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Chapter 6: DNA Replication and Telomere Maintenance 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 6.1 Introduction 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. 6.2 Early insights into the mode of bacterial DNA replication Three possible modes of replication hypothesized based on Watson and Crick’s model: • Semiconservative • Conservative • Dispersive The Meselson-Stahl experiment • 1958 experiment designed to distinguish between semiconservative, conservative, and dispersive replication. • Results were consistent only with semiconservative replication. 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. 6.3 DNA polymerases are the enzymes that catalyze DNA synthesis from 5′ to 3′ 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. • 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. Problem • DNA polymerases can only add nucleotides from 5′→3′ but, the two strands of the double helix are antiparallel. Solution • Semidiscontinuous replication. Semidiscontinuous DNA replication • Major form of replication in eukaryotic nuclear DNA, some viruses, and bacteria. 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. 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. 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. • 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. 6.4 Multi-protein machines mediate bacterial DNA replication 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. 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). 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. Replication is mediated by the replisome 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. 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. 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. • 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. • 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. 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. 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). 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. 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. 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. 6.5 Multi-protein machines trade places during eukaryotic DNA replication 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. • 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. 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. 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. 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. Histone removal at origins of replication • Histone modification and chromatin remodeling factors. • Disassembly of the nucleosomes. • Template DNA is accessible to the replication machinery. 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. 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. 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. 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. • 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) 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. • 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. 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 doublestranded 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′. 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). 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. 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) • Leading strand: switch from DNA polymerase to pol • Lagging strand: switch from pol to pol • Polymerase switching is regulated by PCNA. • 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 . 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. 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. 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. • 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. 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. Maturation of DNA nascent strands • RNA primer removal. • Gap fill-in. • Joining of Okazaki fragments on the lagging strand. 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.” • 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. 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. • 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. 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 H2AH2B dimers. 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. 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. 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 6.6 Alternative modes of circular DNA replication 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. 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. 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. Models for organelle DNA replication • There is no consensus on the mode of replication of organelle DNA. 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. 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 Strand displacement model: • The most widely accepted model. • Replication is unidirectional round the circle and there is one replication fork for each strand. Strand coupled model: • Semidiscontinous, bidirectional replication. • Synthesis of Okazaki fragments on the lagging strand. 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. 6.7 Telomere maintenance: the role of telomerase in DNA replication, aging, and cancer 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. 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. Solution to the end replication problem • Solution reported by Carol Greider and Elizabeth Blackburn in 1985. • Studied Tetrahymena thermophila, a singlecelled eukaryote with over 40,000 telomeres. • Discovered the enzyme telomerase. • Shared the 2009 Nobel prize in physiology or medicine with Jack Szostak. • 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) 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. • 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. Sixth-generation mice lacking telomerase RNA component • Defects in spermatogenesis. • Impaired proliferation of hematopoietic cells. • Premature graying and hair loss. 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 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. Autosomal dominant dyskeratosis congenita • Mutations in telomerase RNA gene in the pseudoknot domain. • Partial loss of function of telomerase RNA. 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?