Lecture PowerPoint to accompany

Molecular Biology
Fourth 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:
– 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
• 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
so very 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 b-
subunit 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 b-
clamp
• Eukaryotic processivity factor, PCNA forms a
trimer, also forms a ring that encircles DNA and
holds DNA polymerase on the template
21-12
 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 21-13
 The b Clamp and Loader

Source: Adapted from Ellison, V. and B. Source: Adapted from Henderson, D.R. and
Stillman, Opening of the clamp: An intimate T.J. Kelly, DNA polymerase III: Running rings
view of an ATP-driven biological machine. Cell around the fork. Cell 84:6, 1996.
106(2001), p. 657, f. 3. 21-14
 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
21-15
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
21-16
 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.
21-17
 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
21-18
 Decatenation: Disentangling
Daughter DNAs
• At the end of replication, circular bacterial
chromosomes form catenanes that 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 right-handed parallel torus catenane with
an even number nodes that is decatenated by
topoisomerase IV
• Linear eukaryotic chromosomes also require
decatenation during DNA replication
21-19
 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 21-20
 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
21-21
Telomere Formation

21-22
 Telomere Structure
• All eukaryotes protect their telomeres from
nucleases and ds break repair enzymes
• Ciliates have TEBP to bind and protect the
3’-single-strand telomeric overhang
• Budding yeast has Cdc13p which recruits
Stn1p and Ten1p that all bind ss telomeric
DNA
• Mammals and fission yeast also have a
protein similar to TEBP binding specifically
to ss telomeric DNA
21-23
Mammalian Telomere Structure
• Mammalian telomeres form looped
structures that protect ss telomeric DNA
• Proteins TRF1 and TRF2 appear to help
telomeric DNA in mammalian cells form a t
loop in which ss 3’-end of telomere
invades the ds telomeric DNA upstream
• TRF1 may help bend the DNA into shape
for strand invasion
• TRF2 binds at point of strand invasion,
may stabilize the displacement loop
21-24