Transcript DNA Replication and Telomere Maintenance

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?