p21CDKN1A does not interfere with loading of PCNA at DNA replication sites, but inhibits subsequent binding of DNA polymerase delta at the G1/S phase transition.
Project Title: Development of high throughput assay for screening of novel DNA replication inhibitors for therapeutic purposes Supervisors: Professor Christian Speck, Professor David Rueda Funding: Tuition fees plus £21,000 pa stipend for 3.5 years Date posted: 09 July 2021 Closing date: 04 August 2021 The student will develop a fluorescence-based assay to identify novel DNA replication inhibitors for anti-cancer therapy. Inhibitors will be consequently characterised for their impact on the multi-step DNA replication process and on cancer cell growth. This interdisciplinary project will train the student in biochemistry, biophysics and drug screening. Project details , LMS 3.5-year Studentships , Apply ...
A number of proteins have been isolated from human cells on the basis of their ability to support DNA replication in vitro of the simian virus 40 (SV40) origin of DNA replication. One such protein, replication factor C (RFC), functions with the proliferating cell nuclear antigen (PCNA), replication protein A (RPA), and DNA polymerase delta to synthesize the leading strand at a replication fork. To determine whether these proteins perform similar roles during replication of DNA from origins in cellular chromosomes, we have begun to characterize functionally homologous proteins from the yeast Saccharomyces cerevisiae. RFC from S. cerevisiae was purified by its ability to stimulate yeast DNA polymerase delta on a primed single-stranded DNA template in the presence of yeast PCNA and RPA. Like its human-cell counterpart, RFC from S. cerevisiae (scRFC) has an associated DNA-activated ATPase activity as well as a primer-template, structure-specific DNA binding activity. By analogy with the phage T4 and ...
TY - JOUR. T1 - The DNA Replication Program Is Altered at the FMR1 Locus in Fragile X Embryonic Stem Cells. AU - Gerhardt, Jeannine. AU - Tomishima, Mark J.. AU - Zaninovic, Nikica. AU - Colak, Dilek. AU - Yan, Zi. AU - Zhan, Qiansheng. AU - Rosenwaks, Zev. AU - Jaffrey, Samie R.. AU - Schildkraut, Carl L.. PY - 2014/1/9. Y1 - 2014/1/9. N2 - Fragile X syndrome (FXS) is caused by a CGG repeat expansion in the FMR1 gene that appears to occur during oogenesis and during early embryogenesis. One model proposes that repeat instability depends on the replication fork direction through the repeats such that (CNG)n hairpin-like structures form, causing DNA polymerase to stall and slip. Examining DNA replication fork progression on single DNA molecules at the endogenous FMR1 locus revealed that replication forks stall at CGG repeats in human cells. Furthermore, replication profiles of FXS human embryonic stem cells (hESCs) compared to nonaffected hESCs showed that fork direction through the repeats is ...
Replication of the two template strands at eukaryotic cell DNA replication forks is a highly coordinated process that ensures accurate and efficient genome duplication. Biochemical studies, principally of plasmid DNAs containing the Simian Virus 40 origin of DNA replication, and yeast genetic studies have uncovered the fundamental mechanisms of replication fork progression. At least two different DNA polymerases, a single-stranded DNA-binding protein, a clamp-loading complex, and a polymerase clamp combine to replicate DNA. Okazaki fragment synthesis involves a DNA polymerase-switching mechanism, and maturation occurs by the recruitment of specific nucleases, a helicase, and a ligase. The process of DNA replication is also coupled to cell-cycle progression and to DNA repair to maintain genome integrity.. ...
Previous genetic studies in yeast suggested that Pol ε plays an important role during chromosomal DNA replication [1, 7-9]. However, because the amino-terminal portion of Pol ε, that is required for its DNA polymerase- and exonuclease activities, is dispensable for yeast DNA replication, repair, and viability [15, 16, 27], the role of Pol ε during DNA replication has remained obscure. This study explores this role using an in vitro Xenopus DNA replication system and wild type and mutant forms of r-xPol ε holoenzyme. Here we show that the DNA replication defect in xPol ε-depleted Xenopus egg extracts is readily corrected by native (n-xPol ε) (data not shown and [22]) or recombinant xPol ε (r-xPol ε) holoenzyme or the p260-p60 Pol ε sub-complex, but not by p260ΔCat holoenzyme, p260 DN, p260 or p260-p12-p17 (Fig. 4). Because the former enzymes are polymerase proficient, while p260ΔCat holoenzyme and p260 DN are polymerase-deficient, although these preparations contained a small amount of ...
Mutations in DNA replication initiator genes in both prokaryotes and eukaryotes lead to a pleiotropic array of phenotypes, including defects in chromosome segregation, cytokinesis, cell cycle regulation and gene expression. For years, it was not clear whether these diverse effects were indirect consequences of perturbed DNA replication, or whether they indicated that DNA replication initiator proteins had roles beyond their activity in initiating DNA synthesis. Recent work from a range of organisms has demonstrated that DNA replication initiator proteins play direct roles in many cellular processes, often functioning to coordinate the initiation of DNA replication with essential cell-cycle activities. The aim of this review is to highlight these new findings, focusing on the pathways and mechanisms utilized by DNA replication initiator proteins to carry out a diverse array of cellular functions.
A complex network of interacting proteins and enzymes is required for DNA replication. Generally, DNA replication follows a multistep enzymatic pathway. At the DNA replication fork, a DNA helicase (DnaB or MCM complex) precedes the DNA synthetic machinery and unwinds the duplex parental DNA in cooperation with the SSB or RPA. On the leading strand, replication occurs continuously in a 5 to 3 direction, whereas on the lagging strand, DNA replication occurs discontinuously by synthesis and joining of short Okazaki fragments. In prokaryotes, the leading strand replication apparatus consists of a DNA polymerase (pol III core), a sliding clamp (beta), and a clamp loader (gamma delta complex). The DNA primase (DnaG) is needed to form RNA primers. Normally, during replication of the lagging-strand DNA template, an RNA primer is removed either by an RNase H or by the 5 to 3 exonuclease activity of DNA pol I, and the DNA ligase joins the Okazaki fragments. In eukaryotes, three DNA polymerases (alpha, ...
A complex network of interacting proteins and enzymes is required for DNA replication. Generally, DNA replication follows a multistep enzymatic pathway. At the DNA replication fork, a DNA helicase (DnaB or MCM complex) precedes the DNA synthetic machinery and unwinds the duplex parental DNA in cooperation with the SSB or RPA. On the leading strand, replication occurs continuously in a 5 to 3 direction, whereas on the lagging strand, DNA replication occurs discontinuously by synthesis and joining of short Okazaki fragments. In prokaryotes, the leading strand replication apparatus consists of a DNA polymerase (pol III core), a sliding clamp (beta), and a clamp loader (gamma delta complex). The DNA primase (DnaG) is needed to form RNA primers. Normally, during replication of the lagging-strand DNA template, an RNA primer is removed either by an RNase H or by the 5 to 3 exonuclease activity of DNA pol I, and the DNA ligase joins the Okazaki fragments. In eukaryotes, three DNA polymerases (alpha, ...
TY - JOUR. T1 - In vitro complementation as an assay for purification of adenovirus DNA replication proteins. AU - Ostrove, J. M.. AU - Rosenfeld, P.. AU - Williams, J.. AU - Kelly, T.. PY - 1983. Y1 - 1983. N2 - As an approach to the purification of adenovirusencoded DNA replication proteins, we have developed in vitro complementation assays that make use of viral mutants defective in DNA replication in vivo. Nuclear extracts prepared from cells infected with H5ts36 or H5ts125, two such mutants belonging to different complementation groups, were found to be defective in viral DNA replication in vitro. However, replication activity could be restored by mixing the two extracts. Replication activity in either extract also could be restored by addition of appropriate replication-deficient fractions purified from cells infected with wild-type adenovirus. By using such assays, H5ts36- and H5ts125-complementing activities were extensively purified. As expected, purified H5ts125-complementing activity ...
DNA replication is a tightly regulated multistep process that requires the sequential action of several protein complexes that select DNA replication origins, recruit on these origins the DNA replication fork helicase that once activated, unwinds and duplicates the DNA. These events must be tightly coupled to cell cycle progression to ensure that DNA replication occurs once and only once per cell cycle.. DNA replication is thus temporally separated into two steps that are controlled by Cyclin-Dependent Kinase (CDK) activity. The first step, which occurs in mitosis and during the G1 phase of the cell cycle, when Cdk activity is low, involves the loading of a double hexameric Mcm2-7 (minichromosome maintenance 2-7) complex on the chromatin as part of the prereplicative complex (pre-RC) (Evrin et al. 2009; Remus et al. 2009; Gambus et al. 2011; Deegan and Diffley 2016). Pre-RC formation requires several loading factors including the hexameric Origin Recognition Complex (ORC-1-6), and Cdc6 and Cdt1 ...
Proper coordination of the functions at the DNA replication fork is vital to the normal functioning of a cell. Specifically the precise coordination of helicase and polymerase activity is crucial for efficient passage though S phase. The Ctf4 protein has been shown to be a central member of the replication fork and links the replicative MCM helicase and DNA polymerase [alpha] primase. In addition, it has been implicated as a member of a complex that promotes replication fork stability, the Fork Protection Complex (FPC), and as being important for sister chromatid cohesion. As such, understanding the role of Ctf4 within the context of a multicellular organism will be integral to our understanding of its potential role in developmental and disease processes. We find that Drosophila Ctf4 is a conserved protein that interacts with members of the GINS complex, Mcm2, and Polymerase [alpha] primase. Using in vivo RNAi knockdown of CTF4 in Drosophila we show that Ctf4 is required for viability, S phase ...
Initiation of DNA replication during the mitotic cell cycle requires the activation of a cyclin-dependent protein kinase (CDK). The B-type cyclins Clb5 and Clb6 are the primary activators of the S phase function of the budding yeast CDK Cdc28. However, in mitotically growing cells this role can be fulfilled by the other B-type cyclins Clb1-Clb4. We report here that cells undergoing meiotic development also require Clb dependent CDK activity for DNA replication. Diploid clb5/clb5 clb6/clb6 mutants are unable to perform premeiotic DNA replication. Despite this defect, the mutant cells progress into the meiotic program and undergo lethal segregation of unreplicated DNA suggesting that they fail to activate a checkpoint that restrains meiotic M phase until DNA replication is complete. We have found that a DNA replication checkpoint dependent on the ATM homolog MEC1 operates in wild-type cells during meiosis and can be invoked in response to inhibition of DNA synthesis. Although cells that lack clb5 and clb6
Dna replication diagram ncert. This process involves multiple steps that have to proceed in a specific sequence to generate the desired product. Dna replication takes place in order to prepare cell for division. Dna replication is an important process that occurs during cell division. Watson and crick dna model. The dna replication in eukaryotes is similar to the dna replication in prokaryotes. Dna replication enzymes have the ability to quicken reactions and build up or break down the items that they act upon. ,br, this creates some additional complications at the replicating fork. There can be dna replication without cell division. Last updated on january 3, 2020 by sagar aryal. Ncert book for class 12 biology chapter 6 molecular basis of inheritance is available for reading or download on this page. (a) dna is a macromolecule since two strands of dna cannot be separated in its entire length (due to very high energy requirement), the replication occur in small replication fork. [image will be ...
Schmid, Jonas. Histone ubiquitination by the DNA damage response is required for efficient DNA replication in unperturbed S-phase. 2018, University of Zurich, Faculty of Science. ...
please provide good explanation1- compare the dna replication process in cells with the process occurring in the pcr, Hire Biology Expert, Ask Academics Expert, Assignment Help, Homework Help, Textbooks Solutions
Supplementary MaterialsPeer Review File 41467_2017_632_MOESM1_ESM. Abstract mRNA-processing (P-) body are cytoplasmic granules that form in eukaryotic cells in response to numerous stresses to serve as sites of degradation and storage of mRNAs. Functional P-bodies are critical for the DNA replication stress response in yeast, yet the repertoire of P-body targets and the mechanisms by which P-bodies promote replication stress resistance are unknown. In this study we identify the complete match of mRNA targets of P-bodies during replication stress induced by hydroxyurea treatment. The key P-body protein Lsm1 controls the large quantity of and mRNAs to prevent their toxic accumulation during replication stress. Accumulation of mRNA causes aberrant downregulation of a network of genes critical for DNA replication stress resistance and prospects to harmful acetaldehyde accumulation. Our data reveal the scope and the targets of regulation by P-body proteins during the DNA replication stress response. ...
To examine the basis for the evolutionary selection for codirectionality of replication and transcription in Escherichia coli, electron microscopy was used to visualize replication from an inducible ColE1 replication origin inserted into the Escherichia coli chromosome upstream (5) or downstream (3) of rrnB, a ribosomal RNA operon. Active rrnB operons were replicated either in the same direction in which they were transcribed or in the opposite direction. In either direction, RNA polymerases were dislodged during replication. When replication and transcription were codirectional, the rate of replication fork movement was similar to that observed in nontranscribed regions. When replication and transcription occurred in opposite directions, replication fork movement was reduced. ...
Chapter 3: DNA Replication Models of DNA replication: Meselson-Stahl Experiment DNA synthesis and elongation DNA polymerases Origin and initiation of DNA replication Prokaryote/eukaryote models (circular/linear chromosomes) Telomere replication Slideshow 29909 by JasminFlorian
Polo-like kinase 1 (Plk1) plays pivotal roles in mitosis; however, little is known about its function in S phase. In this study, we show that inhibition of Plk1 impairs DNA replication and results in slow S-phase progression in cultured cancer cells. We have identified origin recognition complex 2 (Orc2), a member of the DNA replication machinery, as a Plk1 substrate and have shown that Plk1 phosphorylates Orc2 at Ser188 in vitro and in vivo. Furthermore, Orc2-S188 phosphorylation is enhanced when DNA replication is under challenge induced by ultraviolet, hydroxyurea, gemcitabine, or aphidicolin treatment. Cells expressing the unphosphorylatable mutant (S188A) of Orc2 had defects in DNA synthesis under stress, suggesting that this phosphorylation event is critical to maintain DNA replication under stress. To dissect the mechanism pertinent to this observation, we showed that Orc2-S188 phosphorylation associates with DNA replication origin and that cells expressing Orc2-S188A mutant fail to ...
TY - JOUR. T1 - Genetic control of the cell division cycle in yeast. II. Genes controlling DNA replication and its initiation. AU - Hartwell, Leland H.. PY - 1971/7/14. Y1 - 1971/7/14. N2 - Temperature-sensitive mutations occurring in two unlinked complementation groups, cdc4 and cdc8, are recessive and result in a defect in DNA replication at the restrictive temperature. Results obtained with synchronous cultures suggest that cdc4 functions in the initiation of DNA replication and cdc8 functions in the propagation of DNA replication. From the behavior of mutant strains carrying lesions in cdc4, or in cdc8, or in both genes it is concluded that: (1) nuclear division and cell separation in yeast are dependent upon prior DNA replication; (2) a cellular clock controls bud initiation and the running of this clock is independent of the other events in the cycle, DNA replication, nuclear division and cell separation; (3) premature bud initiation is normally prevented as a consequence of the successful ...
TY - JOUR. T1 - Repetitive lagging strand DNA synthesis by the bacteriophage T4 replisome. AU - Spiering, Michelle M.. AU - Nelson, Scott W.. AU - Benkovic, Stephen J.. PY - 2008. Y1 - 2008. N2 - Our studies on the T4 replisome build on the seminal work from the Alberts laboratory. They discovered essentially all the proteins that constitute the T4 replisome, isolated them, and measured their enzymatic activities. Ultimately, in brilliant experiments they reconstituted in vitro a functioning replisome and in the absence of structural information created a mosaic as to how such a machine might be assembled. Their consideration of the problem of continuous leading strand synthesis opposing discontinuous lagging strand synthesis led to their imaginative proposal of the trombone model, an illustration that graces all textbooks of biochemistry. Our subsequent work deepens their findings through experiments that focus on defining the kinetics, structural elements, and protein-protein contacts ...
Flow cytometry, a method for measuring DNA content, also gives information about the cell cycle. Non-cycling cells are said to be in the G0 stage. For cycling cells, it is usual to define four distinct phases of the cell cycle. Mitosis (M phase) is followed by the G1 phase (gap 1). During this phase the cell continuously grows but does not replicate its DNA. When the cell starts to make new DNA it has entered the S (DNA synthesis) phase. The completion of DNA synthesis is followed by the G2 phase (gap 2), during which cell growth continues and proteins are synthesised in preparation for mitosis [1]. Flow cytometry has been shown to be very suitable for determining the DNA replication stages in seeds [2,3,10,11,16,17,18]. In commercial practice, sugar beet seeds are often washed/soaked and treated with fungicides before sowing. These treatments are intended to leach out the soluble inhibitors from the pericarp and to control damping off, which, in turn, improve seed performance in the field ...
AMONG the genetic and epigenetic changes to genomes, changes in ploidy are the most drastic, and as such, polyploidy is not tolerated by most animal species (Li et al. 2009a). A recent study of tetraploid yeast suggests that the deleterious effects of ploidy change are due to the uncoordinated scaling of the spindle pole body, spindle, and kinetochore, thus resulting in genetic instability (GIN) (Storchova et al. 2006). However, ploidy changes occur in every sexual cycle of all eukaryotes and are associated with the inclusion or exclusion of an entire set of chromosome homologs that significantly alters the DNA repair capacity. Little is known about whether DNA damage response is regulated differently in haplophase and diplophase during sexual cycles.. DNA replication stress, induced by oncogene activation, genotoxic stress, or defects in the DNA replication machinery, is believed to cause GIN that accelerates tumorigenesis (Halazonetis et al. 2008). However, DNA replication stress does not ...
Dates: 7 - 10 May 2018 Registration: 26 Mar 18 Abstract: 12 Feb 18 Event webpage: www.embo-embl-symposia.org/symposia/2018/EES18-02/index.html Aim:There has been tremendous progress in the past few years regarding our understanding of DNA replication in eukaryotes, both yeast and mammals. Many important questions in the field are poised to be answered within the next decade. These include understanding DNA replication at the biochemical and three-dimensional protein structure levels. In addition, studies using high throughput technologies at the cellular and organismal levels are poised to answer how accurate replication of the genome is ensured by controlling origin firing in space and time.Several human diseases, including cancer, have already been linked to DNA replication stress, a term that refers to perturbations in DNA replication. Thus, a better understanding of how cells respond to DNA replication stress will help us understand disease development and responses to therapy. By bringing ...
Molecular mechanism of DNA replication. Two distinct Polymerases - a and d, appear to function at the eukaryotic growing fork.Polymerase d (pol. Before replication can start, the DNA has to be made available as a template. This process is called semiconservative replication because one of the old strands is conserved in the new DNA double helix. Notes # Replication of Circular DNA Molecules: Circular DNA molecules occur in both prokaryotes and eukaryotes. Next lesson. DNA replication would not occur without enzymes that catalyze various steps in the process. However, the interaction of the initiation proteins with the ds-DNA is more complex. RNA polymerase requires a number of helper proteins to bind to DNA and initiate RNA Eukaryotic DNA replication, also reviewed in more detail in Chapter 3, Features of Host Cells: Cellular and Molecular Biology Review, is also carried out by DNA polymerases and other proteins within the nucleus. In eukaryotes, the situation is different in a number of ...
Mammalian mitochondria operate multiple mechanisms of DNA replication. In many cells and tissues a strand-asynchronous mechanism predominates over coupled leading and lagging-strand DNA synthesis. However, little is known of the factors that control or influence the different mechanisms of replication, and the idea that strand-asynchronous replication entails transient incorporation of transcripts (aka bootlaces) is controversial. A firm prediction of the bootlace model is that it depends on mitochondrial transcripts. Here, we show that elevated expression of Twinkle DNA helicase in human mitochondria induces bidirectional, coupled leading and lagging-strand DNA synthesis, at the expense of strand-asynchronous replication; and this switch is accompanied by decreases in the steady-state level of some mitochondrial transcripts. However, in the so-called minor arc of mitochondrial DNA where transcript levels remain high, the strand-asynchronous replication mechanism is instated. Hence, replication ...
Chromosome replication in eukaryotic cells is regulated in a highly complex fashion in order to maintain the integrity of the genome from one generation to the next. DNA replication forks are established at different moments in time during S‐phase from multiple origins on each chromosome, yet initiation at each origin can occur just once, so that a single copy of the genome is generated in each round of a typical cell cycle (Blow and Dutta, 2005). This is achieved by dividing the cell cycle into a period when prereplication complexes (pre‐RCs) of proteins essential for initiation are assembled at origins but cannot be activated, and a subsequent and mutually exclusive period when pre‐RCs can be activated but can no longer form (Diffley et al, 1994). At each origin, the pre‐RC is lost during initiation, so that each region of the chromosome is replicated precisely once during each cell cycle.. The key event in the formation of pre‐RCs-also known as the licensing of origins-is the ...
DNA replication is a biological process that occurs in all living organisms and copies their DNA; it is the basis for natural inheritance. The process starts when one double-stranded DNA molecule produces two identical copies of the molecule. The cell cycle (mitosis) also pertains to the DNA replication/reproduction process. DNA replication, in eukaryotes, is controlled within the context of the cell cycle. As the cell grows and divides, it goes through stages in the cell cycle; DNA replication occurs during the S phase (synthesis phase). Whereas bacteria do not go through an exact cell cycle but instead, they continuously copy their DNA. The research conducted in this study was to see if, in fact, that cell size had anything to do with the initiation of DNA replication in bacteria. The objectives and hypothesis was clearly stated in the paper. In order to find the answer to their question, the researchers used Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis) in their experiment. ...
Plays a key role in the initiation and regulation of chromosomal replication. Binds in an ATP-dependent fashion to the origin of replication (oriC) to initiate formation of the DNA replication initiation complex exactly once per cell cycle. Binds the DnaA box (consensus sequence 5-TTATC[CA]A[CA]A-3); subsequent binding of DNA polymerase III subunits leads to replisome formation. The DnaA-ATP form converts to DnaA-ADP; once converted to ADP the protein cannot initiate replication, ensuring only 1 round of replication per cell cycle. DnaA can inhibit its own gene expression as well as that of other genes such as dam, rpoH, ftsA and mioC.
Cyclin-dependent kinases (CDKs) regulate the progression of the cell cycle in eukaryotes. One of the major roles of CDK is to promote chromosomal DNA replication. However, how CDKs promote DNA replication has been a long-standing question, because all the essential CDK substrates in DNA replication have not been identified yet. Recently Sld2 and Sld3 were identified as essential substrates of CDKs in the initiation step of DNA replication in budding yeast. Moreover, bypass of their phosphorylations is sufficient to promote DNA replication. Phosphorylation of Sld2 and Sld3 by CDKs enhances the formation of complex(es) with a BRCT (BRCA1 C-Terminal)-containing replication protein, Dpb11. We further propose that multiple phosphorylation by CDKs controls this process in budding yeast. Even though Sld3 orthologues in multicellular eukaryotes have not been identified, similar complex formation and, therefore, a similar mechanism of initiation control might be employed in eukaryotes.
The T4 bacteriophage dda protein is a DNA-dependent ATPase and DNA helicase that is the product of an apparently nonessential T4 gene. We have examined its effects on in vitro DNA synthesis catalyzed by a purified, multienzyme T4 DNA replication system. When DNA synthesis is catalyzed by the T4 DNA polymerase on a single-stranded DNA template, the addition of the dda protein is without effect whether or not other replication proteins are present. In contrast, on a double-stranded DNA template, where a mixture of the DNA polymerase, its accessory proteins, and the gene 32 protein is required, the dda protein greatly stimulates DNA synthesis. The dda protein exerts this effect by speeding up the rate of replication fork movement; in this respect, it acts identically with the other DNA helicase in the T4 replication system, the T4 gene 41 protein. However, whereas a 41 protein molecule remains bound to the same replication fork for a prolonged period, the dda protein seems to be continual
Maintaining replication fork integrity is vital to preserve genomic stability and avoid cancer. Physical DNA damage and altered nucleotide or protein pools represent replication obstacles, generating replicative stress. Numerous cellular responses have evolved to ensure faithful DNA replication despite such challenges. Understanding those responses is essential to understand and prevent or treat replication-associated diseases, such as cancer.. Re-priming is a mechanism to allow resumption of DNA synthesis past a fork-stalling lesion. This was recently suggested in yeast and explains the formation of gaps during DNA replication on damaged DNA. Using a combination of assays, we indicate the existence of re-priming also in human cells following UV irradiation.. The gap left behind a re-primed fork must be stabilised to avoid replication-associated collapse. Our results show that the checkpoint signalling protein CHK1 is dispensable for stabilisation of replication forks after UV irradiation, ...
The DNA replication checkpoint is a complex signal transduction pathway, present in all eukaryotic cells, that functions to maintain genomic integrity and cell viability when DNA replication is perturbed. In Schizosaccharomyces pombe the major effector of the replication checkpoint is the protein kinase Cds1. Activation of Cds1 is known to require the upstream kinase Rad3 and the mediator Mrc1, but the biochemical mechanism of activation is not well understood. We report that the replication checkpoint is activated in two stages. In the first stage, Mrc1 recruits Cds1 to stalled replication forks by interactions between the FHA domain of Cds1 and specific phosphorylated Rad3 consensus sites in Mrc1. Cds1 is then primed for activation by Rad3-dependent phosphorylation. In the second stage, primed Cds1 molecules dimerize via phospho-specific interactions mediated by the FHA domains and are activated by autophosphorylation. This two-stage activation mechanism for the replication checkpoint allows for rapid
The cohesin complex holds together newly-replicated chromatids and is involved in diverse pathways that preserve genome integrity. We show that in budding yeast, cohesin is transiently recruited to active replication origins and it spreads along DNA as forks progress. When DNA synthesis is impeded, cohesin accumulates at replication sites and is critical for the recovery of stalled forks. Cohesin enrichment at replication forks does not depend on H2A(X) formation, which differs from its loading requirements at DNA double-strand breaks (DSBs). However, cohesin localization is largely reduced in rad50delta mutants and cells lacking both Mec1 and Tel1 checkpoint kinases. Interestingly, cohesin loading at replication sites depends on the structural features of Rad50 that are important for bridging sister chromatids, including the CXXC hook domain and the length of the coiled-coil extensions. Together, these data reveal a novel function for cohesin in the maintenance of genome integrity during S phase. Scc1
DNA replication control is a key process in maintaining genomic integrity. Monitoring DNA replication initiation is particularly important as it needs to be coordinated with other cellular events and should occur only once per cell cycle. Crucial players in the initiation of DNA replication are the ORC protein complex, marking the origin of replication, and the Cdt1 and Cdc6 proteins, that license these origins to replicate by recruiting the MCM2-7 helicase. To accurately achieve its functions, Cdt1 is tightly regulated. Cdt1 levels are high from metaphase and during G1 and low in S/G2 phases of the cell cycle. This control is achieved, among other processes, by ubiquitination and proteasomal degradation. In an overexpression screen for Cdt1 deubiquitinating enzymes, we isolated USP37, to date the first ubiquitin hydrolase controlling Cdt1. USP37 overexpression stabilizes Cdt1, most likely a phosphorylated form of the protein. In contrast, USP37 knock down destabilizes Cdt1, predominantly during ...
DNA-skadande ämnen är vanligt i cancerbehandling, då snabbt växande celler, såsom cancerceller är betydligt känsligare än normala celler för DNA skador. En grupp av ämnen som vanligen används i cancerbehandling är korsbindare av DNA. Dessa ämnen kommer reagera två gånger med DNA och skapa två bindningar mitt emot varandra. DNA strängen, som består av två delar, måste kunna separeras och kopieras (replikation) på ett tillförlitligt sätt för att cellerna ska kunna dela sig och bli flera. DNA strängen måste också kunna dela sig och bli avläst rätt för att nya proteiner ska kunna bildas (transkription). När korsbindarna har bundit till DNA strängarna, hindrar detta deras separation och därigenom förhindras även avläsningen och kopieringen. För att göra undersökningarna av DNA korsbindande ämnen ännu lite svårare, så ger korsbindare flera olika typer av skador. Dels kan det bli flera olika typer av korsbindningar, både mellan två DNA-strängar (ICL) vilket ...
Professor Emeritus Department of Biochemistry Rosalind and Morris Goodman Cancer Centre [email protected] 1979 - PhD, McGill University Research Interests Isolation and characterization of mammalian origins of DNA replication Our primary research interest is in the molecular basis of the mechanisms regulating mammalian DNA replication. Eukaryotic chromosomes are organized into multiple replication units that initiate replication only one per cell cycle. The mechanism that prevents the reinitiation of replication of DNA that has been previously replicated is unknown. Among the major questions about mammalian DNA replication are: 1) whether initiation occurs at specific DNA sequences (replication origins), and 2) what are the molecular features of these sequences. Using the instability of replication loops as a method for the isolation of active replication origins, we have purified and cloned DNA sequences that contain origins of replication. In this manner we have generated libraries of monkey and
Professor Emeritus Department of Biochemistry Rosalind and Morris Goodman Cancer Centre [email protected] 1979 - PhD, McGill University Research Interests Isolation and characterization of mammalian origins of DNA replication Our primary research interest is in the molecular basis of the mechanisms regulating mammalian DNA replication. Eukaryotic chromosomes are organized into multiple replication units that initiate replication only one per cell cycle. The mechanism that prevents the reinitiation of replication of DNA that has been previously replicated is unknown. Among the major questions about mammalian DNA replication are: 1) whether initiation occurs at specific DNA sequences (replication origins), and 2) what are the molecular features of these sequences. Using the instability of replication loops as a method for the isolation of active replication origins, we have purified and cloned DNA sequences that contain origins of replication. In this manner we have generated libraries of monkey and
We showed previously that DNA replication initiates at multiple sites in the 5-kb histone gene repeating unit in early embryos of Drosophila melanogaster. The present report shows evidence that replication in the same chromosomal region initiates at multiple sites in tissue culture cells as well. First, we analyzed replication intermediates by the two-dimensional gel electrophoretic replicon mapping method and detected bubble-form replication intermediates for all fragments restricted at different sites in the repeating unit. Second, we analyzed bromodeoxyuridine-labeled nascent strands amplified by the polymerase chain reaction method and detected little differences in the size distribution of nascent strands specific to six short segments located at different sites in the repeating unit. These results strongly suggest that DNA replication initiates at multiple sites located within the repeating unit. We also found several replication pause sites located at 5 upstream regions of some histone ...
Human genetic variation is distributed nonrandomly across the genome, though the principles governing its distribution are only partially known. DNA replication creates opportunities for mutation, and the timing of DNA replication correlates with the density of SNPs across the human genome. To enable deeper investigation of how DNA replication timing relates to human mutation and variation, we generated a high-resolution map of the human genomes replication timing program and analyzed its relationship to point mutations, copy number variations, and the meiotic recombination hotspots utilized by males and females. DNA replication timing associated with point mutations far more strongly than predicted from earlier analyses and showed a stronger relationship to transversion than transition mutations. Structural mutations arising from recombination-based mechanisms and recombination hotspots used more extensively by females were enriched in early-replicating parts of the genome, though these ...
TY - JOUR. T1 - H3K9me3 demethylase Kdm4d facilitates the formation of pre-initiative complex and regulates DNA replication. AU - Wu, Rentian. AU - Wang, Zhiquan. AU - Zhang, Honglian. AU - Gan, Haiyun. AU - Zhang, Zhiguo. N1 - Publisher Copyright: © 2016 The Author(s).. PY - 2017/1/9. Y1 - 2017/1/9. N2 - DNA replication is tightly regulated to occur once and only once per cell cycle. How chromatin, the physiological substrate of DNA replication machinery, regulates DNA replication remains largely unknown. Here we show that histone H3 lysine 9 demethylase Kdm4d regulates DNA replication in eukaryotic cells. Depletion of Kdm4d results in defects in DNA replication, which can be rescued by the expression of H3K9M, a histone H3 mutant transgene that reverses the effect of Kdm4d on H3K9 methylation. Kdm4d interacts with replication proteins, and its recruitment to DNA replication origins depends on the two prereplicative complex components (origin recognition complex [ORC] and minichromosome ...
A cell-free system has been developed from cells of an Escherichia coli strain, carrying cloned genes 1 (DNA polymerase) and 8 (terminal protein) of bacteriophage PRD1, that catalyzes protein-primed DNA synthesis. DNA synthesis in vitro is entirely dependent upon the addition of PRD1 DNA-terminal protein complex as template, Mg²⁺, and four deoxyribonucleoside triphosphates. The origin and direction of PRD1 DNA replication in vitro were determined by restriction enzyme analysis of ³²P-labeled PRD1 DNA synthesized in this system. Replication starts at either end of the linear PRD1 DNA template. Analysis by alkaline sucrose gradient centrifugation and alkaline agarose gel electrophoresis of DNA synthesized in vitro showed that full-length PRD1 DNA is synthesized. DNA elongation in this system is inhibited by the drug aphidicolin. On the other hand, DNA initiation is inhibited by phenylglyoxal, an arginine-specific α-dicarbonyl reagent. In vitro studies have also demonstrated that linear ...
DNA is the substrate of many cellular processes including DNA replication, transcription and chromatin remodeling. These processes are coordinated to maintain genome integrity and ensure accurate duplication of genetic and epigenetic information. Genome-wide studies have provided evidence of the relationship between transcription and DNA replication timing. A global analysis of DNA replication initiation in T. brucei showed that TbORC1 (subunit of the origin recognition complex, ORC) binding sites are located at the boundaries of transcription units. Although recent studies in T. brucei indicate functional links among DNA replication and transcription, the underlying mechanisms remain unknown. In this study, we adapted an unbiased technology for the identification of replication fork proteins called iPOND (isolation of proteins on nascent DNA) to T. brucei, its first application to a parasite system.The iPOND approach relies on labeling newly replicated DNA with the thymidine analog EdU (5-ethynyl-2′
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DNA damage can stall the DNA replication machinery, leading to genomic instability. Thus, numerous mechanisms exist to complete genome duplication in the absence of a pristine DNA template, but identification of the enzymes involved remains incomplete. Here, we establish that Primase-Polymerase (PrimPol; CCDC111), an archaeal-eukaryotic primase (AEP) in eukaryotic cells, is involved in chromosomal DNA replication. PrimPol is required for replication fork progression on ultraviolet (UV) lightdamaged DNA templates, possibly mediated by its ability to catalyze translesion synthesis (TLS) of these lesions. This PrimPol UV lesion bypass pathway is not epistatic with the Pol h-dependent pathway and, as a consequence, protects xeroderma pigmentosum variant (XP-V) patient cells from UV-induced cytotoxicity. In addition, we establish that PrimPol is also required for efficient replication fork progression during an unperturbed S phase. These and other findings indicate that PrimPol is an important player ...
The genes encoding two of the subunits of the Saccharomyces cerevisiae origin recognition complex (ORC) have been isolated. Characterization of a temperature-sensitive mutation in the gene encoding the 72-kD subunit of ORC (ORC2) indicates that this protein complex functions early in the DNA replication process. Moreover, ORC derived from orc2ts cells is defective for DNA binding. Others have shown a defect in orc2ts cells in transcriptional silencing at the silent mating-type loci. Consistent with this finding, ORC specifically binds to each of the four mating-type silencers identified in yeast. These findings support the hypothesis that ORC acts as an initiator protein at yeast origins of DNA replication and suggest that ORC also functions in the determination of transcriptional domains. ...
In response to replication stress, a phospho-signaling cascade is activated and required for coordination of DNA repair and replication of damaged templates (intra-S phase checkpoint). How phospho-signaling coordinates the DNA replication stress response is largely unknown. We employed state-of-the-art liquid chromatography tandem mass spectrometry (LC-MS/MS) approaches to generate high-coverage and quantitative proteomic and phospho-proteomic profiles during replication stress in yeast, induced by continuous exposure to the DNA alkylating agent methyl methanesulfonate (MMS). We identified 32,057 unique peptides representing the products of 4,296 genes, and 22,061 unique phosphopeptides representing the products of 3,183 genes. 542 phosphopeptides (mapping to 339 genes) demonstrated an abundance change of ≥ 2-fold in response to MMS. The screen enabled detection of nearly all of the proteins known to be involved in the DNA damage response, as well as many novel MMS-induced phosphorylations. We ...
If you are interested in looking at the DNA structure and replication review worksheet, you can do so through many different websites. Acces PDF Dna Replication Worksheet Answers Dna Replication Worksheet Answers This is likewise one of the factors by obtaining the soft documents of this dna replication worksheet answers by online. ____ 1. 4. The new cells then receive the instructions and information needed to function. 3. -What is happening to the DNA molecule in the figure? nucleus before a cell divides. Answer the following questions about DNA replication in complete sentences. 4 0 obj Figure 3. DNA Replication Name _____ Step 1 : After you have completed your DNA keychain, label the bases on the paper model with A, T, G, or C and color the squares to match the bases exactly as they are on your keychain from the bottom to the top. 1. DNA Replication and Transcription Worksheet . Why does DNA need to replicate? Replication occurs in a Theyre the same size and shape, and have the same pattern ...
One of the factors limiting indefinite proliferation of somatic cells is telomere length [1], [2]. Indeed, the inability to fully replicate both strands of a linear DNA molecule is expected to lead to gradual shortening of telomeres in cells that do not express telomerase. Telomere shortening may be even more severe, if the replication machinery fails to reach the telomeric end. Indeed, the highly repetitive primary structure of telomeres [3], the presence of G‐quadruplexes [4], DNA-RNA hybrids [5], [6], and T‐loops [7], as well as the extensive telomeric heterochromatinization [8], challenge the process of terminal DNA replication and make telomeres prone to fork collapse, similar to common fragile sites [9], [10]. Fork collapse within a telomere is unlikely to be resolved by incoming forks or dormant forks, since human telomeres are thought to be devoid of replication origins. Instead, telomere replication is normally dependent on a single origin, located at the subtelomeric regions [11]. ...
One of the factors limiting indefinite proliferation of somatic cells is telomere length [1], [2]. Indeed, the inability to fully replicate both strands of a linear DNA molecule is expected to lead to gradual shortening of telomeres in cells that do not express telomerase. Telomere shortening may be even more severe, if the replication machinery fails to reach the telomeric end. Indeed, the highly repetitive primary structure of telomeres [3], the presence of G‐quadruplexes [4], DNA-RNA hybrids [5], [6], and T‐loops [7], as well as the extensive telomeric heterochromatinization [8], challenge the process of terminal DNA replication and make telomeres prone to fork collapse, similar to common fragile sites [9], [10]. Fork collapse within a telomere is unlikely to be resolved by incoming forks or dormant forks, since human telomeres are thought to be devoid of replication origins. Instead, telomere replication is normally dependent on a single origin, located at the subtelomeric regions [11]. ...
Each cell division, the nuclear DNA must be replicated efficiently and with high accuracy to avoid mutations which can have an effect on cell function. There are three replicative DNA polymerases essential for the synthesis of DNA during replication in eukaryotic cells. DNA polymerase α (Pol α) synthesize short primers required for DNA polymerase δ (Pol δ) and DNA polymerase ε (Pol ε) to carry out the bulk synthesis. The role of Pol δ and Pol ε at the replication fork has been unclear. The aim of this thesis was to examine what role Pol ε has at the replication fork, compare the biochemical properties of Pol δ and Pol ε, and to study the function of the second largest and essential subunit of Pol ε, Dpb2.. To identify where Pol ε replicates DNA in vivo, a strategy was taken where the active site of Pol ε was altered to create a mutator polymerase leaving a unique error-signature. A series of mutant pol ε proteins were purified and analyzed for enzyme activity and fidelity of DNA ...
Biological Function. Although synthesis of the lagging strand involves only half the DNA in the nucleus, the complexity associated with processing Okazaki fragments is about twice that required to synthesize the leading strand. Even in small species such as yeast, Okazaki fragment maturation happens approximately a million times during a single round of DNA replication. Processing of Okazaki fragments is therefore very common and crucial for DNA replication and cell proliferation.. During this process, RNA and DNA primers are removed, allowing the Okazaki fragments to attach to the lagging DNA strand. While this process seems quite simple and repetitive, defects in Okazaki fragment maturation can cause DNA strand breakage which can cause varying forms of chromosome aberrations. Severe defects of Okazaki fragment maturation may halt DNA replication and induce cell death. However, while subtle defects do not affect growth, they do result in future varying forms of genome instabilities. Based on ...
Cdc7-Dbf4 is an essential protein kinase complex required for every single origin firing. As a target of the intra-S checkpoint, Cdc7 kinase activity has also been implicated in the response to replication fork stress, with a role in translesion DNA synthesis (TLS). We have examined the role of Cdc7 in the regulation of replication forks, particularly in response to MMS, which normally stalls replication forks and inhibits late origin firing. We find that replication forks proceed as fast as with no damage along an MMS-damaged template both in cdc7as3 and cdc7-1/mcm5-bob1 cells. However the DNA synthesis in cdc7-1/mcm5-bob1 in MMS is defective, indicated by the slower recovery after MMS by PFGE, suggesting the replication is incomplete. These deregulated forks did not rely on TLS pathway but are dependent on both helicase and E3 ligase function of Rad5 for continued fork progression along MMS-damaged DNA, demonstrating a role for Rad5 at the replication fork. Phosphorylation of MCM2 by DDK was ...
The DNA replication (or origin) licensing machinery ensures precise duplication of the genome and contributes to the regulation of proliferative capacity in metazoa. Using an in vitro fibroblast model system coupled to a cell-free DNA replication assay, we have studied regulation of the origin licensing pathway during exit from and re-entry into the mitotic cell cycle. We show that in the quiescent state (G0) loss of proliferative capacity is achieved in part through down-regulation of the replication licensing factors Cdc6 and Mcm2-7. The origin licensing repressor geminin is absent in quiescent fibroblasts, suggesting that this powerful inhibitor of the licensing machinery is not required to suppress proliferative capacity in G0. Geminin expression is induced at a late stage in the G0-S transition post pre-RC assembly. Ectopic geminin can block re-acquisition of DNA replication competence during re-entry into the cell cycle, indicating that geminin levels must be tightly down-regulated for ...
Interaction of the Escherichia coli Replication Terminator Protein (Tus) with DNA: A Model Derived from DNA-Binding Studies of Mutant Proteins by Surface Plasmon Resonance † Academic Article ...
Abstract: It is critical that chromosomal DNA is precisely duplicated during S phase of the eukaryotic cell cycle, with no sections of DNA left unreplicated or replicated more than once. There is a considerable plasticity in this process because cells license many potential replication origins, of which only a small percentage are used in any one cell cycle, with the others remaining dormant. This means that the usage of replication origins can change under different circumstances. For example, dormant replication origins can be activated when replication forks are inhibited to allow timely completion of the replication programme. A recent paper published in Nature by Courbet et al. [1] illustrates this plasticity of replication origin usage and shows that it is associated with longer-term changes to the organization of chromatin loops. The changes to chromatin organization can then directly affect the way that replication origins are used in subsequent cell cycles.The precise duplication of ...
This thesis analyzes the interaction of two DNA-binding proteins with the plus strand replication origin of bacteriophage f1. The origin has a bipartite structure consisting of a required core origin region and an adjacent A +T- rich enhancer sequence that potentiates replication approximately 100-fold. The core origin binds the initiator protein, and the enhancer contains three binding sites for the E. coli integration host factor (IHF). Both activator proteins bend the DNA sequence to which they bind, implying that together they wrap the origin DNA into a higher order structure that is active in initiation. The replication initiator protein of bacteriophage f1 (gene II protein) is a multifunctional protein that participates in DNA replication at a number of levels. The gene II protein binds to the core origin in a novel two-step fashion. The first binding step involves interaction of two gene II protein molecules with an inverted repeat (β- γ) at the center of the core origin to form a binding
The Rif1 protein, originally identified as a telomere-binding factor in yeast, has recently been implicated in DNA replication control from yeast to metazoans. Here, we show that budding yeast Rif1 protein inhibits activation of prereplication complexes (pre-RCs). This inhibitory function requires t …
Polymerase α is an essential enzyme mainly mediating Okazaki fragment synthesis during lagging strand replication. A specific point mutation in Schizosaccharomyces pombe polymerase α named swi7-1, abolishes imprinting required for mating-type switching. Here we investigate whether this mutation confers any genome-wide defects. We show that the swi7-1 mutation renders cells hypersensitive to the DNA damaging agents methyl methansulfonate (MMS), hydroxyurea (HU) and UV and incapacitates activation of the intra-S checkpoint in response to DNA damage. In addition we show that, in the swi7-1 background, cells are characterized by an elevated level of repair foci and recombination, indicative of increased genetic instability. Furthermore, we detect novel Swi1-, -Swi3- and Pol α- dependent alkylation damage repair intermediates with mobility on 2D-gel that suggests presence of single-stranded regions. Genetic interaction studies showed that the flap endonuclease Fen1 works in the same pathway as Pol ...
In eukaryotic cells, firing of DNA replication origins normally does not recur until after M phase. This characteristic is thought to be due to the properties of initiation proteins like Orc, Cdc6, and Mcms. Using formaldehyde cross-linking, we show that Cdc6p and Mcm7p associate specifically with replication origins during G1 but not during G2 in S. cerevisiae. Mcm7ps association with origins depends on Cdc6p. Ectopic expression of Cdc6p enables it to associate with origins during G2, but this fails to recruit Mcm7p. Our data suggest that the loading of Mcm proteins onto origins is regulated by two mechanisms: first, by Cdc6p occupancy, and second, by S- and M-CDKs, whose activity during S, G2, and M phases prevents Mcm loading.
The nuclear matrix is considered to play a significant role in the DNA replication of eukaryotic cells, although immediate evidence for such a job is inadequate still. GAL4-RAD and PEBP2B1. Moreover, AML1/ETO inhibited Py DNA replication stimulated by GAL4-RAD and Rabbit Polyclonal to KCNK15 PEBP2B1. The inhibition was particular for replication mediated by GAL4-RAD and PEBP2B1, and proportional to the amount of lack of these activators in the nuclear matrix, recommending a requirement of nuclear matrix concentrating on in the arousal of Py DNA replication by RAD. These email address details are the first ever to recommend a molecular hyperlink between your initiation of DNA replication as well as the nuclear matrix area. Accumulating proof suggests an participation of transcription elements in the legislation of DNA replication in eukaryotic cells. The polyomavirus (Py) DNA replication program is fantastic for elucidating the assignments of transcription elements in DNA replication, as Py DNA ...
In eukaryotes, initiation of DNA replication requires the assembly of a multiprotein prereplicative complex (pre-RC) at the origins. We recently reported that a WD repeat-containing protein, origin recognition complex (ORC)-associated (ORCA/LRWD1), plays a crucial role in stabilizing ORC to chromati …
def: A heterotetrameric DNA polymerase complex that catalyzes processive DNA synthesis in the absence of PCNA, but is further stimulated in the presence of PCNA. The complex contains a large catalytic subunit and three small subunits, and is best characterized in Saccharomyces, in which the subunits are named Pol2p, Dpb2p, Dpb3p, and Dpb4p. Some evidence suggests that DNA polymerase epsilon is the leading strand polymerase; it is also involved in nucleotide-excision repair and mismatch repair. [PMID:15814431, PMID:9745046 ...
the nucleus, the complexity associated with processing Okazaki fragments is about twice that required to synthesize the leading strand ... Even in small species such as yeast, Okazaki fragment maturation happens approximately a million times during a single round of DNA replication ... Processing of Okazaki fragments is therefore very common and crucial for DNA replication and cell proliferation ...
Pif1, an evolutionarily conserved helicase, negatively regulates telomere length by removing telomerase from chromosome ends. Pif1 has also been implicated in DNA replication processes Such as Okazaki fragment maturation and replication fork pausing. We find that overexpression of Saccharomyces cervisiae results in dose-dependent. growth inhibition. Strong overexpression causes relocalization of the DNA damage response factors Rfa1 and Mre11 into nuclear foci and activation of the Rad53 DNA damage checkpoint kinase, indicating that. the toxicity is caused by accumulation of DNA-damage. We screened the complete set of similar to 4800 haploid gene deletion mutants and found that moderate overexpression of PIF1, which is only mildly toxic oil its own, causes growth defects in strains with Mutations in genes involved in DNA replication and the DNA damage response. Interestingly, we find that telomerase-deficient strains are also sensitive to PIF1 overexpression. Cur data are consistent with a model ...
The maintenance of genome integrity is critical for the suppression of cancer and premature ageing. Only recently has it become appreciated that DNA replication stress is a crucial driver of genomic instability. The timely progression of replisomes can be disrupted by lesions and secondary structures in the template, by bound proteins and by conflicts with the transcription machinery. A prolonged pause of the replisome then exposes single stranded DNA, which, due to its recombinogenic nature, can lead to genome rearrangements, fragile site expression and cell death. Importantly, some cancers present excessive endogenous levels of replication stress, which can be exploited for their clearance. In this conference, we aim to bring together scientists studying DNA replication and repair, with those interested in how DNA damage can influence cancer and ageing.. Key Sessions. ...
Liver regeneration studies with transgenic mice demonstrated that FoxM1B regulates the onset of hepatocyte DNA replication and mitosis by stimulating expression of cell cycle genes (10, 22, 23, 26). In this study, we used Alb-Cre recombinase to generate a hepatocyte-specific deletion of the Foxm1b gene and demonstrated that Foxm1b is required for normal levels of hepatocyte DNA replication and is essential for mitosis in regenerating liver. We found no significant increase in hepatocyte apoptosis in regenerating Alb-Cre Foxm1b−/− liver (data not shown), suggesting that Foxm1b is required for hepatocyte proliferation but not survival. Reduced DNA replication in regenerating Foxm1b−/− hepatocytes coincided with sustained increase in nuclear staining of the Cdk inhibitor p21 protein between 24 and 40 h after PHx. This increase in nuclear p21 levels and a reduction in Cdc25A phosphatase expression resulted in decreased activation of Cdk2 kinase (Fig. 6G). Cyclin E/A-Cdk2 complex cooperates ...
Purpose.: Diabetic retinopathy fails to halt after cessation of hyperglycemic insult, and a vicious cycle of mitochondria damage continues. The aim of our study was to investigate the effect of termination of hyperglycemia on retinal mtDNA replication, and elucidate the mechanism responsible for the continued mtDNA damage. Methods.: Polymerase gamma 1 (POLG1), the catalytic subunit of the mitochondrial DNA replication enzyme, and the damage to the displacement loop region of mtDNA (D-loop) were analyzed in the retina from streptozotocin-diabetic rats maintained in poor glycemic control (PC, glycated hemoglobin ∼11%) or in good glycemic control (GC, glycated hemoglobin ∼6%) for 6 months, or in PC for three months followed by GC for three months (Rev). To understand the mechanism DNA methylation status of POLG1 promoter was investigated by methylation-specific PCR. The key parameters were confirmed in the isolated retinal endothelial cells exposed to high glucose, followed by normal glucose. ...
Break-induced replication (BIR) repairs one-ended double-strand breaks in DNA similar to those formed by replication collapse or telomere erosion, and it has been implicated in the initiation of genome instability in cancer and other human diseases1,2. Previous studies have defined the enzymes that are required for BIR1-5; however, understanding of initial and extended BIR synthesis, and of how the migrating D-loop proceeds through known replication roadblocks, has been precluded by technical limitations. Here we use a newly developed assay to show that BIR synthesis initiates soon after strand invasion and proceeds more slowly than S-phase replication. Without primase, leading strand synthesis is initiated efficiently, but is unable to proceed beyond 30 kilobases, suggesting that primase is needed for stabilization of the nascent leading strand. DNA synthesis can initiate in the absence of Pif1 or Pol32, but does not proceed efficiently. Interstitial telomeric DNA disrupts and terminates BIR
The studies described in this thesis are concerned with the replication of the small icosahedral phage DNAs. Three aspects of DNA multiplication are presented.;(1) An important problem in DNA replication is the mechanism by which polynucleotide chains are initiated de novo. The dnaG encoded protein (Primase) was shown to catalyze that function. The purification of the enzyme is reported as well as the characterization of the reactions catalyzed by it.;(2) The discovery by Eisenberg et al. (Eisenberg, S., Scott, J. F., and Kornberg, A.(1976) Proc.Natl.Acad.Sci.U.S.A.73,3151) that a combination of the purified E. coli rep, SSb, as well as the DNA polymerase III elongation system and (phi)X174 A proteins catalyzed net synthesis of (+)SS(c) DNA when (phi)X174 RFI DNA was used as a template, led to their proposal of a two-step model of (phi)X174 RF (---,) RF DNA replication.;I report here that cell-free extracts of thermosensitive E. coli mutants in the dnaB, dnaC, and dnaG gene synthesize and ...
Cancers can be categorized into two groups: those whose frequency increases with age, and those resulting from errors during mammalian development. The first group is linked to DNA replication through the accumulation of genetic mutations that occur during proliferation of developmentally acquired stem cells that give rise to and maintain tissues and organs. These mutations, which result from DNA replication errors as well as environmental insults, fall into two categories; cancer driver mutations that initiate carcinogenesis and genome destabilizing mutations that promote aneuploidy through excess genome duplication and chromatid missegregation. Increased genome instability results in accelerated clonal evolution leading to the appearance of more aggressive clones with increased drug resistance. The second group of cancers, termed germ cell neoplasia, results from the mislocation of pluripotent stem cells during early development. During normal development, pluripotent stem cells that originate in
For cells to proliferate, the genome must be replicated exactly once per cell cycle in a timely and accurate manner. Making this task difficult are multiple other genomic processes, such as transcription and DNA repair, that are concurrently operating on the same genomic template. Replication initiates at specific loci called replication origins that must undergo a series of protein loadings before they can begin to replicate. Although this loading schedule takes place at all origins, individual origins fire at distinct and conserved times during S-phase. It has been suggested that origin firing schedules are defined by their propensity to attract rate limiting replication factors from limited pools (where origins with higher propensities replicate earlier and origins with lower propensities replicate later). This model has not been validated and, furthermore, the factors determining an origins propensity to attract replication factors remains poorly understood. In higher eukaryotes, ...
The replisome is a complex molecular machine that carries out replication of DNA. The replisome first unwinds double stranded DNA into two single strands. For each of the resulting single strands, a new complementary sequence of DNA is synthesized. The net result is formation of two new double stranded DNA sequences that are exact copies of the original double stranded DNA sequence. In terms of structure, the replisome is composed of two replicative polymerase complexes, one of which synthesizes the leading strand, while the other synthesizes the lagging strand. The replisome is composed of a number of proteins including helicase, RFC, PCNA, gyrase/topoisomerase, SSB/RPA, primase, DNA polymerase III, RNAse H, and ligase. For prokaryotes, each dividing nucleoid (region containing genetic material which is not a nucleus) requires two replisomes for bidirectional replication. The two replisomes continue replication at both forks in the middle of the cell. Finally, as the termination site ...
offered the super-Chk1 strain; D.M. element mini-chromosome maintenance (MCM)3 that limiting origin licensing affects the features of hematopoietic stem cells and the differentiation of rapidly-dividing erythrocyte precursors. Mcm3-deficient erythroblasts display aberrant DNA replication patterns and fail to total maturation, causing lethal anemia. Our results indicate that hematopoietic progenitors are particularly sensitive to replication stress, and full source licensing ensures their right differentiation and features. The process of genomic duplication starts at replication origins, which are licensed in the G1 phase of the cell division cycle, several hours before their activation in S phase. The licensing process is definitely led by the origin recognition complex (ORC), cell division cycle 6 (CDC6) and Cdc10-dependent transcript 1 (CDT1) proteins, which cooperate to engage the mini-chromosome maintenance (MCM) complex with the DNA. MCM, made up by essential subunits MCM2-7, displays DNA ...
1. Kotsantis P, Petermann E, Boulton SJ. Mechanisms of Oncogene-Induced Replication Stress: Jigsaw Falling into Place. Cancer Discov. 2018;8(5):537-55. doi: 10.1158/2159-8290.CD-17-1461 29653955. 2. Gaillard H, García-Muse T, Aguilera A. Replication stress and cancer. Nat Rev Cancer. 2015;15(5):276-89. doi: 10.1038/nrc3916 25907220. 3. Heller R, C., Kang S, Lam W, M., Chen S, Chan C, S., Bell S, P. Eukaryotic origin-dependent DNA replication in vitro reveals sequential action of DDK and S-CDK kinases. Cell. 2011;146(1):80-91. doi: 10.1016/j.cell.2011.06.012 21729781. 4. Sheu YJ, Stillman B. Cdc7-Dbf4 phosphorylates MCM proteins via a docking site-mediated mechanism to promote S phase progression. Mol Cell. 2006;24(1):101-13. doi: 10.1016/j.molcel.2006.07.033 17018296. 5. Tanaka S, Nakato R, Katou Y, Shirahige K, Araki H. Origin association of Sld3, Sld7, and Cdc45 proteins is a key step for determination of origin-firing timing. Curr Biol. 2011;21(24):2055-63. doi: 10.1016/j.cub.2011.11.038 ...
Proper DNA replication and well-timed cell cycle progression are vital to the normal functioning of a cell. Precise coordination between these mechanisms constituent proteins ensures their processivity while safeguarding against DNA damage. The Ctf4 protein is a central member of the replication fork and links the replicative MCM helicase and polymerase [alpha]-primase. In addition, it has been implicated as a member of a complex that promotes replication fork stability, the Fork Protection Complex (FPC). This investigation represents the first phenotypic analysis of the function of the Ctf4 protein within a multicellular organism model. We show that Ctf4 interacts with Polymerase [alpha], MCM2, Psf1, and Psf2. We also demonstrate that knockdown of this central replication fork component via a GAL4-UAS RNAi system results in a lower frequency of mitosis due to an S-phase delay, endoreplication defects, as well as mitotic bridging in early embryonic development ...
Recombinant Human DNA polymerase eta protein is a Wheat germ Full length protein 1 to 414 aa range and validated in WB, ELISA, SDS-PAGE.
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The life cycle of an adenovirus is divided into early and late phases, separated by the DNA replication process. In the early phase, the virus attaches to a cell with its fibers. The penton base protein interacts with the host cell integrins, and the penton is internalized by the host cell through receptor-mediated endocytosis. The penton is disassembled as it is transported to the nucleus, where the viral particle releases its DNA. The viral DNA takes over as terminal protein attached to the end of the DNA strand initiates transcription. The early genes are responsible for making regulatory proteins, which alter the host proteins to prepare for DNA synthesis, activate other virus genes, and provide protection from the hosts immune system. Viral DNA replication now occurs. The late phase begins when the late genes are expressed during DNA replication. These genes produce proteins that are involved in virus particle assembly. The hosts cellular processes are shut down as transport of mRNA to ...