Lesson 2

DNA Replication
 General Features
of DNA Replication
• Let us first consider the general mechanism of DNA replic
ation. The double-helical model for DNA includes the conc
ept that the two strands are complementary. Thus, each stra
nd can in principle serve as the template for making its ow
n partner. As we will see, this semiconservative model for
DNA replication is the correct one. In addition, molecular
biologists have uncovered the following interesting general
features of DNA replication: It is half discontinuous (made
in short pieces that are later stitched together); it requires
RNA primers; and it is usually bidirectional. Let us look at
each of these features in turn.
Semiconservative Replic
ation
• The Watson–Crick model for DNA replication assumed that as new
strands of DNA are made, they follow the usual base-pairing rules o
f A with T and G with C. The model also proposed that the two pare
ntal strands separate and that each then serves as a template for a ne
w progeny strand. This is called semiconservative replication becau
se each daughter duplex has one parental strand and one new strand.
In other words, one of the parental strands is “conserved” in each da
ughter duplex. However, this is not the only possibility. Another pot
ential mechanism (Figure 20.1b) is conservative replication, in whic
h the two parental strands stay together and somehow produce anoth
er daughter helix with two completely new strands. Yet another poss
ibility is dispersive replication, in which the DNA becomes fragmen
ted so that new and old DNAs coexist in the same strand after replic
ation. This mechanism was envisioned to avoid the formidable prob
lem of unwinding the two DNA strands.
• Three hypotheses for DNA replication. (a) Semiconservative replication gives
two daughter duplex DNAs, each of which contains one old strand (blue) and
one new strand (red). (b) Conservative replication yields two daughter duplexe
s, one of which has two old strands (blue) and one of which has two new stran
ds (red). (c) Dispersive replication gives two daughter duplexes, each of which
contains strands that are a mixture of old and new.
• In 1958, Matthew Meselson and Franklin Stahl pe
rformed a classic experiment to distinguish among
these three possibilities. They labeled E. coli DNA
with heavy nitrogen (15N) by growing cells in a m
edium enriched in this nitrogen isotope. This made
the DNA denser than normal. Then they switched
the cells to an ordinary medium containing primar
ily 14N, for various lengths of time. Finally, they s
ubjected the DNA to CsCl gradient ultracentrifuga
tion to determine the density of the DNA.
• Separation of DNAs by cesium chloride density gradient centrifugation. DNA cont
aining the normal isotope of nitrogen (14N) was mixed with DNA labeled with a h
eavy isotope of nitrogen (15N) and subjected to cesium chloride density gradient c
entrifugation. The two bands had different densities, so they separated cleanly. (a)
A photograph of the spinning rotor under ultraviolet illumination. Note that this is
a photograph through a window in the rotor as it spins. The ultracentrifuge rotor w
as designed to allow the experimenter to check its contents without stopping the ce
ntrifuge. The two dark bands correspond to the two different DNAs that absorb ultr
aviolet light. (b) A graph of the darkness of each band, which gives an idea of the r
elative amounts of the two kinds of DNA.
• Three replication hypotheses. The conserva
tive model (a) predicts that after one genera
tion equal amounts of two different DNAs
(heavy/heavy [H/H] and light/light [L/L])
will occur. Both the semiconservative (b) a
nd dispersive (c) models predict a single ba
ndof DNA with a density halfway between
the H/H and L/L densities.Meselson and St
ahl’s results confi rmed the latter prediction
, so the conservative mechanism was ruled
out. The dispersive model predicts that the
DNA after the second generation will have
a single density, corresponding to molecule
s that are 25% H and 75% L. This should g
ive one band of DNA halfway between the
L/L and the H/L band. The semiconservativ
e model predicts that equal amounts of two
different DNAs (L/L and H/L) will be pres
ent after the second generation. Again, the l
atter prediction matched the experimental r
esults, supporting the semiconservative mo
del.
• Results of CsCl gradient ultracentrifugation experiment that demonstra
tes semiconservative DNA replication. Meselson and Stahl shifted 15
N-labeled E. coli cells to a 14N medium for the number of generations
given at right, then subjected the bacterial DNA to CsCl gradient ultrac
entrifugation. (a) Photographs of the spinning centrifuge tubes under u
ltraviolet illumination. The dark bands correspond to heavy DNA (righ
t) and light DNA (left). A band of intermediate density was also observ
ed between these two and is virtually the only band observed at 1.0 an
d 1.1 generations. This band corresponds to duplex DNAs in which on
e strand is labeled with 15N, and the other with 14N, as predicted by t
he semiconservative replication model. After 1.9 generations, Meselso
n and Stahl observed approximately equal quantities of the intermediat
e band (H/L) and the L/L band. Again, this is what the semiconservativ
e model predicts. After three and four generations, they saw a progress
ive depletion of the H/L band, and a corresponding increase in the L/L
band, again as we expect if replication is semiconservative. (b) Densit
ometer tracings of the bands in panel (a), which can be used to quantif
y the amount of DNA in each band.
• The results of one more round of DNA replication ruled out the disper
sive hypothesis. Dispersive replication would give a product with one-
fourth 15N and three-fourths 14N after two rounds of replication in a
14N medium. Semiconservative replication would yield half of the pr
oducts as H/L and half as L/L (see Figure 20.3b). In other words, the h
ybrid H/L products of the fi rst round of replication would each split a
nd be supplied with new, light partners, giving the 1:1 ratio of H/L to
L/L DNAs. Again, this is precisely what occurred (see Figure 20.4). T
o make sure that the intermediatedensity peak was really a 1:1 mixtur
e of the heavy and light DNA, Meselson and Stahl mixed pure 15N-la
beled DNA with the DNA after 1.9 generations in 14N medium, then
measured the distances among the peaks. The middle peak was center
ed almost perfectly between the other two (50% 6 2% of the distance
between them). Therefore, the data strongly supported the semiconser
vative mechanism.
At Least Semidiscontinuous
Replication
• If we were charged with the task of designing a DNA replicating ma
chine, we might come up with a system such as the one pictured in F
igure 20.5a. DNA would unwind to create a fork, and two new DNA
strands would be synthesized continuously in the same direction as t
he moving fork. However, this scheme has a fatal fl aw. It demands t
hat the replicating machine be able to make DNA in both the 59→39
and 39→59 directions. That is because of the antiparallel nature of t
he two strands of DNA; if one runs 59→39 left to right, the other m
ust run 39→59 left to right. But the DNA synthesizing part (DNA po
lymerase) of all natural replicating machines can make DNA in only
one direction: 59→39. That is, it inserts the 59-most nucleotide first
and extends the chain toward the 39-end by adding nucleotides to th
e 39-end of the growing chain.
• Following this line of reasoning, Reiji Okazaki concluded that both str
ands could not replicate continuously. DNA polymerase could theoreti
cally make one strand (the leading strand) continuously in the 59→39
direction, but the other strand (the lagging strand) would have to be m
ade discontinuously. discontinuity of synthesis of the lagging strand c
omes about because its direction of synthesis is opposite to the
• direction in which the replicating fork is moving. Therefore, as the for
k opens up and exposes a new region of DNA to replicate, the lagging
strand is growing in the “wrong” direction, away from the fork. The o
nly way to replicate this newly exposed region is to restart DNA synth
esis at the fork, behind the piece of DNA that has already been made.
This starting and restarting of DNA synthesis occurs over and over ag
ain. The short pieces of DNA thus created would of course have to be
joined together somehow to produce the continuous strand that is the f
inal product of DNA replication.
• Continuous, semidiscontinuous, and disconti
nuous models of DNA replication. (a) Conti
nuous model. As the replicating fork moves t
o the right, both strands are replicated contin
uously in the same direction, left to right (bl
ue arrows). The top strand grows in the 39→
59 direction, the bottom strand in the 59→39
direction. (b) Semidiscontinuous model. Syn
thesis of one of the new strands (the leading
strand, bottom) is continuous (blue arrow), a
s in the model in panel (a); synthesis of the o
ther (the lagging strand, top) is discontinuou
s (pink arrows), with the DNA being made i
n short pieces. Both strands grow in the 59
→39 direction. (c) Discontinuous model. Bo
th leading and lagging strands are made in sh
ort pieces (i.e., discontinuously; pink arrow
s). Both strands grow in the 59→39 directio
n.
• The model of semidiscontinuous replication makes two pre
dictions that Okazaki’s team tested experimentally: (1) Bec
ause at least half of the newly synthesized DNA appears fi
rst as short pieces, one ought to be able to label and catch t
hese before they are stitched together by allowing only ver
y short periods (pulses) of labeling with a radioactive DNA
precursor. (2) If one eliminates the enzyme (DNA ligase) r
esponsible for stitching together the short pieces of DNA, t
hese short pieces ought to be detectable even with relativel
y long pulses of DNA precursor
• For his model system, Okazaki chose replication of phage
T4 DNA. This had the advantage of simplicity, as well as t
he availability of T4 ligase mutants. To test the first predict
ion, Okazaki and colleagues gave shorter and shorter pulse
s of 3H-labeled thymidine to E. coli cells that were replicat
ing T4 DNA. To be sure of catching short pieces of DNA b
efore they could be joined together, they even administered
pulses as short as 2 sec. Finally, they measured the approxi
mate sizes of the newly synthesized DNAs by ultracentrifu
gation.
• Experimental demonstration of at least semidiscontinuous DNA replication.
(a) Okazaki and his colleagues labeled replicating phage T4 DNA with very sh
ort pulses of radioactive DNA precursor and separated the product DNAs acco
rding to size by ultracentrifugation. At the shortest times, the label went prima
rily into short DNA pieces (found near the top of the tube), as the discontinuou
s model predicted. (b) When these workers used a mutant phage with a defecti
ve DNA ligase gene, short DNA pieces accumulated even after relatively long
labeling times (1 min in the results shown here).
• Already at 2 sec, some labeled DNA was visible in the gra
dient; within the limits of detection, it appeared that all of t
he label was in very small DNA pieces, 1000–2000 nt long
, which remained near the top of the centrifuge tube. With i
ncreasing pulse time, another peak of labeled DNA appear
ed much nearer the bottom of the tube. This was the result
of attaching the small, newly formed pieces of labeled DN
A to much larger, preformed pieces of DNA that were mad
e before labeling began. These large pieces, because they
were unlabeled before the experiment began, did not show
up until enough time had elapsed for DNA ligase to join th
e smaller, labeled pieces to them; this took only a few seco
nds. The small pieces of DNA that are the initial products
of replication have come to be known as Okazaki fragment
s.
• The predominant accumulation of small pieces of labeled DNA could be interpr
eted to mean that replication proceeded discontinuously on both strands, as pictu
red in Figure 20.5c. Indeed, this was Okazaki and colleagues’ interpretation. But
a commonly invoked alternative explanation is that some of the small DNA piec
es are created by a DNA repair system that removes dUMP residues incorporate
d into DNA. UTP is an essential precursor of RNA, but the cell also makes dUT
P, which can be accidentally incorporated into DNA (as dUMP) in place of dTM
P. Two enzymes help to minimize this problem. One of these, dUTPase—the pro
duct of the dut gene, degrades dUTP. The other, uracil N-glycosylase—the prod
uct of the ung gene, removes uracil bases from DNA, creating “abasicsites” that
are subject to breakage as part of the repair process. Thus, this repair process ge
nerates a certain component of short DNA pieces regardless of whether the repli
cating DNA is made continuously or discontinuously. The question is, what prop
ortion of the Okazaki fragments observed in experiments such as those depicted
in Figure 20.6 are due to discontinuous replication and what proportion to the re
pair of misincorporated dUMP residues?
• One way to answer this question would be to look at the sizes of new
ly labeled DNA fragments in dut1 ung2 cells. These cells minimize d
UMP incorporation (because of the presence of dUTPase) and cannot
create abasic sites (because of the absence of uracil N-glycosylase).
Therefore, strand breakage due to dUMP incorporation should be mi
nimized. In fact, this experiment has been done, and most newly labe
led DNAs are still small—Okazaki fragment size. Indeed, it appears t
hat, even in wild-type cells, the amount of dUMP incorporation is qu
ite low—far too low to explain the preponderance of Okazaki fragme
nts observed at short labeling times. These datasuggest a conclusion,
though it is not generally accepted, that replication on both strands o
ccurs discontinuously, at least in E. coli.
Priming of DNA Synthesis
• RNA polymerase initiates transcription simply by starting a new RNA
chain; it puts the fi rst nucleotide in place and then joins the next to it.
But DNA polymerases cannot perform the same trick with initiation o
f DNA synthesis. If we supply a DNA polymerase with all the nucleot
ides and other small molecules it needs to make DNA, then add either
single-stranded or double-stranded DNA with no strand breaks, the po
lymerase will make no new DNA. What is missing?
• We now know that the missing component is a primer, a piece of nucl
eic acid that the polymerase can “grab onto” and extend by adding nu
cleotides to its 39-end. This primer is not DNA, but a short piece of R
NA. First, a replicating fork opens up; next, short RNA primers are m
ade; next, DNA polymerase adds deoxyribonucleotides to these prime
rs, forming DNA, as indicated by the arrows.
• Priming in DNA syn
thesis. (a) The two p
arental strands (blu
e) separate. (b) Shor
t RNA primers (pin
k) are made. (c) DN
A polymerase uses t
he primers as startin
g points to synthesiz
e progeny DNA stra
nds (green arrows).
 Bidirectional Replication
• In the early 1960s, John Cairns labeled replicating E. coli DNA with a radioactive DNA pr
ecursor, then subjected the labeled DNA to autoradiography. Figure shows the results, alon
g with Cairns’s interpretation. The structure represented in Figure is a so-called theta struct
ure because of its resemblance to the Greek letter θ (theta). Because it may not be immedia
tely obvious that the DNA in Figure looks like a theta, Figure provides a schematic diagra
m of the events in the second round of replication that led to the autoradiograph. This draw
ing shows that DNA replication begins with the creation of a “bubble”—a small region wh
ere the parental strands have separated and progeny DNA has been synthesized. As the bub
ble expands, the replicating DNA begins to take on the theta shape. We can now recognize
the autoradiograph as representing a structure shown in the middle of Figure, where the cro
ssbar of the theta has grown long enough to extend above the circular part. The u structure
contains two replicating forks, marked X and Y in Figure. This raises an important questio
n: Does one of these forks, or do both, represent sites of active DNA replication? In other
words, is DNA replication unidirectional, with one fork moving away from the other,which
remains fi xed at the origin of replication? Or is it bidirectional, with two replicating forks
moving in opposite directions away from the origin? Cairns’s autoradiographs were not des
igned to answer this question, but a subsequent study on Bacillus subtilis replication perfor
med by Elizabeth Gyurasits and R.B. Wake showed clearly that DNA replication in that ba
cterium is bidirectional.
• The theta mode of DNA replication in Escherichia coli. (a) An auto
radiograph of replicating E. coli DNA with an interpretive diagram
. The DNA was allowed to replicate for one whole generation and
part of a second in the presence of radioactive nucleotides to label
the DNA. The interpretive diagram to the right uses red to represen
t labeled DNA and blue to represent unlabeled parental DNA. (b)
Detailed description of the theta mode of DNA replication. The col
ors have the same meaning as in panel (a)
• These investigators’ strategy was to allow B. subtilis cells to grow for
a short time in the presence of a weakly radioactive DNA precursor, th
en for a short time with a more strongly radioactive precursor. The lab
eled precursor was the same in both cases: [3H]thymidine. Tritium (3
H) is especially useful for this type of autoradiography because its rad
ioactive emissions are so weak that they do not travel far from their p
oint of origin before they stop in the photographic emulsion and creat
e silver grains. This means that the pattern of silver grains in the autor
adiograph will bear a close relationship to the shape of the radioactive
DNA. It is important to note that unlabeled DNA does not show up in
the autoradiograph. The pulses of label in this experiment were short e
nough that only the replicating bubbles are visible. You should not mi
stake these for whole bacterial chromosomes.
• Experimental demonstration of bidirectional DNA replication. (a) Autoradiograph of replicating Bacillus s
ubtilis DNA. Dormant bacterial spores were germinated in the presence of low radioactivity DNA precurs
or, so the newly formed replicating bubbles immediately became slightly labeled. After the bubbles had gr
own somewhat, a more radioactive DNA precursor was added to label the DNA for a short period. (b) Inte
rpretation of the autoradiograph. The purple color represents the slightly labeled DNA strands produced du
ring the low-radioactivity pulse. The orange color represents the more highly labeled DNA strands produc
ed during the later, high-radioactivity pulse. Because both forks picked up the high radioactivity label, bot
h must have been functioning during the high-radioactivity pulse. DNA replication in B. subtilis is therefo
re bidirectional
• If you look carefully at the above Figure, you will notice that the pa
ttern of silver grains is not uniform. They are concentrated near bot
h forks in the bubble. This extra labeling identifies the regions of D
NA that were replicating during the “hot,” or high-radioactivity, pul
se period. Both forks incorporated extra label, showing that they w
ere both active during the hot pulse. Therefore, DNA replication in
B. subtilis is bidirectional; two forks arise at a fixed starting point
—the origin of replication—and move in opposite directions aroun
d the circle until they meet on the other side. Later experiments em
ploying this and other techniques have shown that the E. coli chro
mosome also replicates bidirectionally.
Rolling Circle Replication
• Certain circular DNAs replicate, not by the theta mode, but by a me
chanism called rolling circle replication. The E. coli phages with sin
gle-stranded circular DNA genomes, such as fX174, use a relatively
simple form of rolling circle replication in which a double stranded
replicative form (RFI) gives rise to many copies of a single-strande
d progeny DNA, as illustrated in simplified form in Figure 20.12. T
he intermediates (steps b and c) give this mechanism the rolling circ
le name because the double-stranded part of the replicating DNA ca
n be considered to be rolling counterclockwise and trailing out the p
rogeny single-stranded DNA, rather like a roll of toilet paper unrolli
ng as it speeds across the floor. This intermediate also somewhat res
embles an upside-down Greek letter σ (sigma), so this mechanism i
s sometimes called the s mode, to distinguish it from the θ mode.
• Schematic representation of rolling circle replication that produces single-stranded circular progeny DNA
s. (a) An endonuclease creates a nick in the positive strand of the double stranded replicative form. (b) Th
e free 39-end created by the nick serves as the primer for positive strand elongation, as the other end of th
e positive strand is displaced. The negative strand is the template. Red denotes newly-synthesized DNA.
(c) Further replication occurs, as the positive strand approaches double length. The circle can be consider
ed to be rolling counterclockwise. (d) The unit length of positive strand DNA that has been displaced is cl
eaved off by an endonuclease and circularized. (e) Replication continues, producing another new positive
strand, using the negative strand as template. This process repeats over and over to yield many copies of t
he circular positive strand.
• The rolling circle mechanism is not confi ned to production of singl
e-stranded DNA. Some phages (e.g., λ) use this mechanism to repli
cate double-stranded DNA. During the early phase of λ DNA replic
ation, the phage follows the θ mode of replication to produce severa
l copies of circular DNA. These circular DNAs are not packaged int
o phage particles; they serve as templates for rolling circle synthesi
s of linear λ DNA molecules that are packaged. The bellow Figure s
hows how this rolling circle operates. Here, the replicating fork loo
ks much more like that in E. coli DNA replication, with (perhaps) c
ontinuous synthesis on the leading strand (the one going around the
circle) and discontinuous synthesison the lagging strand. In λ, the pr
ogeny DNA reaches lengths that are several genomes long before it
is packaged. The multiple-length DNAs are called concatemers. Th
e packaging mechanism is designed to provide each phage head wit
h one genome’s worth of linear DNA, so the concatemer is cut enzy
matically at the cos sites flanking each complete l genome on the co
• Rolling circle model for phage l DNA replication. As the circle rolls
to the right, the leading strand (red) elongates continuously. The lag
ging strand (blue) elongates discontinuously, using the unrolled lead
ing strand as a template and RNA primers for each Okazaki fragme
nt. The progeny double-stranded DNA thus produced grows to man
y genomes in length (a concatemer) before one genome’s worth is c
lipped off and packaged into a phage head.
 Enzymology of DNA
Replication

• Over 30 different polypeptides cooperate in replicating the E. coli D

NA. Let us begin by examining the activities of some of these protein
s and their homologs in other organisms, starting with the DNA poly
merases—the enzymes that make DNA.
Three DNA Polymerases
in E. coli
• Arthur Kornberg discovered the fi rst E. coli DNA polymerase in 195
8. Because we now know that it is only one of three DNA polymerase
s, we call it DNA polymerase I (pol I). In the absence of evidence for
other cellular DNA polymerases, many molecular biologists assumed
that pol I was the polymerase responsible for replicating the bacterial
genome. As we will see, this assumption was incorrect. Nevertheless,
we begin our discussion of DNA polymerases with pol I because it is r
elatively simple and well understood, yet it exhibits the essential char
acteristics of a DNA synthesizing enzyme.
 Pol I
• Although pol I is a single 102-kD polypeptide chain, it is remarkabl
y versatile. It catalyzes three quite distinct reactions. It has a DNA p
olymerase activity, of course, but it also has two different exonuclea
se activities: a 3’→5’, and a 5’→3’exonuclease activity. Why does a
DNA polymerase also need two exonuclease activities? The 3’→5’ a
ctivity is important in proofreading newly synthesized DNA. If pol I
has just added the wrong nucleotide to a growing DNA chain, this n
ucleotide will not base-pair properly with its partner in the parental s
trand and should be removed. Accordingly, pol I pauses and the 3’→
5’ exonuclease removes the mispaired nucleotide, allowing replicati
on to continue. This greatly increases the fidelity, or accuracy, of DN
A synthesis.
• Proofreading in DNA synthesis. (a) An adenine nucleotide
(pink) has been mistakenly incorporated across from a gua
nine. This destroys the perfect base pairing required at the
3’-end of the primer, so the replicating machinery stalls.
(b) This pause then allows Pol I to use its 3’→5’ exonuclea
se function to remove the mispaired nucleotide. (c) With th
e appropriate base-pairing restored, Pol I is free to continu
e DNA synthesis
• The 5’→3’ exonuclease activity allows pol I to degrade a strand ahea
d of the advancing polymerase, so it can remove and replace a strand
all in one pass of the polymerase, at least in vitro. This DNA degrada
tion function is useful because pol I seems to be involved primarily i
n DNA repair (including removal and replacement of RNA primers),
for which destruction of damaged or mispaired DNA (or RNA prime
rs) and its replacement by good DNA is required. The bellow Figure
illustrates this process for primer removal and replacement.
• Removing primers and joining nascent DNA frag
ments. (a) There are two adjacent progeny DNA
fragments, the right-hand one containing an RN
A primer (red) at its 5’-end. The two fragments a
re separated by a single-stranded break called a n
ick. (b) DNA polymerase I binds to the double-st
randed DNA at the nick. (c) The 5’→3’ exonucle
ase and polymerase activities of DNA polymeras
e I simultaneously remove the primer and fill in t
he resulting gap by extending the left-hand DNA
fragment rightward. The polymerase leaves degr
aded primer in its wake. (d) DNA ligase seals the
remaining nick by forming a phosphodiester bon
d between the left-hand and right-hand progeny
DNA fragments.
• Another important feature of pol I is that it can be cleaved by mil
d proteolytic treatment into two polypeptides: a large fragment (t
he Klenow fragment), which has the polymerase and proofreadin
g (3’→5’ exonuclease) activities; and a small fragment with the
5’→3’ exonuclease activity. The Klenow fragment is frequently u
sed in molecular biology when DNA synthesis is required and des
truction of one of the parental DNA strands, or the primer, is und
esirable. For example, the Klenow fragment is often used to perfo
rm DNA end-filling and can also be used to sequence a DNA. O
n the other hand, the whole pol I is used to perform nick translati
on to label a probe in vitro, because nick translation depends on
5’→3’ degradation of DNA ahead of the moving fork.
• In 1969, Paula DeLucia and John Cairns isolated a mutant
with a defect in the polA gene, which encodes pol I. This
mutant (polA1) lacked pol I activity, yet it was viable, stro
ngly suggesting that pol I was not really the DNA replicati
ng enzyme. Instead, pol I seems to play a dominant role in
repair of DNA damage. It fi lls in the gaps left when dama
ged DNA is removed. The fi nding that pol I is not essentia
l spurred a renewed search for the real DNA replicase, and
in 1971, Thomas Kornberg and Malcolm Gefter discovered
two new polymerase activities: DNA polymerases II and II
I (pol II and pol III). We will see that pol III is the actual re
plicating enzyme.
 Pol II and Pol III
• Pol II could be readily separated from pol I by phosphocellulose ch
romatography, but pol III had been masked in wild-type cells by th
e preponderance of pol I. Next, Kornberg, Gefter, and colleagues u
sed genetic means to search for the polymerase that is required for
DNA replication. They tested the pol II and III activities in 15 diffe
rent E. coli strains that were temperature-sensitive for DNA replica
tion. Most of these strains were polA1-, which made it easier to me
asure pol III activity after phosphocellulose chromatography becau
se there was no competing pol I activity. In those few cases where
pol I was active, Gefter and colleagues used N-ethylmaleimide to k
nock out pol III so its activity could be measured as the difference
between the activities in the presence and absence of the inhibitor.
• The most striking fi nding was that there were five strains with mutat
ions in the dnaE gene. In four of these, the pol III activity was very te
mperature-sensitive, and in the fi fth it was slightly temperature-sensi
tive. On the other hand, none of the mutants affected pol II at all. The
se results led to three conclusions: First, the dnaE gene encodes pol II
I. Second, the dnaE gene does not encode pol II, and pol II and pol III
are therefore separate activities. Third, because defects in the gene en
coding pol III interfere with DNA replication, pol III is indispensable
for DNA replication. It would have been nice to conclude that pol II i
s not required for DNA replication, but that was not possible because
no mutants in the gene encoding pol II were tested. However, in sepa
rate work, these investigators isolated mutants with inactive pol II, an
d these mutants were still viable, showing that pol II is not necessary
for DNA replication. Thus, pol III is the enzyme that replicates the E.
coli DNA.
The Pol III Holoenzyme
• The enzyme that carries out the elongation of primers to make both the leadi
ng and lagging strands of DNA is called DNA polymerase III holoenzyme (p
ol III holoenzyme). The “holoenzyme” designation indicates that this is a mu
ltisubunit enzyme, and indeed it is: As the bellow Table illustrates, the holoe
nzyme contains 10 different polypeptides. On dilution, this holoenzyme diss
ociates into several different subassemblies Each pol III subassembly is capa
ble of DNA polymerization, but only very slowly. This suggested that somet
hing important is missing from the subassemblies because DNA replication i
n vivo is extremely rapid. The replicating fork in E. coli moves at the amazin
g rate of 1000 nt/sec. (Imagine the sheer mechanics involved in unwinding p
arental DNA, correctly pairing 1000 nt with partners in the parental DNA str
ands, and forming 1000 phosphodiester bonds every second!) In vitro, the ho
loenzyme goes almost that fast: about 700 nt/sec, suggesting that this is the e
ntity that replicates DNA in vivo. The other two DNA polymerases in the cel
l, pol I and pol II, are not ordinarily found in holoenzyme forms, and they re
plicate DNA much more slowly than the pol III holoenzyme does
• Subunit Composition of E. coli DNA Polymerase III Holoenzyme
 Fidelity of Replication

• The proofreading mechanism of pol III (and pol I) greatly increases the fi delit
y of DNA replication. The pol III core makes about one pairing mistake in one
hundred thousand in vitro—not a very good record, considering that even the E
. coli genome contains over four million base pairs. At this rate, replication wo
uld introduce errors into a significant percentage of genes every generation. Fo
rtunately, proofreading allows the polymerase another mechanism by which to
get the base pairing right. The error rate of this second pass is presumably the s
ame as that of the first pass, or about 10-5. This predicts that the actual error rat
e with proofreading would be 10-5 X 10-5 = 10-10, and that is close to the actual
error rate of the pol III holoenzyme in vivo, which is 10 -10–10-11. This is a tolera
ble level of fidelity. In fact, it is better than perfect fidelity because it allows for
mutations, some of which help the organism to adapt to a changing environmen
t through evolution.
• Consider the implications of the proofreading mechanism,
which removes a mispaired nucleotide at the 3’-end of a D
NA progeny strand. DNA polymerase cannot operate witho
ut a base-paired nucleotide to add to, which means that it c
annot start a new DNA chain unless a primer is already the
re. That explains the need for primers, but why primers ma
de of RNA? The reason seems to be the following: Primers
are made with more errors, because their synthesis is not s
ubject to proofreading. Making primers out of RNA guaran
tees that they will be recognized, removed, and replaced w
ith DNA by extending the neighboring Okazaki fragment.
The latter process is, of course, relatively error-free, becau
se it is catalyzed by pol I, which has a proofreading functio
n.
Strand Separation

• In our discussion of the general features of DNA replication, we hav

e been assuming that the two DNA strands at the fork somehow un
wind. This does not happen automatically as DNA polymerase does
its job; the two parental strands hold tightly to each other, and it tak
es energy and enzyme action to separate them.
 Helicase

• The enzyme that harnesses the chemical energy of ATP to s

eparate the two parental DNA strands at the replicating for
k is called a helicase. We have already seen an example of
helicase action in Chapter 11, in our discussion of the DN
A helicase activity of TFIIH, which unwinds a short region
of DNA to help create the transcription bubble in eukaryot
es. That DNA melting is transient, in contrast to the perma
nent strand separation needed to advance a replicating fork
.
 Single-Strand DNA-Binding Proteins

• Another class of proteins, called single-strand DNA-bindin

g proteins (SSBs), also participate in DNA strand separatio
n during replication. These proteins do not catalyze strand
separation, as helicases do. Instead, they bind selectively t
o single-stranded DNA as soon as it forms and coat it so it
cannot anneal to re-form a double helix. The single strande
d DNA can form by natural “breathing” (transient local sep
aration of strands, especially in A–T-rich regions) or as a re
sult of helicase action, then SSB catches it and keeps it in s
ingle-stranded form.
 Topoisomerases

• Sometimes we refer to the separation of DNA strands as “unzipping.” We sh

ould not forget, when using this term, that DNA is not like a zipper with strai
ght, parallel sides. It is a double helix. Therefore, when the two strands of D
NA separate, they must rotate around each other. Helicase could handle this t
ask alone if the DNA were linear and unnaturally short, but closed circular D
NAs, such as the E. coli chromosome, present a special problem. As the DN
A unwinds at the replicating fork, a compensating winding up of DNA will
occur elsewhere in the circle. This tightening of the helix will create intolera
ble strain unless it is relieved. Cairns recognized this problem in 1963 when
he first observed circular DNA molecules in E. coli, and he proposed a “swi
vel” in the DNA duplex that would allow the DNA strands on either side to r
otate to relieve the strain (Figure 20.20). We now know that an enzyme kno
wn as DNA gyrase serves the swivel function. DNA gyrase belongs to a clas
s of enzymes called topoisomerases that introduce transient single- or doubl
e-stranded breaks into DNA and thereby allow it to change its shape, or topo
logy.
DNA Damage and Repair
• DNA can be damaged in many different ways, and this damage, if left
unrepaired, can lead to mutations: changes in the base sequence of a
DNA. This distinction is worth emphasizing at the outset: DNA dama
ge is not the same as mutation, although it can lead to mutation. DNA
damage is simply a chemical alteration to DNA. A mutation is a chang
e in a base pair. For example, the change from a G–C pair to an ethyl-
G–C pair is DNA damage; the change from a G–C pair to any other na
tural base pair (A–T or T–A or C–G) is a mutation. If a particular kind
of DNA damage is likely to lead to a mutation, we call it genotoxic. In
deed, we will see in the next section that the ethyl-G in our example is
genotoxic because it is likely to mispair with T instead of C during D
NA replication. If this happens, then another round of replication will
place an A across from the mispaired T, and conversion of the normal
G–C pair to an A–T pair (a true mutation) will be complete. Notice th
at this example illustrates the importance of DNA replication in conve
rsion of DNA damage to mutation.
• Damage Caused by Alkylation of Bases
• Damage Caused by Ultraviolet Radiation
• Damage Caused by Gamma and X-Rays
Damage Caused by
Alkylation of Bases
Damage Caused by Ultraviolet Radiati
on
 Repair
• One way to cope with DNA damage is to repair it, or restore it to its
original, undamaged state. There are two basic ways to do this: (1)
Directly undo the damage, or (2) remove the damaged section of D
NA and fill it in with new, undamaged DNA. Let us begin by lookin
g at two methods E. coli cells use to directly undo DNA damage.

• Directly Undoing DNA Damage

• Excision Repair
 Light repair
• In the late 1940s, Albert Kelner was trying to measure the effect of t
emperature on repair of ultraviolet damage to DNA in the bacterium
Streptomyces. However, he noticed that damage was repaired much
faster in some bacterial spores than in others kept at the same temper
ature. Obviously, some factor other than temperature was operating.
Finally, Kelner noticed that the spores whose damage was repaired f
astest were the ones kept most directly exposed to light from a labor
atory window. When he performed control experiments with spores
kept in the dark, he could detect no repair at all. Renato Dulbecco so
on observed the same effect in bacteria infected with UV radiation-d
amaged phages. It now appears that most forms of life share this imp
ortant mechanism of repair, which is termed photoreactivation, or lig
ht repair. However, placental mammals, including humans, do not ha
ve a photoreactivation pathway.
• It was discovered in the late 1950s that photoreactivation is catalyzed
by an enzyme called photoreactivating enzyme or photolyase. Actuall
y, two separate enzymes catalyze the repair of CPDs and (6-4) photop
roducts. The former is called CPD photolyase, or simply photolyase; t
he latter is known as (6-4) photolyase. The CPD photolyase operates b
y the mechanism sketched in the bellow Figure. First, the enzyme dete
cts and binds to the damaged DNA site (a pyrimidine dimer). Then the
enzyme absorbs light in the UV-A to blue region of the spectrum, whi
ch activates it so it can break the bonds holding the pyrimidine dimer t
ogether. This restores the pyrimidines to their original independent sta
te. Finally, the enzyme dissociates from the DNA; the damage is repai
red
• Model for photoreactivation.
(a) Ultraviolet radiation cause
s a pyrimidine dimer to form.
(b) The DNA photolyase enz
yme (red) binds to this region
of the DNA. (c) The enzyme
absorbs near-UV to visible li
ght. (d) The enzyme breaks th
e dimer and finally dissociate
s from the repaired DNA.
Excision Repair
• The percentage of DNA damage products that can be handled by di
rect reversal is necessarily small. Most such damage products invol
ve neither pyrimidine dimers nor O6-alkylguanine, so they must be
handled by a different mechanism. Most are removed by a process
called excision repair. The damaged DNA is fi rst removed, then re
placed with fresh DNA, by one of two mechanisms: base excision r
epair or nucleotide excision repair. Base excision repair is more pre
valent and usually works on common, relatively subtle changes to
DNA bases, such as chemical modifi cations caused by cellular age
nts. Nucleotide excision repair generally deals with more drastic ch
anges to bases, many of which distort the DNA double helix. These
changes tend to be caused by mutagenic agents from outside of the
cell. A good example of such damage is a pyrimidine dimer caused
by UV light.
Base Excision Repair
• Base excision repair in E. coli. (a) DN
A glycosylase extrudes the damaged ba
se (red). (b) DNA glycosylase removes
the extruded base, leaving an apurinic
or apyrimidinic site on the bottom DN
A strand. (c) An AP endonuclease cuts t
he DNA on the 59-side of the AP site.
(d) DNA phosphodiesterase removes th
e AP-deoxyribose phosphate (yellow bl
ock at right) that was left by the DNA g
lycosylase, (e) DNA polymerase I fi lls
in the gap and continues repair synthesi
s for a few nucleotides downstream, de
grading DNA and simultaneously repla
cing it. (f) DNA ligase seals the nick le
ft by the DNA polymerase.
Nucleotide Excision Repai
r
• Bulky base damage, including pyrimidine dimers, can be removed directly,
without help from a DNA glycosylase. In this nucleotide excision repair (N
ER) pathway, the incising enzyme system recognizes the strand with the bu
lky damage and makes cuts on either side of the damage, removing an olig
onucleotide with the damage. The key enzyme E. coli cells use in this proc
ess is called the uvrABC endonuclease because it contains three polypeptid
es, the products of the uvrA, uvrB, and uvrC genes. This enzyme cuts the d
amaged DNA, producing an oligonucleotide that is 12–13 bases long, depe
nding on whether the damage affects one nucleotide (alkylations) or two (p
yrimidine dimers). A more general term for the enzyme system that catalyz
es nucleotide excision repair is excision nuclease, or excinuclease. As we w
ill soon see, the excinuclease in eukaryotic cells removes an oligonucleotid
e about 24–32 nt long, rather than a 12- to 13-mer. In any case, DNA poly
merase fi lls in the gap left by the excised oligonucleotide and DNA ligase
seals the final nick.
• Nucleotide excision repair in E. coli (a) The UvrABC excinuclease cut
s on either side of a bulky damaged base (red). This causes removal
(b) of an oligonucleotide 12 nt long. If the damage were a pyrimidine
dimer, then the oligonucleotide would be a 13-mer instead of a 12-mer
. (c) DNA polymerase I fi lls in the missing nucleotides, using the top
strand as template, and then DNA ligase seals the nick to complete the
task, as in base excision repair.
Self-study

• Probable Roles of Some Eukaryotic DNA Polymerases

• DNA repair in Eukaryotic cells

• Homologous Recombination
REVIEW QUESTIONS
• 1. Compare and contrast the conservative, semiconservative, and dispersive mechanisms of DNA replic
ation.
• 2. Describe and give the results of an experiment that shows that DNA replication is semiconservative.
• 3. Compare and contrast the continuous, discontinuous, and semidiscontinuous modes of DNA replicati
on.
• 4. Describe and give the results of an experiment that shows that DNA replication is at least semidiscon
tinuous.
• 5. What is the evidence for fully discontinuous DNA replication in E. coli cells?
• 6. Describe and give the results of an experiment that measures the size of the primers on Okazaki frag
ments.
• 7. Present electron microscopic evidence that DNA replication of the B. subtilis chromosome is bidirect
ional, whereas replication of the colE1 plasmid is unidirectional.
• 8. Diagram the rolling circle replication mechanism used by the l phage.
• 9. Diagram the proofreading process used by E. coli DNA polymerases.
• 10. What activities are contained in E. coli DNA polymerase I? What is the role of each in DNA replica
tion?
• 11. How does the Klenow fragment differ from the intact E. coli DNA polymerase I? Which enzyme w
ould you use in nick translation? DNA end-fi lling? Why?
• 12. Of the three DNA polymerases in E. coli, which is essential for DNA replication? Present evidence.
• 13. Which pol III core subunit has the DNA polymerase activity? How do we know?