Lecture PowerPoint to accompany
Molecular Biology
Fifth Edition
Robert F. Weaver
Chapter 21
DNA Replication II:
Detailed Mechanism
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
21.1 Initiation
• Initiation of DNA replication means primer
synthesis
• Different organisms use different
mechanisms to make primers
• Different phages infect E. coli using quite
different primer synthesis strategies
• Coliphages were convenient tools to probe
DNA replication as they are so simple they
must rely primarily on host proteins to
replicate their DNAs
21-2
Priming in E. coli
• Primosome refers to collection of proteins
needed to make primers for a given
replicating DNA
• Primer synthesis in E. coli requires a
primosome composed of:
– DNA helicase
– DnaB
– Primase, DnaG
• Primosome assembly at the origin of
replication, oriC, uses multi-step sequence
21-3
Priming at oriC
Source: Adapted from DNA Replication, 2/e, (plate 15) by Arthur Kornberg and Tania Baker.
21-4
Origin of Replication in E. coli
Primosome assembly at oriC occurs as
follows:
– DnaA binds to oriC at sites called dnaA boxes
and cooperates with RNA polymerase and HU
protein in melting a DNA region adjacent to
leftmost dnaA box
– DnaB binds to the open complex and facilitates
binding of primase to complete the primosome
– Primosome remains with replisome, repeatedly
primes Okazaki fragment synthesis on lagging
strand
– DnaB has a helicase activity that unwinds DNA
as the replisome progresses
21-5
Priming in Eukaryotes
• Eukaryotic replication is more complex
than bacterial replication
• Complicating factors
– Bigger size of eukaryotic genomes
– Slower movement of replicating forks
– Each chromosome must have multiple origins
• Started study with a simple monkey virus,
SV40
• Later consider yeast
21-6
Origin of Replication in SV40
• The SV40 origin of replication is adjacent
to the viral transcription control region
• Initiation of replication depends on the viral
large T antigen binding to:
– Region within the 64-bp ori core
– Two adjacent sites
• Exercises a helicase activity that opens up
a replication bubble within the ori core
• Priming is carried out by a primase
associated with host DNA polymerase a
21-7
Origin of Replication in Yeast
• Yeast origins of replication are contained
within autonomously replicating
sequences (ARSs)
• These are composed of 4 important
regions:
– Region A is 15 bp long and contains an 11-bp
consensus sequence highly conserved in
ARSs
– B1 and B2
– B3 may allow for an important DNA bend
within ARS1
21-8
21.2 Elongation
• Once a primer is in place, real DNA
synthesis can begin
• An elegant method of coordinating the
synthesis of lagging and leading strands
keep the Pol III holoenzyme engaged with
the template
• Replication can be highly processive and
rapid
21-9
Speed of Replication
• The Pol III holoenzyme synthesizes DNA
at the rate of about 730 nt/sec in vitro
• The rate in vivo is almost 1000 nt/sec
• This enzyme is highly processive both in
vitro and in vivo
21-10
The Pol III Holoenzyme and
Processivity of Replication
• Pol III core alone is a very poor polymerase,
after assembling 10 nt it falls off the template
• Takes about 1 minute to reassociate with the
template and nascent DNA strand
• Something is missing from the core enzyme
– The agent that confers processivity on
holoenzyme allows it to remain engaged with
the template
– Processivity agent is a “sliding clamp”, the bsubunit of the holoenzyme
21-11
The Role of the b-Subunit
• Core plus the b-subunit can replicate DNA
processively at about 1,000 nt/sec
– Dimer formed by b-subunit is ring-shaped
– Ring fits around DNA template
– Interacts with a-subunit of the core to tether the whole
polymerase and template together
• Holoenzyme stays on its template with the bclamp
• Eukaryotic processivity factor, PCNA forms a
trimer, also forms a ring that encircles DNA and
holds DNA polymerase on the template
21-12
Model of the b dimer/DNA complex
21-13
The Clamp Loader
• The b-subunit needs help from the g complex to
load onto the DNA template
– This g complex acts catalytically in forming this
processive adb complex
– Does not remain associated with the complex during
processive replication
• Clamp loading is an ATP-dependent process
– Energy from ATP changes conformation of the loader
so that d-subunit binds to one of the b-subunits of the
clamp
– This binding opens the clamp and allows it to encircle
DNA
21-14
The b Clamp and Loader
21-15
Lagging Strand Synthesis
• The pol III holoenzyme is double-headed
• There are 2 core polymerases attached through
2 t-subunits to a g complex
– One core is responsible for continuous synthesis of
the leading strand
– Other core performs discontinuous synthesis of the
lagging strand
– The g complex serves as a clamp loader to load the b
clamp onto a primed DNA template
– After loading, b clamp loses affinity for g complex
instead associating with core polymerase
21-16
Model for simultaneous strand synthesis
• The g complex and b
clamp help core
polymerase with
processive synthesis of
an Okazaki fragment
• When fragment
completed, b clamp loses
affinity for core
• Associate b clamp with g
complex which acts to
unload clamp
• Now clamp recycles
21-17
Lagging Strand Replication
Source: Adapted from Henderson, D.R. and T.J. Kelly, DNA polymerase III: Running rings around the
fork. Cell 84:7, 1996.
21-18
21.3 Termination
• Termination of replication is straightforward for
phage that produce long, linear concatemers
• Concatemer grows until genome-sized piece is
snipped off and packaged into phage head
• Bacterial replication – 2 replication forks
approach each other at the terminus region
– Contains 22-bp terminator sites that bind specific
proteins (terminus utilization substance, TUS)
– Replicating forks enter terminus region and pause
– Leaves 2 daughter duplexes entangled
– Must separate or no cell division
21-19
Decatenation: Disentangling Daughter DNAs
• At the end of replication, circular bacterial
chromosomes form catenanes that are
decatenated in a two-step process
– First, remaining unreplicated double-helical
turns linking the two strands are melted
– Repair synthesis fills in the gaps
– Left with a catenane that is decatenated by
topoisomerase IV
• Linear eukaryotic chromosomes also
require decatenation during DNA
replication
21-20
Termination in Eukarytoes
• Unlike bacteria, eukaryotes have a
problem filling the gaps left when RNA
primers are removed at the end of DNA
replication
• If primer on each strand is removed, there
is no way to fill in the gaps
– DNA cannot be extended 3’5’ direction
– No 3’-end is upstream
– If no resolution, DNA strands would get
shorter with each replication
21-21
Telomere Maintenance
• At the ends of eukaryotic chromosomes are special
structures called telomeres
• One strand of telomeres is composed of tandem
repeats of short, G-rich regions whose sequence
varies from one species to another
– G-rich telomere strand is made by enzyme telomerase
– Telomerase contains a short RNA serving as template for
telomere synthesis
• C-rich telomere strand is synthesized by ordinary
RNA-primed DNA synthesis
– This process is like lagging strand DNA replication
• This mechanism ensures that chromosome ends
can be rebuilt and do not suffer shortening with
each round of replication
21-22
Telomere Formation
21-23
Telomere Structure
• All eukaryotes protect their telomeres from
nucleases and ds break repair enzymes
• Eukaryotes from yeast to mammals have a
suite of telomere-binding proteins that
protect the telomeres from degradation,
and also hide the telomere ends from DNA
damage factors that would otherwise
recognize them as chromosome breaks
21-24
Mammalian Telomere Binding Proteins
• In mammals, the group of telomerebinding proteins is known as shelterin,
because it ‘shelters’ the telomere
• Six known mammalian proteins: TRF1,
TRF2, TIN2, POT1, TPP1 and RAP1
• Other proteins besides shelterin binds to
telomeres but they can be distinguished
from the others in three ways: they are
found only at telomeres, they associate
with telomeres throughout the cell cycle
and they function nowhere else in the cell
21-25
Mammalian Telomere Binding Proteins
• TRF1 and 2: bind to the double-stranded
telomeric repeats
• POT1: binds to the single-stranded 3’ tail
of the telomere
• TIN2: organizes shelterin by facilitating
interaction between TRF1 and TRF2 and
tethering POT1, via its partner, TPP1, to
TRF2
21-26
Mammalian Telomere Binding Proteins
• Shelterin affects telomere structure in three ways:
• 1 - it remodels telomeres into t-loops, wherein the
single-stranded 3’-tail invades the doublestranded telomeric DNA, creating a D-loop - in
this way, the 3’-tail is protected
• 2 - it determines the structure of the telomeric
end by promoting 3’-end elongation and
protecting both 3’ and 5’-telomeric ends from
degradation
• 3 - it maintains the telomere length with close
tolerances
21-27
The role of shelterin in suppressing
inappropriate repair and cell cycle arrest
• Unmodified chromosome ends would look like
broken chromosomes and cause two potentially
dangerous DNA repair activities, HDR and NHEJ
• They would also stimulate two dangerous
pathways (the ATM kinase and the ATR kinase)
leading to cell cycle arrest
• Two subunits of shelterin, TRF2 and POT1, block
HDR and NHEJ, as well as repress the two cell
cycle arrest pathways
21-28
Telomere Structure and TelomereBinding Protein in Lower Eukayotes
• Yeasts and ciliated protozoa do not form t-loops,
but their telomeres are still associated with proteins
that protect them
• Fission yeasts have shelterin-like telomere-binding
proteins
• Budding yeasts have only one shelterin relative,
Rap1, which binds to the double-stranded part of
the telomere plus two Rap1-binding proteins and
three proteins that protect the ss 3’-end of the
telomere
21-29
The role of Pot1
• In 2001 proteins that bound to the single-stranded
tails of telomeres were reported in S.pombe and
the gene was named pot1, for the protection of
telomeres
• In S.pombe, Pot1, instead of limiting the growth of
telomeres, as mammalian POT1 does, plays a
critical role in maintaining their integrity
• The loss of Pot1 can cause the loss of telomeres
from this organism
21-30
The role of Pot1
• S.pombe Pot1 binds to telomeres and
protects them from degradation
• Without Pot1, telomeres in this organism are
eliminated
• With time, the few cells that survive without
Pot1 circularize their chromosomes so
telomeres are no longer needed
21-31