DNA replication
Andy Howard
Introductory Biochemistry
8 May 2008
What we’ll discuss
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Prokaryotic DNA replication  Eukaryotic
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Semiconservative
replication
Unwinding of parent DNA
Leading-strand replication
Lagging-strand replication
Okazaki fragments
replication
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Rates
Multiple
starting points
Enzymes
Other
DNA replication
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08 May 2008
iClicker quiz
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1. Which of these DNA sequences
is palindromic?
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(a) TCGATG
(b) TAGGAT
(c ) GGCCCGGG
(d) TACGCGTA
(e) None of the above
DNA replication
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iClicker quiz #2
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Suppose someone introduced a drug that
interfered with deacylation of H1. What effect
would this have on chromatin assembly?
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(a) It would prevent formation of the nucleosome
core particle
(b) It would interfere with assembly of the
structures connecting one core particle to the next
(c) Both (a) and (b)
(d) It would have no effect on chromatin assembly
(e) Like (c), except only in prokaryotes
DNA replication
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Semi-conservative
replication
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A bit of a fanciful term
Photo courtesy
U. Costa Rica
Refers to the fact that, during DNA
replication, each daughter molecule
contains one of the strands of the parent
Thus each daughter contains half (semi)
of the original molecule
This mode of inheritance was predicted
by the Watson/Crick model
DNA replication
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Meselson & Stahl
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1958: showed that DNA really is
replicated this way
DNA grown with 15N has higher density
15N DNA allowed to replicate exactly
once has intermediate density
DNA replication
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The E.coli chromosome
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One circular, double-stranded DNA molecule
of about 4.6*106 bp
Replication begins in only one place, i.e. a
single origin of replication (OriC in E.Coli)
Replication moves both directions until the
two replication efforts meet at the
termination site
Protein machine that accomplishes
replication is the replisome;
one replisome in each direction
Replication forks move 1000 bp/sec; thus
E.coli can be replicated in 38 min
DNA replication
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By contrast:
eukaryotes
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Bigger chromosomes, more of them
Not circular, usually
Fruit-fly chromosomes:
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Human
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Sex chromosome, 2 long autosomes,
one tiny autosome
1.65 * 108 bp, 14000 genes
Drosophila
chromosome
TEM
reconstruction
22 pairs of autosomes, sex chromosome
3.4*109 bp, ~22000 genes
Replication is bidirectional as in E.coli
More than one origin so replication is
comparably fast even though rate is lower
DNA replication
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How replication works
in prokaryotes
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Takes place in the cytosol since there is
no nucleus
Specific enzymes form the molecular
machine to carry out the task
Has to involve separation of the strands
Process divided into initiation,
elongation, and termination
Enzymatic functions identified for each
segment
DNA replication
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Prokaryotic DNA
polymerases
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Several varieties
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DNA polymerase III is the one responsible for
most of the work (but the 3rd discovered);
it’s the biggest and most complex
DNA pol I involved in error correction and
helps with replication of one of the strands
DNA pol II also does DNA repair
Multi-subunit, complex entities
DNA replication
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DNA Pol III
Diagram from Kelman et al
(1998) EMBO J. 17:2436
DNA replication
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Components of DNA Pol III
Subunit
Mr,kDa Gene
Activity
a
130
polC/dnaE
Polymerase
e
27
dnaQ/mutD
3’-5’ exonuclease
q
8.9
holE
b
40
dnaN
Stabilizes
proofreading by e?
Sliding clamp
t
71
dnaX
Dimer, ATPase
Various
Processivity
g complex 15-47
DNA replication
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So how does it work?
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Add 1 nucleotide @ a time to 3’ end of growing chain
Substrate is a dNTP
Watson-Crick bp determines specificity
Enzyme spends 75% of time tossing out wrong bases
Forms phosphodiester linkage
Pol III remains bound to the replication fork
Diagram from
answers.com
DNA replication
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Error correction in DNA pol III
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3’-5’ proofreading recognizes incorrectly paired
bases and repairs most of them
This is an exonuclease activity because it clips
off the last nucleotide in the chain
10-5 inherent error rate drops to 10-7 because
the exonuclease goofs 1% of the time
Separate repair enzymes drop that down to 10-9
DNA replication
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Processivity
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This term refers to the fact that many
nucleotides can be added to a growing chain
following a single association event in which
the polymerase (e.g. E.coli Pol III) associates
with the template DNA.
We describe replication as highly processive if
50,000 bases can be replicated based on a
single association of Pol III with our template.
b subunits slide along, which is how this is
done
g complex is responsible for keeping the
polymerase attached so that this is possible
DNA replication
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Initiation
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Begins in E.coli at a single origin called OriC
DnaA binds to origin—region called DnaA box
Replication fork forms after it binds
Helicases & primases
set up for starting replication
Complementary RNA tag attached at the
replication fork
DNA replication
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Elongation
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DNA polymerase operates in 5’-3’ direction
on both strands
For one strand that’s straightforward
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replication moves in direction of unwinding of the DNA
This is known as leading-strand synthesis
For the other strand it must work opposite to the
unwinding
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therefore it’s more complicated
This is lagging-strand synthesis
DNA replication
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Termination
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Replication needs to know how
to stop
Defined sequence ter is opposite
the origin on the chromosome
Specific enzyme, Tus, involved in
recognizing termination signals
Ter has sequences that play a
role in separating the daughter
chromosomes
Tus-Ter complex;
images courtesy
Memorial Univ.,
Newfoundland
DNA replication
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Leading-strand synthesis
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One base at a time is incorporated by a subunit
of DNA polymerase, complementary to existing
strand
At some point RNA primer is replaced with DNA
Image courtesy U.Pittsburgh
DNA replication
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Lagging-strand
synthesis
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Movement of enzyme is opposite to unwinding
Therefore it must work a few bases (~1000) at a
time and then back up
The segments thus formed on the lagging
strand are known as Okazaki fragments
DNA Pol I removes RNA primer
DNA ligases link together Okazaki fragments
DNA replication
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Primases
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These are DNA-dependent
RNA polymerase enzymes
that initiate DNA synthesis,
particularly on the lagging
strand, where you need to do
that at the beginning of each
Okazaki fragment
DnaG helicase
binding domain
PDB 2R6A
347 kDa trimer of
heterotrimers
Bacillus
stearothermophilus
DNA replication
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Role of
DNA Pol I
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Part of the system for producing a
continuous DNA strand on the lagging
side
Contains both 5’3’ polymerase activity
and 3’5’ proofreading exonuclease
activity
Also has 5’3’ exonuclease activity:
that’s used to remove the RNA primer
DNA replication
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Rates and
sequencing
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Because there’s only one place where
replication can begin, the process has to occur
in discrete steps
The enzymes themselves are efficient, because
they move with the unwinding of the double
helix
Typical rates 1000 nucleotides/ sec
So for E.coli it takes 38 min = 2280 sec to
replicate the entire chromosome
(4.6*106 bp) / [(103 bp/sec)(2 directions)]=
2300 sec
DNA replication
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Where are things
happening?
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Both leading- and lagging-strand
synthesis are catalyzed in both the
clockwise and counterclockwise
directions.
Each DNA Pol III molecule is catalyzing
both leading- and lagging-strand
synthesis.
DNA replication
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Eukaryotic DNA
polymerases
image courtesy
Memorial Univ.,
Newfoundland
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More complex, as you’d expect
More than one initiation point
Therefore even though the enzymes work less
rapidly, they can multiplex the process
The result is that human DNA can be replicated
in roughly the same time scale as E.coli DNA
DNA replication
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Eukaryotic DNA
polymerases
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Several complexes involved:
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a: Elongation and repair
d: Elongation and repair
Human DNA polymerase b:
e: Elongation and repair
From answers.com
b: DNA repair
g: Helps in replicating mitochondrial DNA
chloroplast polymerase
Don’t fall into the trap: these Greek letters don’t
necessarily mean the same thing that they
mean in the context of bacterial replication!
DNA replication
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Specific roles for a and d
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d complex does leading-strand synthesis
and 3’-5’ exonuclease activity, which is
impressively effective
a and d involved in lagging strand
synthesis:
a is DNA polymerase and RNA primase;
d extends segment to complete Okazaki
fragment
DNA replication
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Roles for DNA
polymerase e
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Cryo EM
image from
Asturias et al
(2006) Nature
Struct Mol Biol
13:35;
Big,multi-subunit protein
Similar to DNA pol I from E.coli
It repairs and it fills gaps between
Okazaki fragments
yeast
Largest piece is a polymerase
and does 3’5’ proofreading
DNA replication
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