Abstract DNA replication is a process in which a cell duplicates its genome before cell division, and must proceed accurately and in organized manner to guarantee maintenance of the integrity of the genetic information. DNA polymerases are enzymes that catalyse the synthesis of the new DNA strand by utilizing the parental strand as a template. In addition to chromosomal replication, DNA synthesis and therefore DNA polymerases are also needed in other processes like DNA repair and DNA recombination. The DNA polymerase is an essential DNA polymerase in eukaryotes and is required for chromosomal DNA replication. It has also been implicated in DNA repair, recombination, and in transcriptional and cell cycle control. The regulation of the human enzyme was explored by analysing its expression, phosphorylation and protein-protein interactions. Expression of both the A and B subunits of the human DNA polymerase ε was strongly growth-regulated. After serum-stimulation of quiescent fibroblasts, the steady-state mRNA levels were up-regulated at least 5-fold. In actively cycling cells, however, the steady-state mRNA and protein levels fluctuated less than 2-fold, being highest in G1/S phase. The promoter of the B subunit gene was analysed in detail. The 75 bp core promoter was essentially dependent on the Sp1 transcription factor. Furthermore, mitogenic control of the promoter required an intact E2F binding element, and binding of E2F2, E2F4 and p107 was demonstrated in vitro. A down-regulation element, located immediately downstream from the core promoter, bound E2F1, NF-1 and pRb transcription factors. A model of the promoter function is presented. Topoisomerase IIβ binding protein 1 (TopBP1) was found to be associated with human DNA polymerase ε. TopBP1 contains eight BRCT domains and is homologous to Saccharomyces cerevisiae Dpb11, Schizosaccharomyces pombe Cut5, Drosophila melanogaster Mus101 and the human Breast Cancer susceptibility protein 1 (BRCA1). TopBP1 is a phosphoprotein, whose expression is induced at the G1/S border and is required for chromosomal DNA replication. It co-localizes in S phase with BRCA1 into discrete foci, which do not represent sites of ongoing DNA replication. However, if DNA is damaged or replication is blocked in S phase cells, TopBP1 and BRCA1 re-localize into proliferating cell nuclear antigen (PCNA) containing foci that represent stalled replication forks. Finally, phosphorylation of DNA polymerase ε was described and at least three immunologically distinct and differentially phosphorylated forms were shown to exist. Phosphorylation is on serine and threonine residues and shows a cell cycle dependent fluctuation, but is not affected by DNA damage or by inhibition of DNA replication. BRCA1 co-immunoprecipitates with a hypophosphorylated form of DNA polymerase ε. In contrast, TopBP1 was shown to be associated with a hyperphosphorylated form.

Thesis (Ph. D.)--West Virginia University, 2000. / Title from document title page. Document formatted into pages; contains xi, 185 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2006. / Title from first page of PDF file (viewed Feb. 14, 2008). Includes bibliographical references.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2010.

Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 124-136).

A via de Reparo por Excisão de Nucleotídeos (NER) é responsável pelo reparo das lesões causadas pela luz ultravioleta (UV) e de outras lesões capazes de distorcer a dupla hélice, bloqueando a replicação e a transcrição. Os pacientes que apresentam as síndromes recessivas raras Xeroderma Pigmentosum (XP), tricotiodistrofia (TTD) e síndrome de Cockayne (CS) possuem mutações em algum dos 11 genes relacionados ao NER e à transcrição basal. Mutações na proteína XPD levam ao surgimento de diferentes fenótipos: XP, TTD, XP/CS ou COFS (Cerebro-Oculo-Facio-Skeletal Syndrome), uma forma rara de CS. Os pacientes XP apresentam alta incidência de câncer de pele, o que não ocorre com os pacientes TTD e CS, além de poderem apresentar perda neuronal progressiva, enquanto todos os CS e TTD apresentam uma diminuição na mielinização do cérebro. As neuropatologias são provavelmente associadas a problemas no reparo de danos endógenos no DNA das células nervosas. Diversos trabalhos mostraram o envolvimento do NER no reparo desses danos, os quais pensava-se serem reparados apenas por outro mecanismo, o Reparo por Excisão de Base. Neste trabalho mostramos que fibroblastos de pacientes XP-D, XP-D/CS e TTD, portadores de mutações em XPD, são sensíveis ao estresse oxidativo induzido pelo tratamento com azul de metileno fotoativado, apresentando bloqueio prolongado no ciclo celular e permanência da sinalização de danos ao DNA. A complementação das diferentes linhagens com o gene XPD/ERCC2 foi capaz de restaurar a sobrevivência celular. Foram detectadas diferenças importantes na capacidade de reparo/retomada da transcrição após danos gerados por estresse oxidativo em DNA plasmidial, além da ativação de vias diferentes de morte celular: fibroblastos XP-D apresentam maior capacidade de reparo e apresentam morte por apoptose após estresse oxidativo, enquanto os fibroblastos XP-D/CS e TTD apresentam menor capacidade de reparo ativação de mais de uma via de morte celular (apoptose e necrose), diferenças que podem estar ligadas ao fenótipo dos pacientes. Mutações no gene codificante para a DNA polimerase n, POLH, estão associadas à forma variante de XP (XP-V). Pol n é uma polimerase especializada na síntese translesão (TLS) de fotoprodutos, além de estar implicada na TLS de outros tipos de lesões como bases oxidadas, e em vias não relacionadas à TLS como a hipermutação somática e à replicação de regiões de DNA com arquiteturas não-canônicas. Neste trabalho mostramos que os fibroblastos de pacientes XP-V apresentam sensibilidade ao estresse oxidativo. Mostramos uma indução da proteína pol n em fibroblastos primários após danos genotóxicos, associada ao aumento da capacidade de lidar com a parada na forquilha de replicação, possibilitando a continuidade da replicação do DNA e ao aumento da sobrevivência celular. Mostramos uma diferença na estabilidade genômica nos genes das imunoglobulinas dos pacientes XP-V idosos em comparação com os pacientes jovens e controles de idade pareada, mostrando que a ausência dessa polimerase pode estar ligada ao aumento da instabilidade genômica nesses genes / The Nucleotide Excision Repair (NER) pathway is responsible for the repair of UV photoproducts and other bulky lesions that block both replication and transcription. Patients with the rare recessive disorders Xeroderma Pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne Syndrome (CS) carry mutations in one of the 11 NER genes, linked to repair and basal transcription. Mutations in XPD lead to different phenotypes: XP, TTD, XP/CS or COFS (Cerebro-Oculo-Facio-Skeletal Syndrome), a rare form of CS. XP patients have high incidence of skin cancer, which does not occur in TTD or CS patients, although ther may present neurodegeneration, while all CS and TTD patients have neurodevelopmental symptoms linked to dysmielynation. The pathology of these neurological diseases is probably associated with deficient repair of DNA lesions in nervous cells, generated by endogenous processes. Many groups including ours have demonstrated the involvement of NER in the repair of these lesions, previously thought to be exclusively repaired by Base Excision Repair. In this work we show high sensitivity of both primary and transformed XP-D, XP-D/CS and TTD human fibroblasts in response to oxidative stress generated by photoactivated methylene blue, with prolonged cell cycle arrest and DNA damage signaling. The complementation of the three different cell lines with the XPD/ERCC2 gene was able to restore cell survival. We detected important differences in repair capacity/transcription resumption after damage generated by oxidative stress in plasmid DNA, besides the activation of different cell death pathways: XP-D cells have higher repair capacity and die by apoptosis, while XP-D/CS and TTD cells have little repair capacity and activate more than one death pathway (apoptosis and necrosis). We believe these differences can be related to the patients\' phenotypes. Mutations in DNA polymerase n coding gene, POLH, are associated with the variant form of XP (XP-V). Pol n is a translesion synthesis (TLS) polymerase specialized in the TLS past CPD photoproducts, besides other lesions like oxidized bases, and in other processes like somatic hypermutation and DNA replication in structured regions. In this work we show XP-V human fibroblasts are sensitive to oxidative stress. We detected an induction of pol n after genotoxic stress in primary cells, associated with increased ability to deal with the stalled replication fork, and consequently to DNA replication restart and cell survival. In addition, we detected a difference in genomic stability in immunoglobulin genes in aged XP-V patients in comparison to both young patients and age-matched controls, showing the absence of this polymerase may be linked to increased genomic instability in these genes

A via de Reparo por Excisão de Nucleotídeos (NER) é responsável pelo reparo das lesões causadas pela luz ultravioleta (UV) e de outras lesões capazes de distorcer a dupla hélice, bloqueando a replicação e a transcrição. Os pacientes que apresentam as síndromes recessivas raras Xeroderma Pigmentosum (XP), tricotiodistrofia (TTD) e síndrome de Cockayne (CS) possuem mutações em algum dos 11 genes relacionados ao NER e à transcrição basal. Mutações na proteína XPD levam ao surgimento de diferentes fenótipos: XP, TTD, XP/CS ou COFS (Cerebro-Oculo-Facio-Skeletal Syndrome), uma forma rara de CS. Os pacientes XP apresentam alta incidência de câncer de pele, o que não ocorre com os pacientes TTD e CS, além de poderem apresentar perda neuronal progressiva, enquanto todos os CS e TTD apresentam uma diminuição na mielinização do cérebro. As neuropatologias são provavelmente associadas a problemas no reparo de danos endógenos no DNA das células nervosas. Diversos trabalhos mostraram o envolvimento do NER no reparo desses danos, os quais pensava-se serem reparados apenas por outro mecanismo, o Reparo por Excisão de Base. Neste trabalho mostramos que fibroblastos de pacientes XP-D, XP-D/CS e TTD, portadores de mutações em XPD, são sensíveis ao estresse oxidativo induzido pelo tratamento com azul de metileno fotoativado, apresentando bloqueio prolongado no ciclo celular e permanência da sinalização de danos ao DNA. A complementação das diferentes linhagens com o gene XPD/ERCC2 foi capaz de restaurar a sobrevivência celular. Foram detectadas diferenças importantes na capacidade de reparo/retomada da transcrição após danos gerados por estresse oxidativo em DNA plasmidial, além da ativação de vias diferentes de morte celular: fibroblastos XP-D apresentam maior capacidade de reparo e apresentam morte por apoptose após estresse oxidativo, enquanto os fibroblastos XP-D/CS e TTD apresentam menor capacidade de reparo ativação de mais de uma via de morte celular (apoptose e necrose), diferenças que podem estar ligadas ao fenótipo dos pacientes. Mutações no gene codificante para a DNA polimerase n, POLH, estão associadas à forma variante de XP (XP-V). Pol n é uma polimerase especializada na síntese translesão (TLS) de fotoprodutos, além de estar implicada na TLS de outros tipos de lesões como bases oxidadas, e em vias não relacionadas à TLS como a hipermutação somática e à replicação de regiões de DNA com arquiteturas não-canônicas. Neste trabalho mostramos que os fibroblastos de pacientes XP-V apresentam sensibilidade ao estresse oxidativo. Mostramos uma indução da proteína pol n em fibroblastos primários após danos genotóxicos, associada ao aumento da capacidade de lidar com a parada na forquilha de replicação, possibilitando a continuidade da replicação do DNA e ao aumento da sobrevivência celular. Mostramos uma diferença na estabilidade genômica nos genes das imunoglobulinas dos pacientes XP-V idosos em comparação com os pacientes jovens e controles de idade pareada, mostrando que a ausência dessa polimerase pode estar ligada ao aumento da instabilidade genômica nesses genes / The Nucleotide Excision Repair (NER) pathway is responsible for the repair of UV photoproducts and other bulky lesions that block both replication and transcription. Patients with the rare recessive disorders Xeroderma Pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne Syndrome (CS) carry mutations in one of the 11 NER genes, linked to repair and basal transcription. Mutations in XPD lead to different phenotypes: XP, TTD, XP/CS or COFS (Cerebro-Oculo-Facio-Skeletal Syndrome), a rare form of CS. XP patients have high incidence of skin cancer, which does not occur in TTD or CS patients, although ther may present neurodegeneration, while all CS and TTD patients have neurodevelopmental symptoms linked to dysmielynation. The pathology of these neurological diseases is probably associated with deficient repair of DNA lesions in nervous cells, generated by endogenous processes. Many groups including ours have demonstrated the involvement of NER in the repair of these lesions, previously thought to be exclusively repaired by Base Excision Repair. In this work we show high sensitivity of both primary and transformed XP-D, XP-D/CS and TTD human fibroblasts in response to oxidative stress generated by photoactivated methylene blue, with prolonged cell cycle arrest and DNA damage signaling. The complementation of the three different cell lines with the XPD/ERCC2 gene was able to restore cell survival. We detected important differences in repair capacity/transcription resumption after damage generated by oxidative stress in plasmid DNA, besides the activation of different cell death pathways: XP-D cells have higher repair capacity and die by apoptosis, while XP-D/CS and TTD cells have little repair capacity and activate more than one death pathway (apoptosis and necrosis). We believe these differences can be related to the patients\' phenotypes. Mutations in DNA polymerase n coding gene, POLH, are associated with the variant form of XP (XP-V). Pol n is a translesion synthesis (TLS) polymerase specialized in the TLS past CPD photoproducts, besides other lesions like oxidized bases, and in other processes like somatic hypermutation and DNA replication in structured regions. In this work we show XP-V human fibroblasts are sensitive to oxidative stress. We detected an induction of pol n after genotoxic stress in primary cells, associated with increased ability to deal with the stalled replication fork, and consequently to DNA replication restart and cell survival. In addition, we detected a difference in genomic stability in immunoglobulin genes in aged XP-V patients in comparison to both young patients and age-matched controls, showing the absence of this polymerase may be linked to increased genomic instability in these genes