Chapter 6: DNA Replication and Telomere Maintenance.
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Transcript of Chapter 6: DNA Replication and Telomere Maintenance.
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Chapter 6:
DNA Replication and Telomere Maintenance
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It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.
James D. Watson and Francis Crick, Nature (1953), 171:737
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6.1 Introduction
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DNA replication involves: • The melting apart of the two strands of the
double helix followed by the polymerization of new complementary strands.
• Decisions of when, where, and how to initiate replication to ensure that only one complete and accurate copy of the genome is made before a cell divides.
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6.2 Early insights into the mode of bacterial DNA replication
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Three possible modes of replication hypothesized based on Watson and Crick’s model:
•Semiconservative
•Conservative
•Dispersive
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The Meselson-Stahl experiment
• 1958 experiment designed to distinguish between semiconservative, conservative, and dispersive replication.
• Results were consistent only with semiconservative replication.
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Visualization of replicating bacterial DNA
• Semiconservative mechanism of DNA replication visually verified by J. Cairns in 1963 using autoradiography.
• Bidirectional replication of the E. coli chromosome.
• One origin of replication.
• Replication intermediates are termed theta () structures.
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6.3 DNA polymerases are the enzymes that catalyze DNA
synthesis from 5′ to 3′
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DNA polymerases
• Can only add nucleotides in the 5′→3′ direction.
• Cannot initiate DNA synthesis de novo.
• Require a primer with a free 3′-OH group at the end.
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• Deoxynucleoside 5′ triphosphates (dNTPs) are added one at a time to the 3′ hydroxyl end of the DNA chain.
• The dNTP added is determined by complementary base pairing.
• As phosphodiester bonds form, the two terminal phosphates are lost, making the reaction essentially irreversible.
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Problem • DNA polymerases can only add nucleotides
from 5′→3′ but, the two strands of the double helix are antiparallel.
Solution• Semidiscontinuous replication.
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Semidiscontinuous DNA replication
• Major form of replication in eukaryotic nuclear DNA, some viruses, and bacteria.
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Leading strand synthesis is continuous
• Once primed, continuous replication is possible on the 3′→ 5′ template strand (leading strand).
• Leading strand synthesis occurs in the same direction as movement of the replication fork.
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Leading strand synthesis is continuous
• Discontinuous replication occurs on the 5′→3′ template strand (lagging strand).
• DNA is copied in short segments called “Okazaki fragments” moving in the opposite direction to the replication fork.
• Repetition of primer synthesis and formation of Okazaki fragments.
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Synthesis of both strands occurs concurrently
• Nucleotides are added to the leading and lagging strands at the same time and rate.
• Two DNA polymerases, one for each strand.
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• Fundamental features of DNA replication are conserved from E. coli to humans.
• 1984: A cell-free system allowed scientists to make progress in studying replication in eukaryotic cells.
• Model system: Simian virus 40 (SV40) replication.
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6.4 Multi-protein machines mediate bacterial DNA
replication
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Bacterial DNA polymerases have multiple functions
DNA polymerase I• Primer removal, gap filling between Okazaki fragments,
and nucleotide excision repair pathway.
• Two subunits: Klenow fragment has 5′→3′ polymerase activity; other subunit has both 3′→5′ and 5′→3′ exonuclease activity.
• Unique ability to start replication at a nick in the DNA sugar-phosphate backbone.
• Used extensively in molecular biology research.
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DNA polymerase III• Main replicative polymerase.
DNA polymerase II• Involved in DNA repair mechanisms.
DNA polymerases IV and V• Mediate translesion synthesis (see Chapter 7).
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Initiation of replication• An origin of replication is a site on chromosomal DNA
where a bidirectional replication fork initiates or “fires.”
• Most bacteria have a single, well-defined origin (e.g. oriC in E. coli)
• Some Archaea have as many as three origins (e.g. Sulfolobus).
• Usually A-T rich.
• In E. coli the initiator protein DnaA can only bind to negatively supercoiled origin DNA.
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Major parts of this multi-protein machine are:
• A helicase which unwinds the parental double helix.
• Two molecules of DNA polymerase III.
• A primase that initiates lagging strand Okazaki fragments.
Replication is mediated by the replisome
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Major parts of this multi-protein machine, cont:
• Two sliding clamps that tether DNA polymerase to the DNA.
• A clamp loader that uses ATP to open and close the sliding clamps around the DNA.
• Single-strand DNA binding proteins (SSB) that protect the DNA from nuclease attack.
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Lagging strand synthesis by the replisome:
• As the replication fork advances, the lagging strand polymerase remains associated with the replisome forming a loop.
• The loop grows until the Okazaki fragment is complete.
• DNA polymerase III is released.
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• New clamps are assembled; DNA polymerase III hops aboard to make the next Okazaki fragment.
• This process occurs around the circular genome until the replication forks meet.
• In E. coli, the replication forks meet at a terminus region containing sequence-specific replication arrest sites.
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• DNA polymerase I removes the RNA primers and replaces them with complementary dNTPs.
• DNA ligase catalyzes the formation of a phosphodiester bond between adjacent Okazaki fragments.
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Movement of the replication fork machinery results in:
• Positive supercoiling ahead of the fork.
• Negative supercoiling in the wake of the fork.
• Torsional strain that could inhibit fork movement is relieved by DNA topoisomerase.
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Topoisomerases relax supercoiled DNA
Topoisomers are forms of DNA that have the same sequence but differ in:
• linkage number
• mobility in an electrophoresis gel
Topoisomerases are enzymes that convert (isomerize) one topoisomer of DNA to another by changing the linking number (L).
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Type I topoisomerases cause transient single-stranded breaks in DNA
• Type 1A only relax negative supercoils.
• Type 1B can relax both negative and positive supercoils.
• Do not require ATP.
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Type II topoisomerases cause transient double-stranded breaks in DNA
• Relax both negative and positive supercoils.
• Unknot or decatenate entangled DNA molecules.
• Usually ATP-dependent.
• Bacterial “gyrase” can introduce negative supercoils.
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Is leading strand synthesis really continuous?
• DNA polymerase III can be blocked by a damaged site on the template DNA.
• Sometimes DNA polymerase collides with RNA polymerase and is stalled.
• In both cases, replication can be jumpstarted on the leading strand by formation of a new primer at the replication fork.
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6.5 Multi-protein machines trade places during eukaryotic DNA
replication
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Eukaryotic origins of replication
• Internal sites on linear chromosomes.
• Mice have 25,000 origins, spanning ~150 kb each.
• Humans have 10,000 to 100,000 origins.
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• In the budding yeast Saccharomyces cerevisiae there is a consensus sequence called an autonomous replicating sequence (ARS).
• Mammalian origin sequences are usually AT rich but lack a consensus sequence.
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Mapping eukaryotic DNA replication origins
• Analysis by two-dimensional agarose gel electrophoresis.
• Other techniques allow detection of the start site for DNA synthesis at the nucleotide level.
• Data suggest that there is a single defined start point.
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Selective activation of origins of replication
• The overall rate of replication is largely determined by the number of origins used and the rate at which they initiate.
• During early embryogenesis, origins are uniformly activated.
• At the mid-blastula transition, replication becomes restricted to specific origin sites.
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Replication factories
• Replication forks are clustered in “replication factories.”
• Forty to many hundreds of forks are active in each factory.
• Shown by a pulse-chase technique using BrdU labeling of cells in S-phase and detection with anti-BrdU antibodies.
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Histone removal at origins of replication
• Histone modification and chromatin remodeling factors.
• Disassembly of the nucleosomes.
• Template DNA is accessible to the replication machinery.
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Prereplication complex formation and replication licensing
• DNA replication is restricted to S phase of the cell cycle.
• Origin selection is a separate step from initiation.
• Formation of a prereplication complex.
• Prevents overreplication of the genome.
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Assembly of the origin recognition complex
• The ATP-dependent origin recognition complex (ORC) binds origin sequences.
• Recruits Cdc6 and Mcm proteins.
• The SV40 T antigen functions as a viral ORC.
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The naming of genes involved in DNA replication
• Many genes first characterized in the yeast Saccharomyces cerevisiae.
• Mutations that affect the cell cycle were isolated as conditional, temperature-sensitive mutants.
• At the permissive temperature, the gene product can function.
• At the restrictive temperature, mutant yeast accumulate at a particular point in the cell cycle.
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Assembly of the replication licensing complex
• In association with Cdc6 and Cdt1, ORC loads the licensing protein complex, Mcm2-7.
• Mcm2-7 is a hexameric complex with helicase activity.
• Only licensed origins containing Mcm2-7 can initiate a pair of replication forks.
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• ATP hydrolysis by ORC stimulates prereplication complex assembly.
• Prereplication complex assembly is inhibited when ORC is bound by a nonhydrolyzable analog of ATP (ATP-S)
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Regulation of the replication licensing system by CDKs
• Replication licensing is regulated by the activity levels of cyclin-dependent kinases (CDKs).
• For catalysis, CDKs must associate with a cyclin.
• Cyclins accumulate gradually during interphase and are abruptly destroyed during mitosis.
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• ORC, Cdc6, Cdt1, and Mcm2-7 are downregulated by high CDK activity.
• The mode of downregulation differs for each protein.
• No further Mcm2-7 can be loaded onto origins in S phase, G2, and early mitosis when CDK activity is high.
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Duplex unwinding at replication forks
• DNA helicases are enzymes that use the energy of ATP to melt the DNA duplex.
• They catalyze the transition from double-stranded to single-stranded DNA in the direction of the moving replication fork.
• Mcm2-7 helicase is bound to the leading strand template and moves 3′→5′.
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RNA priming of leading and lagging strand DNA synthesis
• In eukaryotes, the RNA primer is synthesized by DNA polymerase (pol) and its associated primase activity.
• The pol /primase enzyme synthesizes a short strand of 10 bases of RNA, followed by 20-30 bases of initiator DNA (iDNA).
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Polymerase switching
• A key feature of the replication process is the ordered hand-off, or “trading places”, from one protein complex to another.
• Polymerase switching: The hand-off of the DNA template from one polymerase to another.
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Elongation of leading strands and lagging strands
At least 14 different eukaryotic DNA polymerases• Chromosomal DNA replication
DNA pol , pol , pol
• Mitochondrial DNA replication
DNA pol
• Repair processes
All the rest (Chapter 7)
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• Leading strand: switch from DNA polymerase to pol
• Lagging strand: switch from pol to pol
• Polymerase switching is regulated by PCNA.
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• Once DNA pol is recruited to the leading strand, synthesis is continuous.
• Lagging strand synthesis requires repeated cycles of polymerase switching from DNA pol to pol .
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PCNA: a sliding clamp with many protein partners
• PCNA: proliferating cell nuclear antigen.
• Plays an important role in many cellular processes.
• In DNA replication, acts as a sliding clamp to increase DNA polymerase processivity.
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PCNA structure
• PCNA is a ring-shaped trimer.
• In the presence of ATP, the clamp loader RFC opens the trimer and passes DNA into the ring and then reseals it.
• RFC locks onto DNA in a screw-cap-like arrangement.
• The RFC spiral matches the minor grooves of the DNA double helix.
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Proofreading
• Replicative polymerases are high fidelity but not perfect: 10-4 to 10-5 errors per base pair.
• Proofreading exonuclease activity reduces the error rate to 10-7 to 10-8 errors per base pair.
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• DNA polymerase has a hand-shaped structure.
• 5′→3′ polymerase activity is within the fingers and thumb.
• 3′→5′ exonuclease activity is at the base of the palm.
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Nucleotide selectivity largely depends on the geometry of Watson-Crick base pairs
• The abnormal genometry of mismatched base pairs results in steric hindrance at the active site.
• Base-base hydrogen bonding also contributes to fidelity.
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Maturation of DNA nascent strands
• RNA primer removal.
• Gap fill-in.
• Joining of Okazaki fragments on the lagging strand.
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Two different pathways proposed for RNA primer removal:
1.Ribonuclease H1 nicks the RNA primer and the primer is degraded by FEN-1 (flap endonuclease 1)
2.DNA pol causes strand displacement and FEN-1 removes the entire RNA containing 5′ “flap.”
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• FEN-1 is a structure-specific 5′ nuclease with both exonuclease and endonuclease activity.
• PCNA-coordinated rotary handoff mechanism of DNA from DNA pol to FEN-1.
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Gap fill-in and joining of the Okazaki fragments
• The remaining gaps left by primer removal are filled in by DNA polymerase or .
• End product is a nicked double-stranded DNA.
• Nicks are sealed by DNA ligase I.
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• In association with PCNA, DNA ligase I joins the Okazaki fragments by catalyzing the formation of new phosphodiester bonds.
• DNA binding domain encircles DNA and interacts with the minor groove.
• Stabilizes distorted structure with A-form helix upstream of the gap.
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Histone deposition
• Nucleosomes re-form within approximately 250 bp behind the replication fork.
• Chromatin assembly factor 1 (CAF-1) brings histones to the DNA replication fork in association with PCNA.
• Histones H3 and H4 form a complex and are deposited first, followed by two histone H2A-H2B dimers.
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Two models for nucleosome assembly after DNA replication:
• The tetrameric model: histones H3 and H4 are deposited on DNA as parental or newly synthesized tetramers.
• The dimeric model: histones H3 and H4 are deposited on DNA as parental or newly synthesized dimers.
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Topoisomerase untangles the newly synthesized DNA
• In eukaryotes, replication continues until one fork meets a fork from the adjacent replicon.
• The progeny DNA molecules remain intertwined.
• Toposiomerase II is required to resolve the two separate progeny genomes.
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Topoisomerase-targeted anti-cancer drugs
• Target rapidly growing cells.
• Act either as inhibitors of at least one step in the catalytic cycle or as poisons.
• Topoisomerase I is a target for a number of anti-cancer drugs.
e.g. camptothecin
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6.6 Alternative modes of circular DNA replication
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Rolling circle replication
• Multiplication of many bacterial and eukaryotic viral DNAs, bacterial F factors during mating, and in certain cases of gene amplification.
• A phosphodiester bond is broken in one of the strands of a circular DNA.
• Synthesis of a new circular strand occurs by addition of dNTPs to the 3′ end using the intact strand as a template.
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Phage X174 replication
• When one round of replication is complete, a full-length, single-stranded circle of DNA is released.
• The process repeats over and over to yield many copies of the phage genome.
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Xenopus oocyte ribosomal DNA (rDNA) amplification
• In oocytes of the South African clawed frog, rDNA is amplified to form extrachromosomal circles.
• The double stranded DNA replicates to form many rDNA repeat units in length, then one repeat’s worth is cleaved off by a nuclease.
• DNA ligase joins the end to form a circle.
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Models for organelle DNA replication
• There is no consensus on the mode of replication of organelle DNA.
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Models for chloroplast DNA (cpDNA) replication
• A subject of debate particularly since there is controversy over whether cpDNA is linear or circular.
• Some evidence for a strand displacement model.
• Other models include a theta replication intermediate, rolling circle replication, and recombination-dependent replication.
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Models for mitochondrial DNA (mtDNA) replication
• DNA polymerase is used exclusively for mtDNA replication.
• Two models for replication have been proposed:
1. The strand displacement model
2. The strand coupled model
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Strand displacement model:
• The most widely accepted model.
• Replication is unidirectional round the circle and there is one replication fork for each strand.
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Strand coupled model:
• Semidiscontinous, bidirectional replication.
• Synthesis of Okazaki fragments on the lagging strand.
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RNase MRP and cartilage-hair hypoplasia
• RNase MRP is an RNP that plays a role in:
– Cleavage of RNA primers in mtDNA replication.
– Nucleolar processing of pre-rRNA.
• Mutations in the RNA component cause a rare form of dwarfism called cartilage-hair hypoplasia.
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6.7 Telomere maintenance: the role of telomerase in DNA
replication, aging, and cancer
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The end replication problem
• When the final primer is removed from the lagging strand, an 8-12 nucleotide region is left unreplicated.
• Predicts that chromosomes would get shorter with each round of replication.
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Telomeres
• Eukaryotic chromosomes end with tandem repeats of a simple G-rich sequence.
Humans: TTAGGG
Tetrahymena: TTGGGG
• Seal the ends of chromosomes.
• Confer stability by keeping the chromosomes from ligating together.
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Solution to the end replication problem
• Solution reported by Carol Greider and Elizabeth Blackburn in 1985.
• Studied Tetrahymena thermophila, a single-celled eukaryote with over 40,000 telomeres.
• Discovered the enzyme telomerase.
• Shared the 2009 Nobel prize in physiology or medicine with Jack Szostak.
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• Telomerase is a ribonucleoprotein (RNP) complex with reverse transcriptase activity.
• Contains an essential RNA component that provides the template for telomere repeat synthesis.
– RNA: Telomerase RNA component (TERC)– Protein: Telomerase reverse transcriptase
(TERT)
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Maintenance of telomeres by telomerase
• Telomerase elongates the 3′ end of the template for the lagging strand (G-rich overhang).
• A pseudoknot in telomerase RNA is important for processivity of repeat additions.
• Repeated translocation and elongation steps results in chromosome ends with an array of tandem repeats.
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• Elongation of the shorter lagging strand (C-rich strand) occurs by the normal replication machinery.
• Alternatively, the 3′ overhang folds into a t-loop structure, which prevents telomerase access.
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Other modes of telomere maintenance
• Telomerase-mediated telomere maintenance is widespread among eukaryotes from ciliates to yeast to humans.
• A striking exception is the fruitfly Drosophila melanogaster, which maintains telomeres by the addition of large retrotransposons.
• In human and fungi, telomeres can also be maintained by a recombination-based mechanism.
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Regulation of telomerase activity
• Telomere length regulation involves the accessibility of telomeres to telomerase.
• Length control involves a number of factors including:– Proteins POT1, TRF1, and TRF2– t-loop formation
• A telomere-specific protein complex forms called shelterin.
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Model for length control
• POT1 binds to the TRF1 complex on the double-stranded portion of telomeres.
• TRF1 (and TRF2) “count” the number of G-rich repeats.
• Transfer of POT1 to the 3′ overhang.
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When the telomere is long enough:
• POT1 levels are high at the 3′ overhang.
• The action of telomerase is blocked.
When the telomere is too short:
• Little or no POT1 is present at the 3′ end.
• Telomerase is no longer inhibited.
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A model for t-loop formation
• The 3′ single-stranded DNA tail invades the double-stranded telomeric DNA.
• A loop forms in which the 3′ overhang is base paired to the C strand sequence.
• The t-loop may aid in preventing telomerase access.
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Telomerase, aging, and cancer• In most unicellular organisms, telomerase has
a “housekeeping function.”
• In most human somatic cells, not enough telomerase is expressed to maintain a constant telomere length: Progressive shortening of telomeres.
• High levels of telomerase activity in ovaries, testes, rapidly dividing somatic cells, and cancer cells.
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Telomerase and aging: the Hayflick limit
• The Hayflick limit is the point at which cultured cells stop dividing and enter an irreversible state of cellular aging (senescence).
• Proposed to be a consequence of telomere shortening.
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Telomere shortening: a molecular clock for aging?
• Telomerase: A target for anti-aging therapy or anti-cancer therapy?
• Cellular senescence may be a mechanism to protect multicellular organisms from cancer.
• Cancer cells become immortalized and thus can grow uncontrolled.
• In most cancer cells, telomerase has been reactivated.
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Direct evidence for a relationship between telomere shortening and aging
• Evidence from experiments in human cells in culture and in transgenic mice.
• However, there are reports of instances where short telomere length does not correlate with entry into cellular senescence.
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1. Effect of experimental activation of telomerase on normal human somatic cells
• Experiment carried out in telomerase-negative normal human cell types.
• Demonstrated a link between telomerase activity and cellular immortality.
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2. Insights from telomerase-deficient mice
Cells from mice engineered to lack a telomerase RNA component:
• Progressive telomere shortening after 300 cell divisions.
• After 450 divisions, cell growth stopped.
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Sixth-generation mice lacking telomerase RNA component
• Defects in spermatogenesis.
• Impaired proliferation of hematopoietic cells.
• Premature graying and hair loss.
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Dyskeratosis congenita: loss of telomerase activity
• Premature aging syndrome.
• Problems in tissues where cells multiply rapidly and where telomerase is normally expressed.
• Two forms of dyskeratosis congenita:– X-linked recessive– Autosomal dominant
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X-linked recessive dyskeratosis congenita
• Mutations in dyskerin gene.
• Dyskerin is a pseudouridine synthase that binds to small nucleolar RNAs and to telomerase RNA.
• Patients with dyskerin mutations have 5-fold less telomerase activity than unaffected siblings.
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Autosomal dominant dyskeratosis congenita
• Mutations in telomerase RNA gene in the pseudoknot domain.
• Partial loss of function of telomerase RNA.
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3. Gene therapy for liver cirrhosis
• Inhibition of liver cirrhosis in mice by telomerase gene delivery.
• Why hasn’t this gene therapy strategy progressed to human trials?