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.

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

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 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′.

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 H2A-H2B 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 single-celled 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?