several diseases, the most prevalent of which is cancer. Several neurodegenerative and accelerated aging diseases are also characterized by genomic instability. The number and complexity of DNA repair pathways that human cells possess underscores the importance of genomic stability. These pathways ensure that damaged DNA is repaired and that a cells complement of DNA remains stable upon cell division. How one particular type of DNA alteration, a DNA loop, is processed in human cells was the focus of this study. We have employed an in vitro system to study defined DNA loop substrates by human nuclear extracts. The influence of either a 5 or 3 nick, the range of loop sizes processed, and the role of DNA mismatch repair, DNA nucleotide excision repair, and the Werner Syndrome helicase proteins were variables tested. The results indicate tha t DNA loops containing between 5 to 12 nucleotides are processed in a strand - specific manner when either a 5 or 3 nick is present , with repair being targeted solely to the nicked strand . This repair occurs by both mismatch repair dependent and independent pathways. The processing of DNA loops containing 30 nucleotides in length is directed either by a 5 nick, or by the loop itself, but not by a 3 nick. The nick independent pathway results solely in loop removal. The large loop pathway is independent of mismatch repair, nucleotide excision repair, and the WRN helicase/exonuclease protein. Both of the 5 nick directed pathways occur by excision that initiates at the pre- existing nick and proceeds towards the loop along the shortest path between the nick and loop. DNA resynthesis occurs using either DNA polymerase , , or and also initiates at the pre-existing 5 nick. The 3 nick directed intermediate loop repair pathway proceeds in a similar fashion, likely after a nick is made 5 to the loop region on the strand that contained the pre-existing nick. DNA synthesis inhibition has only a minor affect on the nick independent loop removal pathway as only a short tract of DNA surrounding the loop site is processed. In total, the results point to at least 3 novel pathways that process DNA loops that likely contribute to total genomic stability.

The aims of this study were to undertake the first comprehensive in vitro, in vivo and clinical investigation into the effects of the PARP-1 inhibitor, AG014699, in human cancers defective in homologous recombination (HR) DNA double strand break (DSB) repair. HR deficient cells were 9-fold more sensitive to AG014699 than HR proficient cells (mean LC50 = 3.26 μM vs. 29.68; P < 0.0001), confirming the theory of synthetic lethality. BRCA1 methylated UACC3199 breast cancer cells were also sensitive to AG014699 with mean LC50 significantly lower than the HR proficient cells (7.6 μM vs. 29.68; P = 0.002). AG014699 inhibited PARP activity by > 95% and induced DNA DSBs in all 11 cell lines studied. Evidence of HR (by Rad51 foci) was observed only in cells with functional BRCA1/2. A prolonged schedule of AG014699 (10 mg/kg daily for five days of a seven-day cycle for six cycles) more effectively delayed the growth of BRCA2 mutated xenografts than a ten day AG014699 schedule (tumour growth delay (TGD) = 27.5 vs. 12.5 days; P = 0.02). AG014699 significantly delayed UACC3199 tumour growth compared to untreated controls (mean time to relative tumour volume 5 = 35.8 vs. 25.2 days; P = 0.05); confirming in vitro findings that BRCA1 methylated cancer cells are sensitive to PARP inhibition. Clinical trial data from 38 patients demonstrated that AG014699 is non-toxic and efficacious with a clinical benefit rate of 34%. Higher baseline PARP-1 activity was associated with response to AG014699. The major findings of these studies are: the confirmation of the selective cytotoxicity of PARP inhibitors in BRCA mutated cancers; the results in UACC3199 cells which suggest that cancers with other HR defects could benefit from single agent PARP inhibitors, and finally the concept that length of exposure to (not just degree of) PARP inhibition is important for single agent anti-tumour activity. Furthermore, these data have formed the basis for a major amendment to the clinical trial; the result of which is eagerly awaited.

Thesis (M.S.)--University of Kentucky, 2009.
Title from document title page (viewed on May 6, 2009). Document formatted into pages; contains: v, 27 p. : ill. Includes abstract and vita. Includes bibliographical references (p. 25-26).

The human ribosomal RNA genes are critically important for cell metabolism and viability. They code for the catalytic RNAs which, encased in a housing of more than 80 ribosomal proteins, link together amino acids by peptide bonds to generate all cellular proteins. Because the RNAs are not repeatedly translated, as is the case with messenger RNAs, multiple copies are required. The genes which code for the human ribosomal RNAs (rRNAs) are arranged as clusters of tandemly repeated sequences. Three of four catalytic RNAs are spliced from a single transcript. The genes are located on the short arms of the five acrocentric chromosomes (13, 14, 15, 21, and 22). The genes for the fourth rRNA are on chromosome 1q42, also arranged as a cluster of tandem repeats. The repeats are extremely similar in sequence, which makes them ideal for misalignment, non‐allelic homologous recombination (NAHR), and genomic destabilization during meiosis , replication, and damage repair. In this dissertation, I have used pulse‐field gel electrophoresis and in‐blot Southern hybridization to explore the physical structure of the human rRNA genes and determine their stability and heritability in normal, healthy individuals. I have also compared their structure in solid tumors compared to normal, healthy tissue from the same patient to determine whether dysregulated homologous recombination is an important means of genomic destabilization in cancer progression. Finally, I used the NCI‐60 panel of human cancer cell lines to compare the results from the pulsed‐field analysis, now called the gene cluster instability (GCI) assay, to two other indicators of homologous‐recombination-mediated genomic instability: sister chromatid exchange, and 5‐hydroxymethyl‐2’deoxyuridine sensitivity.

ATP plays a critical role in the regulation of many enzyme processes. In this work, I have focused on the ATP mediated regulation of the recombination processes catalyzed by the E. coliRecA and the human Rad51 proteins. The RecA protein is a multifunctional enzyme, which plays a central role in the processes of recombinational DNA repair, homologous genetic recombination and in the activation of the cellular SOS response to DNA damage. Each of these functions requires a common activating step, which is the formation of a RecA-ATP-ssDNA nucleoprotein filament. The binding of ATP results in the induction of a cooperative, high affinity ssDNA binding state within RecA (Menetski & Kowalczykowski, 1985b; Silver & Fersht, 1982). Data presented here identifies Gln194 as the NTP binding site "γ-phosphate sensor", in that mutations introduced at this residue disrupt all ATP induced RecA activities, while basal enzyme function is maintained. Additionally, we have dissected the parameters contributing to cooperative nucleoprotein filament assembly in the presence of cofactor. We show that the dramatic increase in the affinity of RecA for ssDNA in the presence of ATP is a result of a significant increase in the cooperative nature of filament assembly and not an increase in the intrinsic affinity of a RecA monomer for ssDNA. Previous work using both mutagenesis and engineered disulfides to study the subunit interface of the RecA protein has demonstrated the importance of Phe217 for the maintenance of both the structural and functional properties of the protein (Skiba & Knight, 1994; Logan et al., 1997; Skiba et al., 1999). A Phe217Tyr mutation results in a striking increase in cooperative filament assembly. In this work, we identify Phe217 as a key residue within the subunit interface and clearly show that Phe217 is required for the transmission of ATP mediated allosteric information throughout the RecA nucleoprotein filament. The human Rad51 (hRad51) protein, like its bacterial homolog RecA, catalyzes genetic recombination between homologous single and double stranded DNA substrates. This suggests that the overall process of homologous recombination may be conserved from bacteria to humans. Using IAsys biosensor technology, we examined the effect of ATP on the binding of hRad51 to ssDNA. Unlike RecA, we show that hRad51 binds cooperatively and with high affinity to ssDNA both in the presence and absence of nucleotide cofactor. These results show that ATP plays a fundamentally different role in hRad51 vs.RecA mediated processes. In summary, through the work presented in this dissertation, we have defined the critical molecular determinants for ATP mediated allosteric regulation within RecA. Furthermore, we have shown that ATP is not utilized by Rad51 in the same manner as shown for RecA, clearly defining a profound mechanistic difference between the two proteins. Future studies will define the requirement for ATP in hRad51 mediated processes.

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム
Kyoto University (京都大学)
0048
新制・課程博士
博士(医科学)
甲第21696号
医科博第100号
新制||医科||7(附属図書館)
京都大学大学院医学研究科医科学専攻
(主査)教授 齊藤 博英, 教授 清水 章, 教授 Shohab YOUSSEFIAN
学位規則第4条第1項該当

L'intégrité de l'information génétique est essentielle aux fonctions cellulaires et pour éviter l'instabilité génomique, qui est une des caractéristique du cancer. Suite à des cassures double brin (Double Strand Breaks; DSBs), la voie de signalisation de réponse aux dommages de l'ADN est activée dans la cellule qui comprend deux sous voies de signalisation : la jonction d'extrémités non-homologues et la recombinaison homologue. Le complexe TREX-2 associé au pore nucléaire est impliqué dans l'export des ARNm. Chez la levure, TREX-2 est impliqué dans le maintien de la stabilité génomique. Nous nous sommes intéressés au rôle de TREX-2 dans la réparation de DSBs dans les cellules humaines. La déplétion du complexe TREX-2 entraine une réparation de l'ADN par recombinaison homologue insuffisante. De plus, nos résultats démontrent que la protection contre les dommages de l'ADN par TREX-2 dépend aussi de l'équilibre entre H2B an H2Bub1 contrôlé par le module de deubiquitination de SAGA
The maintenance of proper genetic information is essential to avoid genomic instability, which is a hallmark of cancer. In response to Double Strand Breaks (DSBs), cells initiate the DNA Damage Response (DDR), that acts through two main sub-pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). The nuclear pore-associated TREX-2 complex is involved in mRNA export and has been implicated, in yeast, in genome stability maintenance. Here we investigated the role of TREX-2 in DSB repair in human cells. We find that loss of the scaffold subunit of TREX-2 (GANP) results in DNA repair deficiency by HR. Moreover, we showed that the mechanism through which TREX-2 protects human cells from DNA damage is dependent on an interplay with the co-activator complex SAGA that regulates H2Bub1 histone mark. Our results demonstrate a functional cross-talk between human TREX-2 and the SAGA deubiquitination activity that is important to ensure correct DSB repair during HR

Repair of DNA double-strand breaks via homologous recombination is an essential pathway for vertebrate cell development and maintenance of genome integrity throughout the organism’s lifetime. The Rad51 enzyme provides the central catalytic function of homologous recombination while many other proteins are involved in regulation and assistance of Rad51 activity, including a group of five proteins referred to as Rad51 paralogs (Rad51B, Rad51C, Rad51D, Xrcc2, Xrcc3). At the start of my work, cellular studies of human Rad51 (HsRad51) had shown only that it forms distinct nuclear foci in response to DNA damage. Additionally, no information regarding the cellular localization, potential DNA damage-induced redistribution or cellular functions for any of the Rad51 paralog proteins was available. Therefore, the goals of this work were to (1) present a more complete description of the cellular localization and DNA damage-induced redistribution of Rad51 and the two paralog proteins known to specifically associate with Rad51, Rad51C and Xrcc3, and (2) to define specific functional roles for Rad51C and Xrcc3 in mediating Rad51 activity. I focused on the use of cellular, RNAi and immunofluorescence methods to study endogenous Rad51, Rad51C and Xrcc3 in human cells. In my initial studies we showed for the first time that Xrcc3 forms distinct foci in both the nucleus and cytoplasm independent of DNA damage, that the distribution of these foci did not change significantly throughout the time course of DNA damage and repair, and that Xrcc3 focus formation is independent of Rad51. Additionally, and unlike most previously published images of nuclear Rad51, we found that the majority of DNA damage-induced nuclear Rad51 foci do not colocalize with gamma H2AX, a histone marker used to indicate the occurrence of DNA double strand breaks. As a consequence of these initial outcomes, a significant amount of effort was devoted to developing and optimizing immunofluorescence methods. Importantly, we developed a purification method for commercially available monoclonal antibodies against Rad51C and Xrcc3 that significantly improved their reactivity and specificity. My next study concentrated on Rad51C. Similar to Xrcc3, we found for the first time that Rad51C forms distinct nuclear and cytoplasmic foci independent of DNA damage and Rad51. An additional and surprising outcome was our discovery that Rad51C plays an important role in regulating the ubiquitination and proteasome-mediated degradation of Rad51. While biochemical functions for Rad51 paralog proteins had been suggested in the literature, this was the first demonstration of a specific biochemical function for Rad51C that contributes directly to the Rad51 activity in the homologous recombination pathway. Our improved immunofluorescence methods allowed us to see the accumulation of Rad51, Rad51C and Xrcc3 at the nuclear periphery early in response to DNA damage, suggesting the existence of a DNA damage-dependent trafficking mechanism that promoted movement of these proteins from the cytoplasm to the nucleus. This led to further studies in which we show distinct co-localization of cytoplasmic Rad51 with actin as well as alpha and beta tubulin. Using both immunofluorescence and sub-cellular fractionation methods our recent results strongly suggest that trafficking of Rad51 to the nucleus in response to DNA damage is regulated at least in part by its association with cytoskeletal proteins, and involves movement of both existing pools of Rad51 and newly synthesized protein. In a particularly exciting development, in collaboration with Leica Microsystems and Dr. Joerg Bewersdorf at The Jackson Laboratory, Bar Harbor, ME., I have been able to exploit a new technology, 4Pi microscopy, to provide the first images of endogenous nuclear proteins using this method. Results presented in this thesis have added significantly to a more complete understanding of cellular localization Rad51, Rad51C and Xrcc3, and have provided important insights into possible mechanisms of cellular trafficking of Rad51 in response to response to DNA damage. Additionally, we have defined a specific function for Rad51C in its regulation of Rad51 ubiquitination. These findings open several new avenues of investigation for furthering our understanding of the cellular and molecular functions of proteins with critical roles in the maintenance of genome integrity in human cells.

Indiana University-Purdue University Indianapolis (IUPUI)
DNA double strand break (DSB) is one of the most threatening of all types of DNA damages as it leads to a complete breakage of the chromosome. The cell has evolved several mechanisms to repair DSBs, one of which is break-induced replication (BIR). BIR repair of DSBs occurs through invasion of one end of the broken chromosome into a homologous template followed by processive replication of DNA from the donor molecule. BIR is a key cellular process and is implicated in the restart of collapsed replication forks and several chromosomal instabilities. Recently, our lab demonstrated that the fidelity of DNA synthesis associated with BIR in yeast Saccharomyces Cerevisiae is extremely low. The level of frameshift mutations associated with BIR is 1000-fold higher as compared to normal DNA replication. This work demonstrates that BIR stimulates base substitution mutations, which comprise 90% of all point mutations, making them 400-1400 times more frequent than during S-phase DNA replication. We show that DNA Polymerase δ proofreading corrects many of the base substitutions in BIR. Further, we demonstrate that Pif1, a 5’-3’ DNA helicase, is responsible for making BIR efficient and also highly mutagenic. Pif1p is responsible for the majority of BIR mutagenesis not only close to the DSB site, where BIR is less stable but also at chromosomal regions far away from the DSB break site, where BIR is fast, processive and stable. This work further reveals that, at positions close to the DSB, BIR mutagenesis in the absence of Pif1 depends on Rev3, the catalytic subunit of translesion DNA Polymerase ζ. We observe that mutations promoted by Pol ζ are often complex and propose that they are generated by a Pol ζ- led template switching mechanism. These complex mutations were also found to be frequently associated with gross chromosomal rearrangements. Finally we demonstrate that BIR is carried out by unusual conservative mode of DNA synthesis. Based on this study, we speculate that the unusual mode of DNA synthesis associated with BIR leads to various kinds of genomic instability including mutations and chromosomal rearrangements.

Indiana University-Purdue University Indianapolis (IUPUI)
Break-induced replication (BIR) is a mechanism to repair double-strand breaks (DSBs) that possess only a single end that can find homology in the genome. This situation can result from the collapse of replication forks or telomere erosion. BIR frequently produces various genetic instabilities including mutations, loss of heterozygosity, deletions, duplications, and template switching that can result in copy-number variations (CNVs). An important type of genomic rearrangement specifically linked to BIR is half crossovers (HCs), which result from fusions between parts of recombining chromosomes. Because HC formation produces a fused molecule as well as a broken chromosome fragment, these events could be highly destabilizing. Here I demonstrate that HC formation results from the interruption of BIR caused by a defective replisome or premature onset of mitosis. Additionally, I document the existence of half crossover instability cascades (HCC) that resemble cycles of non-reciprocal translocations (NRTs) previously described in human tumors. I postulate that HCs represent a potent source of genetic destabilization with significant consequences that mimic those observed in human diseases, including cancer.