Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2004. / Includes bibliographical references (leaves 83-100). Also available in electronic version. Access restricted to campus users.

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2002. / Includes bibliographical references (leaves 74-80). Also available in electronic version. Access restricted to campus users.

Thesis (Ph. D.)--University of Toledo, 2008. / Typescript. "Submitted as partial fulfillment of the requirements for the Doctor of Philosophy in Chemistry." Includes bibliographical references (leaves 245-252).

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references (leaves 60-71).

Replication Protein A (RPA), the eukaryotic single-stranded DNA-binding complex, is essential for multiple processes in cellular DNA metabolism including, but not limited to, DNA replication, DNA repair and recombination. The `canonical' RPA is composed of three subunits (RPA1, RPA2, and RPA3). In addition to the three canonical subunits, there is a human homolog to the RPA2 subunit, termed RPA4, which can substitute for RPA2 in complex formation. The resulting RPA complex has been termed `alternative' RPA (aRPA). The normal function of aRPA is not known; however, previous studies have shown that it does not support S-phase progression in vivo. The goal of this thesis was to characterize the function of aRPA in DNA replication, DNA repair and recombination and profile its expression in human tissues. The studies presented in this thesis show that the aRPA complex has solution and DNA binding properties indistinguishable from the canonical RPA complex as determined by gel mobility shift assays. However, aRPA was unable to support DNA replication and inhibited canonical RPA function in a cell-free simian virus 40 system. aRPA inhibited both initiation and elongation of DNA synthesis in the SV40 system. Two regions of RPA4, the putative L34 loop and the C-terminal winged helix domain, were responsible for inhibiting SV40 DNA replication. The mechanism of SV40 DNA replication inhibition during initiation and elongation was characterized using assays for DNA polymerase α and DNA polymerase δ. aRPA was shown to have reduced interaction with DNA polymerase α and was not able to efficiently stimulate DNA synthesis by DNA polymerase α on aRPA coated single-stranded DNA. However, aRPA stimulated DNA synthesis by DNA polymerase δ in the presence of PCNA and RFC even though a reduced interaction was observed between aRPA and polymerase δ. The role of aRPA in DNA repair was also investigated. aRPA interacted with both Rad52 and Rad51 but had a reduced interaction with Rad51. However, aRPA was still able to stimulate Rad51-dependent strand exchange. aRPA also supported the dual incision/excision reaction of nucleotide excision repair. aRPA was less efficient in nucleotide excision repair than canonical RPA and this reduction was attributed to reduced interactions with the repair factor XPA. In contrast, aRPA exhibited higher affinity for damaged DNA than canonical RPA. The expression of RPA4 and RPA2 was determined by quantitative PCR in established cell lines, human normal tissues and human tumor tissue. RPA4 was shown to be expressed in all normal tissues examined but the level of expression was tissue specific. Additionally, RPA4 expression was decreased in all tumor tissues examined and was at the limit of detection in established cell lines. Taken together, the results presented in this thesis suggest that aRPA is a `non-proliferative' form of RPA that functions to maintain the genomic stability of non-dividing cells.

Orientador: Maria Isabel Nogueira Cano / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T10:53:20Z (GMT). No. of bitstreams: 1 SiqueiraNeto_JairLagede_D.pdf: 2960762 bytes, checksum: 91f4b8aed472a4d4c93cf040e8c534e7 (MD5) Previous issue date: 2007 / Resumo: A leishmaniose é uma parasitose humana emergente e ainda não controlada, causada por protozoários pertencentes ao gênero Leishmania. Atualmente, a doença atinge mais de 12 milhões de pessoas, não existindo ainda métodos eficientes para seu controle e erradicação. Por estas razões a Organização Mundial da Saúde classifica a leishmaniose como doença de categoria I e incentiva o desenvolvimento de novos métodos para controlar a doença para buscar novos alvos para drogas contra parasita. No presente trabalho, estudou-se as proteínas LaRBP38 e LaRPA-1 previamente identificadas por se associarem in vitro com a simples-fita telomérica rica em ¿G¿ de L. amazonensis. Os telomeros são extremidades físicas dos cromossomos de eucariotos, formados por complexos nucleoproteicos. São responsáveis por conferir estabilidade aos cromossomos, envitando a degradação pela maquina de reparo celular e a fusão entre extremidades cromossomais. Instabilidades no telômero causam normalmente danos irreparáveis à célula podendo levar à senescência e morte celular. As proteínas que se mantém complexadas ao telômero são responsáveis por mantê-lo funcioal. Cada proteína desempenha um papel importante, seja na proteção, processo replicativo ou manutenção da estabilidade estrutural do telômero, sendo portanto, alvos potenciais para o desenvolvimento de terapias antiparasitárias. A proteína RPA é conservada em toda escala evolutiva e cumpre importantes papéis nas maquinarias de replicação, recombinação e reparo do DNA genômico ...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: Leishmaniasis is an emerging and non-controlled human disease, caused by protozoan belonging to the Leishmania genera. More than 12 million people are infected and there are no efficient methods for the controlling or eradication of the disease. For those reasons, the World Health Organization classifies leishmaniasis as category I disease and encourages the development of new methods to control the disease and to find new targets for drugs against the parasite. In the present work, we studied the proteins LaRBP38 and LaRPA-1, prior identifield by in vitro assays as proteins that associates with the Leishmania amazonensis G-rich single-stranded telomeric DNA. Telomeres are the physical ends of eukaryote chromosomes formed by proteins and DNA complexes. They are responsible for the chromosome stability, avoiding degradation by the rapair machinery end-to-end fusion. Telomere instability may cause irreversible damage in the cell, leading to senescence and cell death. The proteins that interact with the telomeres are responsible for the functional dynamics of theses structures. Each protein has an important role in the protection, replication process or in the stability maintenance. Therefore, telomeric protein could be considered good targets for the development of new therapies. RPA is an evolutionary conserved protein and plays important roles in replication, recombination and repair machineries. At the telomeres, RPA recruits telomerase, the protein responsible for telomeric elengation ...Note: The complete abstract is available with the full electronic digital thesis or dissertations / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(農学) / 甲第18333号 / 農博第2058号 / 新制||農||1023(附属図書館) / 学位論文||H26||N4840(農学部図書室) / 31191 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 奥野 哲郎, 教授 佐久間 正幸, 准教授 吉田 天士 / 学位規則第4条第1項該当

RPA (replication protein A) is an essential factor for DNA DSB (double-strand break) repair and cell cycle checkpoint activation. The 32 kDa subunit of RPA undergoes hyperphosphorylation in response to cellular genotoxic insults. However, the potential involvement of hyperphosphorylated RPA in DSB repair and check-point activation remains unclear. Using co-immunoprecipitation assays, we showed that cellular interaction of RPA with two DSB repair factors, Rad51 and Rad52, was predominantly mediated by the hyperphosphorylated species of RPA in cells after UV and camptothecin treatment. Moreover, Rad51 and Rad52 displayed higher affinity for the hyperphosphorylated RPA than native RPA in an in vitro binding assay. Checkpoint kinase ATR (ataxia telangiectasia mutated and Rad3-related) also interacted more efficiently with the hyperphosphorylated RPA than with native RPA following DNA damage. Consistently, immunofluorescence microscopy demonstrated that the hyperphosphorylated RPA was able to co-localize with Rad52 and ATR to form significant nuclear foci in cells. Our results suggest that hyperphosphorylated RPA is preferentially localized to DSB repair and the DNA damage checkpoint complexes in response to DNA damage.

Replication protein A (RPA) is a eukaryotic single-stranded DNA-binding protein consisting of three subunits of 70-, 32-, and 14-kDa (RPA70, RPA32, RPA14, respectively). It is a protein essential for most cellular DNA metabolic pathways. Checkpoint proteins Rad9, Rad1, and Hus1 form a clamp-like complex which plays a central role in the DNA damage-induced checkpoint response. In this report, we presented the evidence that Rad9-Rad1-Hus1 (9-1-1) complex directly interacted with RPA in human cells, and this interaction was mediated by the binding of Rad9 protein to both RPA70 and RPA32 subunits. In addition, the cellular interaction of 9-1-1 with RPA or hyperphosphorylated RPA was stimulated by UV irradiation or camptothecin treatment in a dose-dependent manner. Such treatments also resulted in the colocalization of the nuclear foci formed with the two complexes. Consistently, knockdown of the RPA expression in cells by the small interference RNA (siRNA) blocked the DNA damage-dependent chromatin association of 9-1-1, and also inhibited the 9-1-1 complex formation. Taken together, our results suggest that 9-1-1 and RPA complexes collaboratively function in DNA damage responses, and that the RPA may serve as a regulator for the activity of 9-1-1 complex in the cellular checkpoint network.

Human RPA (replication protein A), a single-stranded DNA-binding protein, is required for many cellular pathways including DNA repair, recombination and replication. However, the role of RPA in nucleotide excision repair remains elusive. In the present study, we have systematically examined the binding of RPA to a battery of well-defined ssDNA (single-stranded DNA) substrates using fluorescence spectroscopy. These substrates contain adducts of (6-4) photoproducts, N-acetyl-2-aminofluorene-, 1-amino-pyrene-, BPDE (benzo[a]pyrene diol epoxide)- and fluorescein that are different in many aspects such as molecular structure and size, DNA disruption mode (e.g. base stacking or non-stacking), as well as chemical properties. Our results showed that RPA has a lower binding affinity for damaged ssDNA than for non-damaged ssDNA and that the affinity of RPA for damaged ssDNA depends on the type of adduct. Interestingly, the bulkier lesions have a greater effect. With a fluorescent base-stacking bulky adduct, (+)-cis-anti-BPDE-dG, we demonstrated that, on binding of RPA. the fluorescence of BPDE-ssDNA was significantly enhanced by up to 8-9-fold. This indicated that the stacking between the BPDE adduct and its neighbouring ssDNA bases had been disrupted and there was a lack of substantial direct contacts between the protein residues and the lesion itself. For RPA interaction with short damaged ssDNA, we propose that, on RPA binding, the modified base of ssDNA is looped out from the surface of the protein, permitting proper contacts of RPA with the remaining unmodified bases.