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Chapter 5
• DNA Replication,
Repair, and
Recombination
Outline of this chapter
• The maintenance of DNA sequences
• DNA replication mechanisms
• The initiation and completion of DNA
replication in chromosomes
• DNA repair
• General recombination
• Site-specific recombination
The maintenance of DNA
sequences
It is extremely important to keep the
mutation rate low in all organisms.
Mutation and mutation rate
• Mutation rate is the rate at which observable
changes occur in DNA sequences.
• Although human and E. coli are very
different organisms, mutation rates of E.
coli and human are approximately the same
(10-9 per cell generation).
The estimation of mutation rate
• The estimate of mutation rate can be done
by looking at a gene that is not required for
the survival of the organism. For example,
the change of sequence of lactose-utilizing
gene in E. coli grown under glucosecontaining medium or the change of
fibrinopeptide in mammals.
Mutation rate is often
underestimated
• Some mutation can happen undetected
(‘silent’) if they don’t affect amino acid
sequence or biological function of protein.
• Also, mutations that dramatically affect
biological function of the protein will
vanish from population before they can be
detected (‘preferential death’).
Unit evolutionary
time
• When we plotted the amino
acid differences in a
particular protein for
several pairs of species
against the time that has
elapsed since the pair of
species diverged from a
common ancestor, the slope
of this straight line is what
we call the “unit
evolutionary time”.
Unit evolutionary time
• Unit evolutionary time reflects differences
in the probability that an amino acid change
will be harmful for each protein.
• Some proteins like histone H4 are
completely intolerant to mutation.
The effect of mutation rate to all
organisms
• Mutation rate has limit the number of
essential proteins for any organisms to
60,000. If mutation rate is 10 percent higher
(10-8 per cell generation), we will probably
be fruit flies.
How is mutation rate being kept
low enough?
• Mutation rate is kept low enough by several
mechanisms, including
- Proofreading of DNA polymerase
- DNA repair
• At this moment we will discuss the
proofreading of DNA polymerase
The first step of proofreading:
correct nucleotide has a higher
affinity for the moving polymerase
The second step:
exonucleolytic
proofreading
• After an incorrect
nucleotide is
covalently added to
the chain, DNA
polymerase cannot
continue the
polymerization
reaction until the
incorect nucleotide is
removed.
Exonucleolytic
proofreading
• This is because DNA
polymerase has an
absolute requirement
for a base-paired 3’OH end of a primer
strand to continue its
polymerization
reaction.
Exonucleolytic proofreading
• Most of the DNA polymerase has 3’-to-5’
exonuclease activity. This activity resides either in
a separate domain of the same protein or in a
separate subunit.
Exonucleolytic
proofreading is
probably why
DNA is not
synthesized from
3’-to-5’
DNA replication mechanisms
DNA replication is semiconservative
and asymmetrical.
DNA replication is
semiconservative
• Because each of the
two new DNA strands
inherit one new strand
and one old strand, we
called this
“semiconservative”.
DNA replication is
semiconservative
1958, Matthew Meselson and Franklin Stahl
DNA replication is
semiconservative
DNA replication is
semiconservative
How is DNA being replicated?
The structure of DNA makes its
replication asymmetrical
• Because DNA is reverse and
complementary (‘antiparallel’) and there is
no 3’-to-5’ DNA polymerase, the replication
of DNA must be progressed asymmetrically,
with one strand moving faster (‘leading’)
while the other moving slower (‘lagging’).
The lagging strand is lagging because it
is synthesized discontinuously
• Lagging strand DNA synthesis is delayed because
it must wait for the leading strand to expose the
template strand on which each Okazaki fragment
is synthesized.
The Okazaki fragment
• Okazaki fragments are the short DNA
fragments produced during lagging strand
DNA synthesis. They will be ligated
together by ligase shortly after completion.
• Prokaryotes like E. coli has Okazaki
fragment of 1000~2000 nucleotides long
while eukaryotes like us has shorter
Okazaki fragments (100~200 nucleotides 
nucleosomes?).
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When Okazaki
used ligase mutant
of bacteriophage
T4 to perform the
same experiment,
he saw all the
fragments are short
fragments (Okazaki
fragment).
The replication “fork”(fig. 5-6)
In the early
1960s John
Cairns used
radioactive DNA
precursor to label
replicating E.
coli DNA. The
results were like
this picture.
weak label
Strong
label
J. Huberman and A. Tsai
Drosophila melanogaster
Analyses carried out in the early 1960s on
whole replicating chromosomes revealed a
localized region of replication that moves
progressively alone the parental DNA
double helix, forming a Y-shaped structure.
DNA primase initiate new DNA
synthesis
Because no DNA polymerase can
initiate DNA synthesis anew, a
different protein must initiate it.
DNA primase
• DNA primase
synthesize RNA
primers to serve as
initiation site for DNA
polymerase.
• In eukaryotes, this
RNA primer is about
10 nucleotides long. In
prokaryotes, it is about
12 nucleotide in length.
RNA primer is
degraded after
synthesis
• After DNA is
synthesized, RNA primer
is being degraded and
replaced by DNA (strand
replacement synthesis).
DNA helicase opens up the DNA
double helix
It is very difficult to make double
stranded DNA unwind without any
help.
Most of the DNA helicase is
hexameric
Energy is required for
the catalysis of DNA
helicase
• DNA helicases were first
isolated as proteins that
hydrolyzed ATP when they
are bound to single strand
DNA.
Single-strand DNA binding
proteins (SSB) stabilize exposed
single-stranded region
Without SSB, single stranded region
will form short hairpins that
hindering replication
SSB act cooperatively to bind
single-stranded DNA
The processivity of DNA
polymerase is enhanced by
sliding clamp
Processivity
• It means the tendency of a polymerase to
stick with the replicating job once it starts.
• When we said this polymerase is highly
processive, meaning that once it starts
replicating DNA, it won’t stop for a long
time.
Sliding clamp
b clamp
PCNA
• The sliding clamp form
a ring around the DNA
helix.
• One side of the ring
binds to DNA
polymerase while the
whole ring slide freely
along the DNA as the
polymerase moves.
Sliding clamp is loaded by clamp
loader, which is a five-protein
complex
Clamp loading process requires
ATP hydrolysis
• Clamp loader
interact with sliding
clamp from the same
side that DNA
polymerase does.
• Upon ATP
hydrolysis, sliding
clamp is loaded on to
a primer-template
junction.
Sliding clamp has different sides
Used clamp can be recycled by
clamp loader
Clamp loader can be clamp
unloader
• The g-complex can unload b-clamp from
replicated DNA.
• The action of g-complex at this situation is also
ATP-dependent.
In prokaryotes, the leading and
lagging strand DNA replication
machines are associated.
How is mutation rate kept low
enough – mismatch repair
• When an incorrect nucleotide was
incorporated in synthesizing DNA strand
undetected, it becomes a mismatch on the
DNA.
• Usually it will be caught by mismatch repair
mechanism.
Mismatch repair
• The most important thing for mismatch
repair is the right strand cannot be repair
according to the wrong strand sequence.
• In prokaryotes like E. coli, newly
synthesized strand is distinguished from the
old strand by the methylation of A residues
in the DNA of the sequence of GATC.
Mismatch repair in E. coli
• MutS, MutL and MutH will recognize the
methylated A on the GATC, then Mut H makes a
nick 5’ of the mismatch….
Mismatch repair in E. coli
• Exonuclease I, MutL, MutS, and helicase removes
DNA 3’ of the nick, including the incorrect
nucleotide. Because helicase is used in this
reaction so ATP is required.
Mismatch repair in E. coli
• DNA polymerase III fills the gap (with help from
SSB) and DNA ligase seals the nick.
Mismatch repair in eukaryotes
• While E. coli used methylated A in GATC
sequences to distinguish old and new strands,
eukaryotes like yeast and Drosophila do not have
their DNA methylated.
• However, newly synthesized DNA strands are
known to be preferentially nicked (Okazaki
fragments?), so this is used to distinguish old and
new strands in eukaryotes.
Mismatch repair in eukaryotes
• Eukaryotes also have
MutL and MutS.
• MutS binds to a
mismatched base
pair, while MutL
scans the nearby
DNA for a nick.
Mismatch repair in eukaryotes
• Once a nick is found,
MutL triggers the
degradation of the
nicked strand all the
way back through the
mismatch.
The winding problem that arises
during DNA replication
As replication fork DNA unwinds,
other parts of DNA are overwound.
The winding
problem
• For circular or
extremely long
DNA, the unwinding
of replication results
in overwinding of
other parts of the
DNA.
The winding
problem
• As the DNA is being
replicated, the tension of
the rest of DNA
increases. If the tension
is not relieved, DNA
replication will come to a
halt eventually because
the tension is too big for
helicase to unwind.
DNA topoisomerase
• DNA topoisomerase can relieve the tension
of the overwound DNA.
• DNA topoisomerase can be viewed as a
reversible nuclease that adds itself
covalently to a DNA backbone phosphate.
It breaks one or both strands of DNA, turn it
around, ligate it back as it leaves.
• There are two types of DNA
topoisomerases, type I and II.
Topoisomerase I
• Topoisomerase I
produces a transient
single-strand nick.
Topoisomerase I
• This break
allows the two
sections of
DNA helix on
either side of
the nick to
rotate freely
Topoisomerase I
• Afterwards, the
nick is sealed by
topoisomerase
itself.
• Topoisomerase I
does not require
ATP hydrolysis to
complete its
action.
Topoisomerase II
• Topoisomerase II breaks
both strands of the DNA.
• It is activated by sites on
chromosomes where two
double helices cross over
each other.
The initiation and completion of
DNA replication in chromosomes
How DNA replication is initiated and
how cells carefully regulate this
process
Replication origin
• Replication origin is the position at which the
DNA helix is first opened during replication.
• Simple organisms have origins that contain DNA
sequences several hundred nucleotide pairs in
length.
• Because A-T base pair is held together with fewer
hydrogen bonds than G-C base pair, regions of
DNA enriched in A-T pairs are typically found at
replication origins.
Basic process of replication
initiation
Bacterial chromosomes have a
single replication origin
• Bacteria like E. coli (4.6x106
bps) has a single origin of
replication and the replication
proceed bidirectionally with a
speed approximately 500-1000
nt per second.
Replication initiation in bacteria
Replication initiation in bacteria
Replication initiation in bacteria
helicase
Replication initiation in bacteria
Replication initiation is tightly
controlled
• Initiation occurs only when sufficient nutrients are
available for the bacterium to complete an entire round of
replication.
• Also, newly synthesized DNA with hemimethylated
origins cannot be used for replication initiation.
Pulse-chase studies revealed how
eukaryotes replicate their chromosomes
How eukaryotes replicate their
chromosomes
• Result suggested that
multiple replication
forks must proceed at
the same time, since
replication fork is
estimated to travel at
about 50 nt per
second.
DNA replication in eukaryotes
• Replication origins tend to be activated in clusters
(replication units, 20-80 origins).
• New replication units seem to be activated at
different times during the cell cycle until all of the
DNA is replicated.
• Individual origins are spaced at intervals of
30~300 kbp.
• Replication also proceed bidirectionally from each
replication origin.
DNA replication in eukaryotes
• Eukaryotes only replicate their DNA during
a specific part of the cell cycle (S phase).
• Typical mammalian S phase is about 8
hours long (yeast: 40 minutes) while to
finish replicating one origin takes only 1
hour. Therefore, the replication origins are
not all activated simultaneously.
The timing of replication is related to the
packing of the DNA in chromatin
• Pulse-chase with
bromodeoxyuridine
(BrdU) found that
different regions of
each chromosome are
replicated in a
reproducible order
during S phase.
The timing of replication is related to the
packing of the DNA in chromatin
• Observation done on female mammalian cell
showed that heterochromatin tends to be replicated
very late in the S phase. Later on, the observation
on “housekeeping genes” also suggested that
genes that are transcriptionally active (therefore
least condensed) will be replicated first.
• Chromosome condensation does not affect the
speed of replication. It only affects the timing of
replication initiation.
Yeast replication
origin
• ARS
(autonomously
replicating
sequence) was
found as yeast
replication
origin.
Yeast replication origin (ARS)
• Each chromosome containing multiple ARS that
are spaced 30 kb apart.
Minimum sequence of ARS
• Minimum sequence requirement for a functional
ARS was done by deletion studies on the original
ARS sequences found.
• The ORC-binding site is where the origin
recognition complex bound.
How is DNA replication
triggered?
• ORC-origin form a stable complex
throughout the cell cycle.
• During G1 phase, a prerepicative complex
(Mcm helicase and Cdc6 helicase loading
factor) is assembled on each ORC.
• S phase is triggered when a protein kinase is
activated that assembles the rest of the
replication machinery.
How does the mechanism ensure
that a replication origin is used
only once during each cell cycle?
• After the rest of the replication machinery is
assembled, Mcm helicase will start moving,
forming two replication forks for each
origin.
• The protein kinase that triggers S phase will
prevent all further assembly of the Mcm
helicase into prereplicative complexes until
it is inactivated (M phase).
Mammalian replication origin
• Mammalian replication origin, such as b-globin
gene cluster, contains several thousand
nucleotides.
• DNA sequences that are distant from origin affect
the activity of replication origin (decondensing
effect).
Mammalian replication origin
• An ORC complex similar to yeast ORC is
probably existed in mammals. Also,
homologs of yeast Cdc6 and Mcm are
found.
• However, the binding sites for the ORC
proteins seem to be less specific in
mammals.
Eukaryotic DNA replication
involves nucleosomes
• Eukaryotic chromosomes are composed of
the mixture of DNA and protein.
• So chromosome duplication requires not
only the DNA be replicated but also that
new chromosomal proteins (histones) be
synthesized and assembled onto the DNA
behind each replication fork.
Histones remain associated with
DNA after replication fork passes
• By some unknown mechanism involving
chromatin-remodeling proteins, the replication
apparatus can pass through parental nucleosomes
without displacing them from the DNA.
• Both DNA helices inherit half of the old histones.
The structure of nucleosome
The synthesis of histones
• Vertebrate cells have about 20 sets of each
histone genes (H1, H2A, H2B, H3, and H4).
• Histones are synthesized during S phase.
Increased transcription and decreased
mRNA degradation makes the level of
histone mRNA to increase fiftyfold.
• Histone mRNA is quickly degraded after
DNA synthesis is stopped.
The assembly
of nucleosome
• Nucleosomes are
assembled by
chromatin assembly
factor (CAF).
• Newly synthesized
histones H3 and H4
are rapidly acetylated
on their N-terminal
tails.
The assembly of nucleosome
• The acetylated H3
and H4 will be
deacetylated after
they are
assembled into
chromatin.
The termination problem
• Prokaryotes have circular DNA which will
become entangled at the end of DNA
replication.
• Eukaryotes have linear DNA which will
have insufficient space for lagging strand
synthesis to start a new primer. The
consequences of this is shorter and shorter
chromosomes after generation.
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In E. coli the terminus
region consists of 6
termination sites, named
TerA-TerF, and
proteins called
(terminus
utilization
substance) binds to it.
When replication forks enter the terminus region
they pause, resulting two entangled daughter
duplex.
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Type I topo
catenane
Type II topo
Telomere and telomerase
• Eukaryotes have special nucleotide
sequences at the ends of their chromosomes,
which are incorporated into telomeres, and
attract an enzyme called telomerase.
• Telomere DNA sequences are similar in
many organisms. In humans, this sequence
is GGGTTA, extending about 10,000
nucleotides.
Telomerase
• Telomerase
synthesizes
telomere DNA
using an RNA
template that is a
component of the
enzyme itself.
The 3’ end at each telomere is
always slightly longer than
the 5’ end.
The end of telomere
DNA is protected
• The protruding 3’
end has been shown
to loop back to tuck
the single-stranded
terminus into the
duplex DNA of the
telomeric repeat
sequence.
The “tucked” 3’ overhang of
telomere
Telomere length is used as a
counting device of cell age
• Experiments showed that cells that
proliferate indefinitely have homeostatic
mechanisms that maintain the number of
these repeats within a limited range.
Yeast cells, which can divide
indefinitely, control the length of their
telomeres within a limited range
Telomeres and replicative cell
senescence
• Human fibroblasts normally proliferate for
about 60 cell divisions in culture, then it
will cease to divide, this phenomenon is
called replicative cell senescence.
• According to a hypothesis, somatic cells
like fibroblast has telomerases that are
turned off, so each time a it divides, about
50-100 nucleotides are lost.
Telomeres and replicative cell
senescence
• Eventually, descendent cells cease to divide
because their chromosomes are defective.
• This hypothesis was proven to be correct in
fibroblast supplied with active fibroblast.
• However, mouse with defective telomerase
does not age prematurely. However, these
mice do have a pronounced tendency to
develop tumors (dyskeratosis congenita).