DNA Replication in Eukaryotes

DNA Replication in Eukaryotes tài liệu, giáo án, bài giảng , luận văn, luận án, đồ án, bài tập lớn về tất cả các lĩnh vự...

REVIEW ARTICLE Origins of DNA replication in the three domains of life Nicholas P. Robinson and Stephen D. Bell MRC Cancer Cell Unit, Hutchison MRC Research Centre, Hills Road, Cambridge, UK The origin of origins In a now classic 1963 paper, Jacob, Brenner & Cuzin proposed that, in a manner analogous to the inter- action of trans-acting regulators with cis-acting opera- tors in control of gene expression, an initiator factor would act at a replicator sequence in the chromosome to control and facilitate DNA replication [1]. However, in contrast to the then prevalent models for negative regulation of gene expression, it was proposed that the replication initiator factor would act positively to pro- mote replication at the replicator, or as it is now named, origin of replication. In the following 40 years much has been learnt about the nature of initiators and origins of replication, particularly in simple model systems. However, many of the molecular details of the basis of origin selection remain poorly understood, particularly in higher eukaryotes. Bacteria In bacteria the origin of replication is termed oriC, and typically a single origin exists per bacterial chro- mosome [2]. In Escherichia coli, oriC is located between the gldA and mioC genes. The  250 bp oriC region contains multiple repeated sequences containing a nine base pair consensus element termed the DnaA box [3]. Other bacteria also possess single origins of replication with multiple DnaA boxes although both the precise number and distribution of these boxes vary between species [4]. Interestingly, in many bac- teria the origin of replication is found adjacent to the gene for DnaA itself, suggesting a mechanism for the coordinate control of origin activity and levels of initi- ator proteins [4]. An individual consensus DnaA box is bound by a monomer of the DnaA protein and this interaction induces a sharp bend in the binding site [5]. However, in natural bacterial origins there are multiple DnaA boxes and these orchestrate complex cooper- ative binding events to DnaA boxes with varying degrees of conformity to the consensus sequence. A particularly interesting ramification of this is that a DnaA box with poor conservation to the consensus may not be able to bind DnaA on its own. However, binding to this ‘weak’ site can be facilitated by binding of DnaA to an adjacent high affinity consensus site [4]. Keywords Cdc6; DnaA; DNA Replication; MCM; ORC Correspondence S. D. Bell, MRC Cancer Cell Unit, Hutchison MRC Research Centre, Hills Road, Cambridge, CB2 2XZ, UK Fax: +44 1223 763296 Tel: +44 1223 763311 E-mail: sb419@hutchison-mrc.cam.ac.uk (Received 8 April 2005, revised 11 May 2005, accepted 13 May 2005) doi:10.1111/j.1742-4658.2005.04768.x Replication of DNA is essential for the propagation of life. It is somewhat surprising then that, despite the vital nature of this process, cellular organ- isms show a great deal of variety in the mechanisms that they employ to ensure appropriate genome duplication. This diversity is manifested along classical evolutionary lines, with distinct combinations of replicon architec- ture and replication proteins being found in the three domains of life: the Bacteria, the Eukarya and the Archaea. Furthermore, although there are mechanistic parallels, even within a given domain of life, the way origins of replication are defined shows remarkable variation. Abbreviations ACS, DNA Replication in Eukaryotes DNA Replication in Eukaryotes Bởi: OpenStaxCollege Eukaryotic genomes are much more complex and larger in size than prokaryotic genomes The human genome has three billion base pairs per haploid set of chromosomes, and billion base pairs are replicated during the S phase of the cell cycle There are multiple origins of replication on the eukaryotic chromosome; humans can have up to 100,000 origins of replication The rate of replication is approximately 100 nucleotides per second, much slower than prokaryotic replication In yeast, which is a eukaryote, special sequences known as Autonomously Replicating Sequences (ARS) are found on the chromosomes These are equivalent to the origin of replication in E coli The number of DNA polymerases in eukaryotes is much more than prokaryotes: 14 are known, of which five are known to have major roles during replication and have been well studied They are known as pol α, pol β, pol γ, pol δ, and pol ε The essential steps of replication are the same as in prokaryotes Before replication can start, the DNA has to be made available as template Eukaryotic DNA is bound to basic proteins known as histones to form structures called nucleosomes The chromatin (the complex between DNA and proteins) may undergo some chemical modifications, so that the DNA may be able to slide off the proteins or be accessible to the enzymes of the DNA replication machinery At the origin of replication, a pre-replication complex is made with other initiator proteins Other proteins are then recruited to start the replication process ([link]) A helicase using the energy from ATP hydrolysis opens up the DNA helix Replication forks are formed at each replication origin as the DNA unwinds The opening of the double helix causes over-winding, or supercoiling, in the DNA ahead of the replication fork These are resolved with the action of topoisomerases Primers are formed by the enzyme primase, and using the primer, DNA pol can start synthesis While the leading strand is continuously synthesized by the enzyme pol δ, the lagging strand is synthesized by pol ε A sliding clamp protein known as PCNA (Proliferating Cell Nuclear Antigen) holds the DNA pol in place so that it does not slide off the DNA RNase H removes the RNA primer, which is then replaced with DNA nucleotides The Okazaki fragments 1/5 DNA Replication in Eukaryotes in the lagging strand are joined together after the replacement of the RNA primers with DNA The gaps that remain are sealed by DNA ligase, which forms the phosphodiester bond Telomere replication Unlike prokaryotic chromosomes, eukaryotic chromosomes are linear As you’ve learned, the enzyme DNA pol can add nucleotides only in the 5' to 3' direction In the leading strand, synthesis continues until the end of the chromosome is reached On the lagging strand, DNA is synthesized in short stretches, each of which is initiated by a separate primer When the replication fork reaches the end of the linear chromosome, there is no place for a primer to be made for the DNA fragment to be copied at the end of the chromosome These ends thus remain unpaired, and over time these ends may get progressively shorter as cells continue to divide The ends of the linear chromosomes are known as telomeres, which have repetitive sequences that code for no particular gene In a way, these telomeres protect the genes from getting deleted as cells continue to divide In humans, a six base pair sequence, TTAGGG, is repeated 100 to 1000 times The discovery of the enzyme telomerase ([link]) helped in the understanding of how chromosome ends are maintained The telomerase enzyme contains a catalytic part and a built-in RNA template It attaches to the end of the chromosome, and complementary bases to the RNA template are added on the 3' end of the DNA strand Once the 3' end of the lagging strand template is sufficiently elongated, DNA polymerase can add the nucleotides complementary to the ends of the chromosomes Thus, the ends of the chromosomes are replicated 2/5 DNA Replication in Eukaryotes The ends of linear chromosomes are maintained by the action of the telomerase enzyme Telomerase is typically active in germ cells and adult stem cells It is not active in adult somatic cells For her discovery of telomerase and its action, Elizabeth Blackburn ([link]) received the Nobel Prize for Medicine and Physiology in 2009 Elizabeth Blackburn, 2009 Nobel Laureate, is the scientist who discovered how telomerase works (credit: US Embassy Sweden) Telomerase and Aging Cells that undergo cell division continue to have their telomeres shortened because most somatic cells not make telomerase This essentially means that telomere shortening 3/5 DNA Replication in Eukaryotes is associated with aging With the advent of modern medicine, preventative health care, and healthier lifestyles, the human life span has increased, and there is an increasing demand for people to look younger and have a better quality of life ...Initiation of JC virus DNA replication in vitro by human and mouse DNA polymerase a-primase Richard W. P. Smith 1, * and Heinz-Peter Nasheuer 1,2 1 Abteilung Biochemie, Institut fu ¨ r Molekulare Biotechnologie, Jena, Germany; 2 National University of Ireland, Galway, Department of Biochemistry, Galway, Ireland Host species specificity of the polyomaviruses simian virus 40 (SV40) and mouse polyomavirus (PyV) has been shown to be determined by the host DNA polymerase a-primase complex involved in the initiation of both viral and host DNA replication. Here we demonstrate that DNA repli- cation of the related human pathogenic polyomavirus JC virus (JCV) can be supported in vitro by DNA polymerase a-primase of either human or murine origin indicating that the mechanism of its strict species specificity differs from that of SV40 and PyV. Our results indicate that this may be due to differences in the interaction of JCV and SV40 large T antigens with the DNA replication initiation complex. Keywords: DNA replication; initiation; DNA polymerase a-primase; species specificity; polyomavirus. Polyomavirus DNA replication has served as a model system to study eukaryotic DNA replication [1,2]. JC virus (JCV) belongs to the polyomavirus family and is the causative agent of progressive multifocal leukoencephalo- pathy in immunocompromised humans (reviewed in [3–7]). JCV exhibits a highly restricted host range and this species specificity appears to be governed by host encoded DNA replication factors as hamster glial cells, which support viral early gene transcription, nevertheless fail to replicate JCV DNA [8]. JCV is closely related to simian virus 40 (SV40) and to mouse polyomavirus (PyV), both of which show clear species specificities as lytic infection is limited to primate and mouse cells, respectively [9]. The species specificities of both SV40 and PyV are regulated at the level of initiation of DNA replication [10], a process that has been extensively studied both in vivo and in vitro owing to the development of cell-free DNA replication systems [2,11–16]. Polyomavirus DNA replication is carried out by the host cell machinery supplemented with a single essential viral protein, large T antigen (TAg), which recognizes and partially unwinds the viral replication origin, recruits host proteins such as replication protein A (RPA) and DNA polymerase a-primase, and functions as the replicative helicase [2,17,18]. Species specificity of both SV40 and PyV DNA replication can be reproduced in vitro using DNA carrying the viral core origin and purified replication enzymes [14,19–21]. For both SV40 and PyV it has been clearly demonstrated that the host factor responsible for species specificity is DNA polymerase a-primase, which initiates DNA replication in all eukaryotes [19,22–27]. DNA polymerase a-primase consists of four subunits with apparent molecular masses of 180, 68, 58 and 48 kDa of which the largest and smallest subunits are a DNA polymerase and a primase, respectively [28–30]. SV40 DNA replication in vitro was recently shown to require a functional interaction between the SV40 TAg and the C-terminus of the p180 subunit of human DNA poly- merase a-primase [26]. The genome of JCV is 69% homologous to that of REVIE W Open Access Initiation of bacteriophage T4 DNA replication and replication fork dynamics: a review in the Virology Journal series on bacteriophage T4 and its relatives Kenneth N Kreuzer 1* , J Rodney Brister 2 Abstract Bacteriophage T4 initiates DNA replication from specialized structures that form in its genome. Immediately after infection, RNA-DNA hybrids (R-loops) occur on (at least some) replication origins, with the annealed RNA serving as a primer for leading-strand synthesis in one direction. As the infection progresses, replication initiation becomes dependent on recombination proteins in a process called recombination-dependent replication (RDR). RDR occurs when the replication machinery is assembled onto D-loop recombination intermediates, and in this case, the invading 3’ DNA end is used as a primer for leading strand synthesis. Over the last 15 years, these two modes of T4 DNA replication initiation have been studied in vivo using a variety of approaches, including replication of plasmids with segments of the T4 genome, analysis of replication intermediates by two-dimensional gel electrophoresis, and genomic approaches that measure DNA copy number as the infection progresses. In addition, biochemical approaches have reconstituted replication from origin R-loop structures and have clarified some detailed roles of both replication and recombination proteins in the process of RDR and related pathways. We will also discuss the parallels between T4 DNA replication modes and similar events in cellular and eukaryotic organelle DNA replication, and close with some current questions of interest concerning the mechanisms of replication, recombination and repair in phage T4. Introduction Studies during the last 15 years have provided strong evidence that T4 DNA replication initiates from s pecia- lized structures, namely R-loops for origin-dependent replication and D-loops for recombination-dependent replication (RDR). The roles of many of the T4 replica- tion and recombination proteins in these processes are now understood in detail, and the transition from o ri- gin-dependent replication to RDR has been ascribed to both down-regulation of origin transcripts and activa- tion of the UvsW helicase, which unwinds origin R-loops. One of the interesting themes that emerged in studies of T4 DNA metabolism is the extensive overlap between different modes of replication initiation and the processes of DNA repair, recombination, and replication fork restart. As discussed in more detail below, the distinction between origin-d ependent and recombination-dependent replication is blurred by the involvement of recombina- tion proteins in certain aspects of origin replication. Another example of overlap is the finding that repair o f double-strand breaks (DSBs) in phage T4 infections occurs by a mechanism that is very closely related to the process of RDR. The close interconnections between recombination and replication are not unique to phage T4 - it has become obvious that the process of homolo- gous recombination and particular recombination pro- teins play critical roles in cellular DNA replication and the maintenance of genomic stability [1-4]. * Correspondence: kenneth.kreuzer@duke.edu 1 Department of Biochemistry, Duke University Medical Center, Durham, NC 27710 USA Full list of author information is available at the end of the article Kreuzer and Brister Virology Journal 2010, 7:358 http://www.virologyj.com/content/7/1/358 © 2010 Kreuzer and Brister; lic ensee BioMed Central Ltd. This is an Ope n Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unre stricted use, distribution, and reproduction in any medium, provi ded the original work is properly cite d. Origin-dependent replication Most chromosomes that have been studied include defined loci where DNA synthesis is initiated. Such ori- gins of replication have unique physical Structural analysis of bacteriophage T4 DNA replication: a review in the Virology Journal series on bacteriophage T4 and its relatives Mueser et al. Mueser et al. Virology Journal 2010, 7:359 http://www.virologyj.com/content/7/1/359 (3 December 2010) REVIEW Open Access Structural analysis of bacteriophage T4 DNA replication: a review in the Virology Journal series on bacteriophage T4 and its relatives Timothy C Mueser 1* , Jennifer M Hinerman 2 , Juliette M Devos 3 , Ryan A Boyer 4 , Kandace J Williams 5 Abstract The bacteriophage T4 encodes 10 proteins, known collectively as the replisome, that are responsible for the repli- cation of the phage genome. The replisomal proteins can be subdivided into three activities; the replicase, respon- sible for duplicating DNA, the primosomal prot eins, responsible for unwinding and Okazaki fragment initiation, and the Okazaki repair proteins. The replicase includes the gp43 DNA polymerase, the gp45 processivity clamp, the gp44/62 clamp loader complex, and the gp32 single-stranded DNA binding protein. The primosomal proteins include the gp41 hexameric helicase, the gp61 primase, and the gp59 helicase loading protein. The RNaseH, a 5’ to 3’ exonuclease and T4 DNA ligase comprise the activities necessary for Okazaki repair. The T4 provides a model sys- tem for DNA replication. As a consequence, significant effort has been put forth to solve the crystallographic struc- tures of these replisomal proteins. In this review, we discuss the structures that are available and provide comparison to related proteins when the T4 structures are unavailable. Three of the ten full-length T4 replisomal proteins have been determined; the gp59 helicase loading protein, the RNase H, and the gp45 processivity clamp. The core of T4 gp32 and two proteins from the T4 related phage RB69, the gp43 polymerase and the gp45 clamp are also solved. The T4 gp44/62 clamp loader has not been crystallized bu t a compa rison to the E. coli gamma complex is provided. The structures of T4 gp41 helicase, gp61 primase, and T4 DNA ligase are unknown, structures from bacteriophage T7 proteins are discussed instead. To better understand the functionality of T4 DNA replication, in depth structural analysis will require complexes between proteins and DNA substrates. A DNA primer template bound by gp43 polymerase, a fork DNA substrate bound by RNase H, gp43 polymerase bound to gp32 protein, and RNase H bound to gp32 have been crystallographically determined. The preparation and crystallization of complexes is a significant challenge. We discuss alternate approaches, such as small angle X-ray and neutron scat- tering to generate molecular envelopes for modeling macromolecular assemblies. Bacteriophage T4 DNA Replication The semi-conservative, semi-discontinuous process of DNA replicat ion is conserved in all life forms. The par- ental anti-parallel DNA strands are separated and copied following hydrogen bonding rules for the keto form of each base as proposed by Watson and Crick [1]. Pro- gen y cel ls therefore inherit one parental strand and one newly synthesized strand comprising a new duplex DNA genome. Protection of the integrity of genom ic DNA is vital to the survival of all organisms. In a masterful dichotomy, the genome encodes proteins that are also the caretakers of the genome. RNA can be viewed as the evolutionary center of this juxtaposition of DNA and protein. Viruses have also played an intriguing role in the evolutionary process, perhaps from t he inception of DNA in primordial times to modern day lateral gene transfer. Simply defined, viruses are encapsulated geno- mic information. Possibly an ancient encapsulated virus became the nucleus of an ancient prokaryote, a symbio- tic relationship comparable to mitochondria, as some have recently proposed [2-4]. This early relationship has evolved into highly complex eukaryotic cellular pro- cesses of replication, recombination and repair requiring multiple signaling pathways BioMed Central Page 1 of 15 (page number not for citation purposes) Retrovirology Open Access Research APOBEC3G induces a hypermutation gradient: purifying selection at multiple steps during HIV-1 replication results in levels of G-to-A mutations that are high in DNA, intermediate in cellular viral RNA, and low in virion RNA Rebecca A Russell 1 , Michael D Moore 2 , Wei-Shau Hu 2 and Vinay K Pathak* 1 Address: 1 Viral Mutation Section, HIV Drug Resistance Program, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland 21702, USA and 2 Viral Recombination Section, HIV Drug Resistance Program, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland 21702, USA Email: Rebecca A Russell - rebecca.russell@path.ox.ac.uk; Michael D Moore - kenny.moore@path.ox.ac.uk; Wei-Shau Hu - whu@ncifcrf.gov; Vinay K Pathak* - vpathak@ncifcrf.gov * Corresponding author Abstract Background: Naturally occurring Vif variants that are unable to inhibit the host restriction factor APOBEC3G (A3G) have been isolated from infected individuals. A3G can potentially induce G-to- A hypermutation in these viruses, and hypermutation could contribute to genetic variation in HIV- 1 populations through recombination between hypermutant and wild-type genomes. Thus, hypermutation could contribute to the generation of immune escape and drug resistant variants, but the genetic contribution of hypermutation to the viral evolutionary potential is poorly understood. In addition, the mechanisms by which these viruses persist in the host despite the presence of A3G remain unknown. Results: To address these questions, we generated a replication-competent HIV-1 Vif mutant in which the A3G-binding residues of Vif, Y 40 RHHY 44 , were substituted with five alanines. As expected, the mutant was severely defective in an A3G-expressing T cell line and exhibited a significant delay in replication kinetics. Analysis of viral DNA showed the expected high level of G- to-A hypermutation; however, we found substantially reduced levels of G-to-A hypermutation in intracellular viral RNA (cRNA), and the levels of G-to-A mutations in virion RNA (vRNA) were even further reduced. The frequencies of hypermutation in DNA, cRNA, and vRNA were 0.73%, 0.12%, and 0.05% of the nucleotides sequenced, indicating a gradient of hypermutation. Additionally, genomes containing start codon mutations and early termination codons within gag were isolated from the vRNA. Conclusion: These results suggest that sublethal levels of hypermutation coupled with purifying selection at multiple steps during the early phase of viral replication lead to the packaging of largely unmutated genomes, providing a mechanism by which mutant Vif variants can persist in infected individuals. The persistence of genomes containing mutated gag genes despite this selection pressure indicates that dual infection and complementation can result in the packaging of hypermutated genomes which, through recombination with wild-type genomes, could increase viral genetic variation and contribute to evolution. Published: 13 February 2009 Retrovirology 2009, 6:16 doi:10.1186/1742-4690-6-16 Received: 23 December 2008 Accepted: 13 February 2009 This article is available from: http://www.retrovirology.com/content/6/1/16 © 2009 Russell et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Retrovirology 2009, 6:16 http://www.retrovirology.com/content/6/1/16 Page 2 of 15 (page number not for citation purposes) Background The APOBEC3 proteins APOBEC3G (A3G) and APOBEC3F (A3F) are potent inhibitors of Vif-deficient HIV-1 [1-5]. However, in the presence of HIV-1 Vif the A3G and A3F proteins are targeted for proteasomal ... Strand elongation DNA pol III Pol δ, pol ε Sliding clamp Sliding clamp PCNA 4/5 DNA Replication in Eukaryotes Section Summary Replication in eukaryotes starts at multiple origins of replication The.. .DNA Replication in Eukaryotes in the lagging strand are joined together after the replacement of the RNA primers with DNA The gaps that remain are sealed by DNA ligase, which... synthesized in short stretches called Okazaki fragments The RNA primers are replaced with DNA nucleotides; the DNA remains one continuous strand by linking the DNA fragments with DNA ligase The