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Alternativas da replicação do DNA: vias de controle e dinâmica das forquilhas em trypanosomas.; DNA replication alternatives: control pathways and fork dynamic in trypanosomas.

Calderano, Simone Guedes
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 03/12/2013 PT
Relevância na Pesquisa
66.22%
A replicação do DNA tem início nas origens de replicação que são licenciadas na transição das fases M/G1, pelo complexo de pré-replicação (CPR), e ativadas apenas na fase S. Existem diversas origens de replicação no genoma, mas apenas parte destas origens é disparada em diferentes momentos de S, havendo assim origens early (disparadas no início de S) e late (disparadas mais tardiamente). Em trypanosomas as origens de replicação são reconhecidas por um CPR formado por Orc1/Cdc6 e pelo complexo MCM2-7. Em T. cruzi observamos que existem dois mecanismos diferentes para controlar a replicação do DNA. Durante o ciclo celular da forma epimastigota, as proteínas do CPR são sempre expressas e ligadas ao DNA, mas durante o ciclo de vida Orc1/Cdc6 se liga ao DNA apenas nas formas que replicam, e Mcm7 não é expressa nas que não replicam. Também foi analisado o perfil das forquilhas de replicação em T. brucei utilizando a técnica de SMARD onde vimos que a velocidade da forquilha é semelhante a dos demais eucariontes, além de encontrarmos a primeira origem de replicação late.; The DNA replication starts at the origins of replication, which are licensed at M/G1 transition, by the pre replication complex (PRC), and are activated just at S phase. There are many origins of replication along genome...

DNA replication stress-induced loss of reproductive capacity in S. cerevisiae and its inhibition by caloric restriction

Weinberger, Martin; Marques, Belém Sampaio; Ludovico, Paula; Burhans, William C.
Fonte: Taylor & Francis Publicador: Taylor & Francis
Tipo: Artigo de Revista Científica
Publicado em //2013 ENG
Relevância na Pesquisa
66.13%
In many organisms, attenuation of growth signaling by caloric restriction or mutational inactivation of growth signaling pathways extends lifespan and protects against cancer and other age-related diseases. The focus of many efforts to understand these effects has been on the induction of oxidative stress defenses that inhibit cellular senescence and cell death. Here we show that in the model organism S. cerevisiae, growth signaling induces entry of cells in stationary phase into S phase in parallel with loss of reproductive capacity, which is enhanced by elevated concentrations of glucose. Overexpression of RNR1 encoding a ribonucleotide reductase subunit required for the synthesis of deoxynucleotide triphosphates and DNA replication suppresses the accelerated loss of reproductive capacity of cells cultured in high glucose. The reduced reproductive capacity of these cells is also suppressed by excess threonine, which buffers dNTP pools when ribonucleotide reductase activity is limiting. Caloric restriction or inactivation of the AKT homolog Sch9p inhibits senescence and death in stationary phase cells caused by the DNA replication inhibitor hydroxyurea or by inactivation of the DNA replication and repair proteins Sgs1p or Rad27p. Inhibition of DNA replication stress represents a novel mechanism by which caloric restriction promotes longevity in S. cerevisiae. A similar mechanism may promote longevity and inhibit cancer and other age-related diseases in humans.

RNA Primer Handoff in Bacteriophage T4 DNA Replication: THE ROLE OF SINGLE-STRANDED DNA-BINDING PROTEIN AND POLYMERASE ACCESSORY PROTEINS*

Nelson, Scott W.; Kumar, Ravindra; Benkovic, Stephen J.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 15/08/2008 EN
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66.12%
In T4 phage, coordinated leading and lagging strand DNA synthesis is carried out by an eight-protein complex termed the replisome. The control of lagging strand DNA synthesis depends on a highly dynamic replisome with several proteins entering and leaving during DNA replication. Here we examine the role of single-stranded binding protein (gp32) in the repetitive cycles of lagging strand synthesis. Removal of the protein-interacting domain of gp32 results in a reduction in the number of primers synthesized and in the efficiency of primer transfer to the polymerase. We find that the primase protein is moderately processive, and this processivity depends on the presence of full-length gp32 at the replication fork. Surprisingly, we find that an increase in the efficiency of primer transfer to the clamp protein correlates with a decrease in the dissociation rate of the primase from the replisome. These findings result in a revised model of lagging strand DNA synthesis where the primase remains as part of the replisome after each successful cycle of Okazaki fragment synthesis. A delay in primer transfer results in an increased probability of the primase dissociating from the replication fork. The interplay between gp32, primase...

Multiple ATP Binding Is Required to Stabilize the “Activated” (Clamp Open) Clamp Loader of the T4 DNA Replication Complex*

Pietroni, Paola; von Hippel, Peter H.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 17/10/2008 EN
Relevância na Pesquisa
66.06%
Most DNA replication systems include a sliding clamp that encircles the genomic DNA and links the polymerase to the template to control polymerase processivity. A loading complex is required to open the clamp and place it onto the DNA. In phage T4 this complex consists of a trimeric clamp of gp45 subunits and a pentameric loader assembly of four gp44 and one gp62 subunit(s), with clamp loading driven by ATP binding. We measure this binding as a function of input ligand concentration and show that four ATPs bind to the gp44/62 complex with equal affinity. In contrast, the ATPase rate profile of the clamp-clamp loader complex exhibits a marked peak at an input ATP concentration close to the overall Kd (∼30 μm), with further increases in bound ATP decreasing the ATPase rate to a much lower level. Thus the progressive binding of the four ATPs triggers a conformational change in the complex that markedly inhibits ATPase activity. This inhibition is related to ring opening by using a clamp that is covalently cross-linked across its subunit interfaces and thus rendered incapable of opening. Binding of this clamp abolishes substrate inhibition of the ATPase but leaves ATP binding unchanged. We show that four ATP ligands must bind to the T4 clamp loader before the loader can be fully “activated” and the clamp opened...

Yeast DNA Replication Protein Dpb11 Activates the Mec1/ATR Checkpoint Kinase*

Navadgi-Patil, Vasundhara M.; Burgers, Peter M.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 19/12/2008 EN
Relevância na Pesquisa
66.1%
The Saccharomyces cerevisiae Mec1-Ddc2 protein kinase (human ATR-ATRIP) initiates a signal transduction pathway in response to DNA damage and replication stress to mediate cell cycle arrest. The yeast DNA damage checkpoint clamp Ddc1-Mec3-Rad17 (human Rad9-Hus1-Rad1: 9-1-1) is loaded around effector DNA and thereby activates Mec1 kinase. Dpb11 (Schizosaccharomyces pombe Cut5/Rad4 or human TopBP1) is an essential protein required for the initiation of DNA replication and has a role in checkpoint activation. In this study, we demonstrate that Dpb11 directly activates the Mec1 kinase in phosphorylating the downstream effector kinase Rad53 (human Chk1/2) and DNA bound RPA. However, DNA was not required for Dpb11 to function as an activator. Dpb11 and yeast 9-1-1 independently activate Mec1, but substantial synergism in activation was observed when both activators were present. Our studies suggest that Dpb11 and 9-1-1 may partially compensate for each other during yeast checkpoint function.

A DNA Polymerase-α·Primase Cofactor with Homology to Replication Protein A-32 Regulates DNA Replication in Mammalian Cells*S⃞

Casteel, Darren E.; Zhuang, Shunhui; Zeng, Ying; Perrino, Fred W.; Boss, Gerry R.; Goulian, Mehran; Pilz, Renate B.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 27/02/2009 EN
Relevância na Pesquisa
66.19%
α-Accessory factor (AAF) stimulates the activity of DNA polymerase-α·primase, the only enzyme known to initiate DNA replication in eukaryotic cells (Goulian, M., Heard, C. J., and Grimm, S. L. (1990) J. Biol. Chem.265 ,13221 -132302165497). We purified the AAF heterodimer composed of 44- and 132-kDa subunits from cultured cells and identified full-length cDNA clones using amino acid sequences from internal peptides. AAF-132 demonstrated no homologies to known proteins; AAF-44, however, is evolutionarily related to the 32-kDa subunit of replication protein A (RPA-32) and contains an oligonucleotide/oligosaccharide-binding (OB) fold domain similar to the OB fold domains of RPA involved in single-stranded DNA binding. Epitope-tagged versions of AAF-44 and -132 formed a complex in intact cells, and purified recombinant AAF-44 bound to single-stranded DNA and stimulated DNA primase activity only in the presence of AAF-132. Mutations in conserved residues within the OB fold of AAF-44 reduced DNA binding activity of the AAF-44·AAF-132 complex. Immunofluorescence staining of AAF-44 and AAF-132 in S phase-enriched HeLa cells demonstrated punctate nuclear staining, and AAF co-localized with proliferating cell nuclear antigen, a marker for replication foci containing DNA polymerase-α·primase and RPA. Small interfering RNA-mediated depletion of AAF-44 in tumor cell lines inhibited [methyl-3H]thymidine uptake into DNA but did not affect cell viability. We conclude that AAF shares structural and functional similarities with RPA-32 and regulates DNA replication...

Timeless Maintains Genomic Stability and Suppresses Sister Chromatid Exchange during Unperturbed DNA Replication*S⃞

Urtishak, Karen A.; Smith, Kevin D.; Chanoux, Rebecca A.; Greenberg, Roger A.; Johnson, F. Brad; Brown, Eric J.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 27/03/2009 EN
Relevância na Pesquisa
66.15%
Genome integrity is maintained during DNA replication by coordination of various replisome-regulated processes. Although it is known that Timeless (Tim) is a replisome component that participates in replication checkpoint responses to genotoxic stress, its importance for genome maintenance during normal DNA synthesis has not been reported. Here we demonstrate that Tim reduction leads to genomic instability during unperturbed DNA replication, culminating in increased chromatid breaks and translocations (triradials, quadriradials, and fusions). Tim deficiency led to increased H2AX phosphorylation and Rad51 and Rad52 foci formation selectively during DNA synthesis and caused a 3-4-fold increase in sister chromatid exchange. The sister chromatid exchange events stimulated by Tim reduction were largely mediated via a Brca2/Rad51-dependent mechanism and were additively increased by deletion of the Blm helicase. Therefore, Tim deficiency leads to an increased reliance on homologous recombination for proper continuation of DNA synthesis. Together, these results indicate a pivotal role for Tim in maintaining genome stability throughout normal DNA replication.

Csm3, Tof1, and Mrc1 Form a Heterotrimeric Mediator Complex That Associates with DNA Replication Forks

Bando, Masashige; Katou, Yuki; Komata, Makiko; Tanaka, Hirokazu; Itoh, Takehiko; Sutani, Takashi; Shirahige, Katsuhiko
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
66.1%
Mrc1 (mediator of replication checkpoint), Tof1 (topoisomerase I interacting factor), and Csm3 (chromosome segregation in meiosis) are checkpoint-mediator proteins that function during DNA replication and activate the effector kinase Rad53. We reported previously that Mrc1 and Tof1 are constituents of the replication machinery and that both proteins are required for the proper arrest and stabilization of replication forks in the presence of hydroxyurea. In our current study, we show that Csm3 is a component of moving replication forks and that both Tof1 and Csm3 are specifically required for the association of Mrc1 with these structures. In contrast, the deletion of mrc1 did not affect the association of Tof1 and Csm3 with the replication fork complex. In agreement with previous observations in yeast cells, the results of a baculovirus coexpression system showed that these three proteins interact directly with each other to form a mediator complex in the absence of replication forks.

Single-Molecule Studies of Eukaryotic DNA Replication

Loveland, Anna Barbara
Fonte: Harvard University Publicador: Harvard University
Tipo: Thesis or Dissertation
EN_US
Relevância na Pesquisa
66.24%
DNA replication is a fundamental cellular process. However, the structure and dynamics of the eukaryotic DNA replication machinery remain poorly understood. A soluble extract system prepared from Xenopus eggs recapitulates eukaryotic DNA replication outside of a cell on a variety of DNA templates. This system has been used to reveal many aspects of DNA replication using a variety of ensemble biochemical techniques. Single-molecule fluorescence imaging is a powerful tool to dissect biochemical mechanisms. By immobilizing or confining a substrate, its interaction with individual, soluble, fluorescently-labeled reactants can be imaged over time and without the need for synchrony. These molecular movies reveal binding parameters of the reactant and any population heterogeneity. Moreover, if the experiments are imaged in wide-field format, the location or motion of the labeled species along the substrate can be followed with nanometer accuracy. This dissertation describes the use and development of novel single-molecule fluorescence imaging techniques to study eukaryotic DNA replication. A biophysical characterization of a replication fork protein, PCNA, revealed both helical and non-helical sliding modes along DNA. Previous experiments demonstrate that the egg extracts efficiently replicate surface-immobilized linear DNA. This finding suggested replication of DNA could be followed as motion of the replication fork along the extended DNA. However...

The Ubiquitin Ligase (CRL4^{Cdt2}) Targets Thymine DNA Glycosylase for Destruction during DNA Replication and Repair

Slenn, Tamara Jeannine
Fonte: Harvard University Publicador: Harvard University
Tipo: Thesis or Dissertation
EN_US
Relevância na Pesquisa
66.23%
The E3 ubiquitin ligase (CRL4^{Cdt2}) targets proteins for destruction during DNA replication and following DNA damage (Havens and Walter, 2011). Its substrates contain "PIP degrons" that mediate substrate binding to the processivity factor PCNA at replication forks and damage sites. The resulting PCNA-PIP degron complex forms a docking site for (CRL4^{Cdt2}), which ubiquitylates the substrate on chromatin. Several (CRL4^{Cdt2}) substrates are known, including Cdt1, multiple CDK inhibitors, Drosophila E2f1, human Set8, S. pombe Spd1, and C. elegans (Poleta) (Havens and Walter, 2011). An emerging theme is that (CRL4^{Cdt2}) targets proteins whose presence in S phase is toxic. Here, I used Xenopus egg extract to characterize a new (CRL4^{Cdt2}) substrate, thymine DNA glycosylase (TDG). TDG is a base excision repair protein that targets G-U and G-T mispairs, which arise from cytosine and 5-methylcytosine deamination (Cortazar et al., 2007). Thus, TDG may function in epigenetic gene regulation via DNA demethylation, in addition to its canonical DNA repair function. A yet unknown E3 ubiquitin ligase triggers TDG destruction during S phase (Hardeland et al., 2007). Understanding TDG proteolysis in S phase is relevant to the regulation of DNA replication...

Binding and Cleavage Specificities of Human Dna2 and FEN1 Define Their Enzymatic Activities during DNA Replication and Repair

Gloor, Jason William ; Bambara, Robert A.
Fonte: University of Rochester Publicador: University of Rochester
Tipo: Tese de Doutorado
ENG
Relevância na Pesquisa
66.17%
Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biochemistry and Biophysics, 2012.; During eukaryotic nuclear DNA replication, genomic DNA is synthesized discontinuously on the lagging strand. In humans, these discontinuous Okazaki fragments are initiated by 8-10 nucleotides (nt) of RNA followed by 150-200 nt of DNA. Upon encountering the previously synthesized Okazaki fragment, the replicative polymerase displaces the RNA primer into a 5’ flap. Most flaps are processed while short by flap endonuclease 1 (FEN1). In some instances, certain flaps lengthen requiring sequential cleavage by Dna2, a nuclease/helicase, and FEN1. Both nucleases require a free 5’ flap-end to employ a flap threading mechanism for cleavage. While Dna2 cleaves flaps within 5-6 nt of their base, FEN1 creates nicked double-stranded DNA intermediates for subsequent ligation. Furthermore, Dna2 also processes 5’ flap substrates, independent of FEN1, during double strand break (DSB) repair and telomere maintenance in order to generate a significantly different intermediate structure. In this study, we use recombinant human proteins to demonstrate that Dna2 and FEN1 substrate specificities define their enzymatic activities during DNA replication and repair. Here we present mechanistic details of FEN1 substrate interaction...

SCF-FBXO31 E3 ligase targets DNA replication factor Cdt1 for proteolysis in the G2 phase of cell cycle to prevent Re-replication

Johansson, P.; Jeffery, J.; Al-Ejeh, F.; Schulz, R.B.; Callen, D.F.; Kumar, R.; Khanna, K.K.
Fonte: American Society for Biochemistry and Molecular Biology Inc. Publicador: American Society for Biochemistry and Molecular Biology Inc.
Tipo: Artigo de Revista Científica
Publicado em //2014 EN
Relevância na Pesquisa
66.06%
FBXO31 was originally identified as a putative tumor suppressor gene in breast, ovarian, hepatocellular, and prostate cancers. By screening a set of cell cycle-regulated proteins as potential FBXO31 interaction partners, we have now identified Cdt1 as a novel substrate. Cdt1 DNA replication licensing factor is part of the pre-replication complex and essential for the maintenance of genomic integrity. We show that FBXO31 specifically interacts with Cdt1 and regulates its abundance by ubiquitylation leading to subsequent degradation. We also show that Cdt1 regulation by FBXO31 is limited to the G2 phase of the cell cycle and is independent of the pathways previously described for Cdt1 proteolysis in S and G2 phase. FBXO31 targeting of Cdt1 is mediated through the N terminus of Cdt1, a region previously shown to be responsible for its cell cycle regulation. Finally, we show that Cdt1 stabilization due to FBXO31 depletion results in re-replication. Our data present an additional pathway that contributes to the FBXO31 function as a tumor suppressor.; Pegah Johansson, Jessie Jeffery, Fares Al-Ejeh, Renèe B. Schulz, David F. Callen, Raman Kumar, and Kum Kum Khanna

Durch Staphylococcus-aureus-Alphatoxin permeabilisierte, mit Simian Virus 40 infizierte CV1-Zellen als Modellsystem zum Studium der DNA-Replikation höherer Zellen; Staphylococcus aureus alphatoxine permeabilized, simian virus 40 infected CV1 cells as a model system to study DNA replication in mammalian cells

Eger, Frank
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
DE_DE
Relevância na Pesquisa
66.24%
Die eukaryontische Replikation wurde mit Hilfe der Permeabilisierung durch Staphylococcus-Alphatoxin unter Verwendung des Modellsystems der DNA-Replikation des Simian Virus 40 (SV40) unter hypoxischen und normoxischen Bedingungen untersucht. Das bei Eger 1995 benutzte Kalium-Glutamat-PIPES-Puffersystem für permeabilisierte Zellen wurde weiter optimiert, um die SV40-Replikation in vivo so gut wie möglich zu imitieren. Die Nachweisverfahren für SV40-DNA-Replikationsintermediate und -produkte wurden erweitert und verfeinert, die Etablierung und Aufrechterhaltung von Hypoxie (u.a. durch Zugabe von Ascorbat, Ascorbatoxidase und NaHCO3 im 37°C-Wärmeraum) wurde verbessert. Die erhaltenen Daten stützen folgende Feststellungen: 1. In permeabilisierten Zellen findet sowohl Prä-Initiation als auch Initiation statt. 2. Die DNA-Elongation in permeabilisierten Zellen ist der in vivo vergleichbar. 3. Der hypoxisch arretierte Zustand der SV40-DNA-Replikation in intakten Wirtszellen läßt sich sowohl durch normoxische als auch durch hypoxische Permeabilisierung hypoxischer Zellen aufheben. Durch Reoxigenierung kurz vor Permeabilisierung werden die stärksten Effekte beobachtet. 4. In hypoxischen, permeabilisierten, SV40-infizierten CV1-Zellen ist die Etablierung eines Zustands der DNA-Replikation möglich...

Untersuchung des molekularen Mechanismus der schnellen Regulation der DNA-Replikation an mit Alpha-Toxin aus S. aureus permeabilisierten Kulturzellen; Examination of the molecular mechanism of the fast regulation of DNA-replication using cell culture cells, which are permeabilized by alpha toxin from Staphylococcus aureus

Martin, Peter
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
DE_DE
Relevância na Pesquisa
66.23%
HeLa-Zellen können durch alpha -Toxin aus Staphylococcus aureus für niedermolekulare Substanze bis ungefähr 2 kD permeabel gemacht werden. Durch die Bereitstellung eines geeigneten Mediums kann in HeLa-Zellen, die auf diese Weise permeabilisiert wurden, Replikation stattfinden. Nach der Permabilisierung replizieren HeLa-Zellen im Vergleich zu intakten Zellen verlangsamt. Die einzelnen Schritte der Replikation – Initiation, Elongation, Termination – finden jedoch in geregeltem Ablauf wie in vivo statt. Die Replikation in permeabilisierten HeLa-Zellen kann durch Hemmung der prozessiven DNA-Polymerisation unterbunden werden. Durch Hemmung anderer biochemischer Stoffwechselwege kann jedoch kein Einfluss auf die Replikation genommen werden. Nach der Permeabilisierung ist die Replikation also nicht mehr in gleicher Weise wie in vivo regulierbar. Bei der radioaktiven Markierung von replizierender DNA in permeabilisierten HeLa-Zellen akkumuliert eine Klasse kurzkettiger Moleküle. Die Länge dieser Nukleotidketten beträgt von 16–100 Nukleotide. Es treten diskrete Größenklassen von Molekülen auf. Diese können bei verlängerter Inkubation in gereifte DNA eingebaut werden. Auch enthalten diese Moleküle einen alkalilabilen Anteil. Bei diesen Intermediaten handelt es sich wahrscheinlich um Primer der Replikation. Der Unterschied in der Regulierbarkeit zwischen intakten HeLa-Zellen und permeabilisierten Zellen konnte nicht auf direkte Effekte der Zerstörung der Zellwand zurückgeführt werden. Auch konnte unter Zuhilfenahme einer großen Zahl von in Frage kommenden Parametern keine Möglichkeit gefunden werden...

CHARACTERIZING THE FUNCTIONAL DOMAINS OF THE BACULOVIRUS LATE EXPRESSION FACTOR 3 (LEF-3) INVOLVED IN NUCLEAR LOCALIZATION AND DNA REPLICATION

Au, VICTORIA
Fonte: Quens University Publicador: Quens University
Tipo: Tese de Doutorado Formato: 28589973 bytes; application/pdf
EN; EN
Relevância na Pesquisa
66.09%
Transient replication assays have identified a late expression factor 3, LEF-3, to be essential for DNA replication and late gene expression in the baculovirus species, AcMNPV. Although its specific role in these two processes has not been determined, this single-stranded DNA binding protein is multi-functional. LEF-3 forms a homo-oligomer, binds single-stranded DNA, interacts with components of the viral replication complex and is required to transport the helicase protein P143 into the nucleus of infected cells. Various regions within LEF-3 were deleted to determine the domain essential to its function and the N-terminal amino acids 1-125 were found to be sufficient for late gene expression. This N-terminal region includes the 56 amino acid region of LEF-3 required for nuclear transport. In order to define this domain, the effect of site-specific mutagenesis of LEF-3 on its intracellular distribution was determined. Fluorescence microscopy of expression plasmid transfected cells demonstrated that amino acids 14 to 37 formed the core nuclear localization signal (NLS), but the flanking amino acids may act as regulatory elements. Comparison with other group 1 Alphabaculoviruses suggested that this core region contained a functionally duplicated NLS. The AcMNPV LEF-3 also functioned in mammalian cells indicating that the protein nuclear import systems in insect and mammalian cells are conserved. Mutagenesis of two conserved cysteine residues located at amino acids 82 and 106 were not essential for nuclear localization or for interaction with P143. However...

Human GINS, a conserved DNA replication factor and candidate cancer marker

Marinsek, Nina
Fonte: University of Cambridge; MRC Cancer Cell Unit; Department of Oncology Publicador: University of Cambridge; MRC Cancer Cell Unit; Department of Oncology
Tipo: Thesis; doctoral; PhD
EN
Relevância na Pesquisa
66.11%
The GINS complex (a heterotetramer of Sld5, Psf1, Psf2 and Psf3) is a highly conserved DNA replication factor required for the initiation and elongation of DNA replication. GINS is believed to associate with Cdc45 and MCM proteins on replicating DNA. The interaction between GINS and MCM is also conserved in archaea. In my thesis, I explore the subcellular localisation of the GINS complex in relation to the MCM proteins and sites of DNA replication by high-resolution confocal microscopy. For these studies, I generated and carefully validated purified rabbit polyclonal and mouse monoclonal antibodies; these show a specific staining pattern by immunohistochemistry, immunoblotting and immunofluorescence. At high-resolution, all GINS antibodies produced a focal nuclear pattern, similar to that seen for the MCMs. However, confusingly, colocalisation between GINS and MCMs and between the GINS subunits themselves is poor. Investigations are continuing to understand this conundrum. Given the value of MCM proteins as specific and sensitive markers for cancer screening, I investigated whether GINS subunits also have potential diagnostic value. Sld5 and Psf3 expression is restricted to the proliferative compartment in normal tissue, but is found in the majority of cells in a wide range of dysplastic and malignant tissues...

DNA Replication of the Male X Chromosome Is Influenced by the Dosage Compensation Complex in Drosophila melanogaster

DeNapoli, Leyna
Fonte: Universidade Duke Publicador: Universidade Duke
Tipo: Dissertação
Publicado em //2013
Relevância na Pesquisa
66.26%

Abstract

DNA replication is an integral part of the cell cycle. Every time a cell divides, the entire genome has to be copied once and only once in a timely manner. In order to accomplish this, DNA replication begins at many points throughout the genome. These start sites are called origins of replication, and they are initiated in a temporal manner throughout S phase. How these origins are selected and regulated is poorly understood. Saccharomyces cerevisiae and Schizosaccharomyces pombe have autonomously replicating sequences (ARS) that can replicate plasmids extrachromosomally and function as origins in the genome. Metazoans, however, have shown no evidence of ARS activity.

DNA replication is a multistep process with several opportunities for regulation. Potential origins are marked with the origin recognition complex (ORC), a six subunit complex. In S. cerevisiae, ORC binds to the ARS consensus sequence (ACS), but no sequence specificity is seen in S. pombe or in metazoans. Therefore, factors other than sequence play a role in origin selection.

In G1, the pre-replicative (pre-RC) complex assembles at potential origins. This involves the recruitment of Cdc6 and Cdt1 to ORC, which then recruits MCM2-7 to the origin. In S phase...

Papillomavirus DNA replication

Ferran, Maureen; McBride, Alison
Fonte: Humana Press: Methods in Molecular Medicine Publicador: Humana Press: Methods in Molecular Medicine
Tipo: Artigo de Revista Científica Formato: 35612 bytes; application/pdf
EN_US
Relevância na Pesquisa
66.18%
Papillomavirus genomes replicate and are maintained as stable extrachromosomal plasmid DNA (episomes) in many cell lines. This process requires the viral E1 and E2 proteins and the origin of replication. The minimal origin of replication consists of an E1 binding site, an E2 binding site, and an AT rich region that probably facilitates origin unwinding. The E1 protein is an ATP-dependent helicase that specifically binds to and unwinds the origin. The E2 protein is the major transcriptional transactivator of the virus but it is also required for viral DNA replication. The E2 protein probably plays more of an auxiliary role in DNA replication; it has been shown to cooperatively bind to the origin with the E1 protein, to alleviate repression of replication by nucleosomes, and to interact with cellular replication proteins (RPA). To date, the most successful antiviral targets have been directed against viral-specific enzymes. Therefore, the ATPase and helicase activities of the E1 protein are attractive targets. Papillomavirus DNA replication may also be inhibited by compounds that interfere with the ability of E1 to bind DNA or to interact with the E2 protein.

How important is DNA replication for mutagenesis?

Huttley, Gavin Austin; Jakobsen, I; Easteal, Simon
Fonte: Society for Molecular Biology Evolution Publicador: Society for Molecular Biology Evolution
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
66.16%
Rates of mutation and substitution in mammals are generally greater in the germ lines of males. This is usually explained as resulting from the larger number of germ cell divisions during spermatogenesis compared with oogenesis, with the assumption made that mutations occur primarily during DNA replication. However, the rate of cell division is not the only difference between male and female germ lines, and mechanisms are known that can give rise to mutations independently of DNA replication. We investigate the possibility that there are other causes of male-biased mutation. First, we show that patterns of variation at ~5,200 short tandem repeat (STR) loci indicate a higher mutation rate in males. We estimate a ratio of male-to- female mutation rates of ~1.9. This is significantly greater than 1 and supports a greater rate of mutation in males, affecting the evolution of these loci. Second, we show that there are chromosome-specific patterns of nucleotide and dinucleotide composition in mammals that have been shaped by mutation at CpG dinucleotides. Comparable patterns occur in birds. In mammals, male germ lines are more methylated than female germ lines, and these patterns indicate that differential methylation has played a role in male-biased vertebrate evolution. However...

A universal protein�protein interaction motif in the eubacterial DNA replication and repair systems

Dalrymple, Brian Paul; Kongsuwan, Kritaya; Wijffels, Gene; Dixon, Nicholas; Jennings, Peter Andrew
Fonte: National Academy of Sciences (USA) Publicador: National Academy of Sciences (USA)
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
66.08%
The interaction between DNA polymerases and sliding clamp proteins confers processivity in DNA synthesis. This interaction is critical for most DNA replication machines from viruses and prokaryotes to higher eukaryotes. The clamp proteins also participate in a variety of dynamic and competing protein-protein interactions. However, clamp-protein binding sequences have not so far been identified in the eubacteria. Here we show from three lines of evidence, bioinformatics, yeast two-hybrid analysis, and inhibition of protein-protein interaction by modified peptides, that variants of a pentapeptide motif (consensus QL[SD]LF) are sufficient to enable interaction of a number of proteins with an archetypal eubacterial sliding clamp (the β subunit of Escherichia coli DNA polymerase III holoenzyme). Representatives of this motif are present in most sequenced members of the eubacterial DnaE, PoIC, PoIB, DinB, and UmuC families of DNA polymerases and the MutS1 mismatch repair protein family. The component tripeptide DLF inhibits the binding of the α (DnaE) subunit of E. coli DNA polymerase III to β at μM concentration, identifying key residues. Comparison of the eubacterial, eukaryotic, and archaeal sliding clamp binding motifs suggests that the basic interactions have been conserved across the evolutionary landscape.