Lecture PowerPoint to accompany

Molecular Biology
Fourth Edition

Robert F. Weaver

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

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 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 (Dna B)
– DnaB
– Primase, DnaG
• Primosome assembly at the origin of
replication, oriC uses multi-step sequence
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 Priming at oriC

Source: Adapted from DNA Replication, 2/e, (plate 15) by Arthur Kornberg and Tania Baker. 21-4
 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

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 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

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 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

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 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 -
subunit of the holoenzyme 21-8
 The Role of the -Subunit
• Core plus the -subunit can replicate DNA
processively at about 1,000 nt/sec
– Dimer formed by -subunit is ring-shaped
– Ring fits around DNA template
– Interacts with -subunit of the core to tether the whole
polymerase and template together
• Holoenzyme stays on its template with the -clamp
• Eukaryotic processivity factor PCNA (proliferating
cell nuclear antigen) forms a trimer, also forms a
ring that encircles DNA and holds DNA polymerase
on the template
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 Lagging Strand Synthesis
• The pol III holoenzyme is double-headed
• There are 2 core polymerases attached through
2 -subunits to a  complex
– One core is responsible for continuous synthesis of
the leading strand
– Other core performs discontinuous synthesis of the
lagging strand
– The  complex serves as a clamp loader to load the 
clamp onto a primed DNA template
– After loading,  clamp loses affinity for  complex
instead associating with core polymerase
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Simultaneous Strand Synthesis
• The  complex and 
clamp help core
polymerase with
processive synthesis of
an Okazaki fragment
• When fragment
completed,  clamp loses
affinity for core
• Associate  clamp with 
complex which acts to
unload clamp
• Now clamp recycles
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 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.
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 21.3 Termination
• Termination of replication is straight forward 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

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• https://www.youtube.com/watch?v=sGyZ2
s3FOWghttps://www.youtube.com/watch?
v=sGyZ2s3FOWg

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