Translesion DNA Polymerases Remodel the Replisome and Alter the Speed of the

Replicative Helicase
Author(s): Chiara Indiani, Lance D. Langston, Olga Yurieva, Myron F. Goodman and Mike
O'Donnell
Source: Proceedings of the National Academy of Sciences of the United States of America,
Vol. 106, No. 15 (Apr. 14, 2009), pp. 6031-6038
Published by: National Academy of Sciences
Stable URL: https://www.jstor.org/stable/40482038
Accessed: 10-07-2020 15:37 UTC

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Translesion DNA polymerases remodel the replisome
and alter the speed of the replicative helicase
Chiara Indiani3, Lance D. Längstem3, Olga Yurieva3, Myron F. Goodman0, and Mike O'Donnella'1
aLaboratory of DNA Replication, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, Box 228, New York, NY 10065;
and bDepartments of Biological Sciences and Chemistry, University of Southern California, Los Angeles, CA 90089
This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2006.
Contributed by Mike O'Donnell, February 11, 2009 (sent for review January 22, 2009)
All cells contain specialized translesion DNA polymerases that
replicate past sites of DNA damage. We find that Escherichia coli
translesion DNA polymerase II (Pol II) and polymerase IV (Pol IV)
function with DnaB helicase and regulate its rate of unwinding,
slowing it to as little as 1 bp/s. Furthermore, Pol II and Pol IV freely
exchange with the polymerase III (Pol III) replicase on the ß-clamp
and function with DnaB helicase to form alternative repl ¡somes,
even before Pol III stalls at a lesion. DNA damage-induced levels of
Pol II and Pol IV dominate the clamp, slowing the helicase and
stably maintaining the architecture of the replication machinery
while keeping the fork moving. We propose that these dynamic
actions provide additional time for normal excision repair of
lesions before the replication fork reaches them and also enable
the appropriate translesion polymerase to sample each lesion as it
is encountered.
DNA repair | DNA replication | replication fork | replisome sliding clamp
Chromosomes
ing are duplicated
helicase, primase, and DNA by replisome
polymerase machines
activités (1). contain-
Replicative DNA polymerases are tethered to DNA by ring-
shaped sliding clamp proteins. The Escherichia coli polymerase
III (Pol III) holoenzyme replicase contains 10 different subunits
consisting of a clamp loader that binds 2 molecules of Pol III,
each tethered to DNA by the ß-sliding clamp for processive
leading and lagging strand synthesis (2). Pol III also binds tightly
to the hexameric DnaB helicase that encircles the lagging strand
template. The Pol III-toDnaB interaction is mediated by the
r-subunit within Pol III (3). This tight connection couples Pol III
motion to DnaB unwinding and increases the rate of unwinding
by DnaB helicase from a basal level of 35 bp/s to a rate in excess
of 500 bp/s (3). These tight connections of Pol III to DnaB and
the ß-clamp provide the replisome with sufficient stability, in
principle, to replicate the entire genome. In practice, however,
replication of long chromosomal DNA is an uneven path with a
variety of obstacles along the way, including protein blocks and
damaged templates.
In the presence of high levels of DNA damage, >40 gene
products are induced by the SOS response that act to restore
genomic integrity and assure cell survival (4). Among the
SOS-induced gene products are 3 translesion DNA polymerases,
Pols II, IV, and V (encoded by polB, dinB, and umuCD,
respectively), that perform potentially mutagenic DNA synthesis
across template lesions (5). Pols IV and V are members of the
Y family of error-prone DNA polymerases; they lack a proof-
reading 3 '-5' exonuclease (6, 7). Pol II is a B-family polymerase;
it has high fidelity and contains proofreading activity (8). Pols II
and IV are present during normal cell growth and may be
involved in repairing low levels of DNA damage that occur
routinely in the cell (9-11). Pol V, however, is closely associated
with mutagenesis and is not detectable in cells under normal
conditions (7, 12, 13).
Pol II and Pol IV are among the first genes that are up-
regulated in the SOS response, whereas the levels of Pol V are
www.pnas.org/cgi/doi/IO.IOya/pnas.OQOIAOBlOe
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very slow to rise, peaking 45 min after SOS induction (4, 9, 11,
12). In vitro studies have shown that these DNA polymerases
function with the ß-clamp for lesion bypass and trade places on
ß with a stalled Poll III replicase, as originally demonstrated in
bacteriophage T4 (14-17). Once the lesion is bypassed, the
high-fidelity Pol III can switch back on the ß-clamp to reestablish
the fast moving E. coli replisome (650 nt per s) (14, 17). In this
view of translesion synthesis, the TLS polymerase only occupies
the ß-clamp during the short time interval of lesion bypass.
It seems unlikely that TLS polymerases would be able to
replace Pol III in the context of a moving replisome, because
TLS polymerases are very slow and have low fidelity. These
characteristics are inconsistent with the rapid speed and high
fidelity required for chromosome replication. It is possible that
TLS polymerases are excluded from replisomes by the tight
connection between Pol III and the DnaB helicase via the
r-subunit. Additional insurance against TLS polymerase entry
into the replisome is the known action of DnaB helicase when it
becomes uncoupled from Pol III, whereupon it continues for
only «1 kb before DnaB stops unwinding (18-20). Presumably,
DnaB dissociates from DNA when it is uncoupled from leading-
strand Pol III action. Once DnaB dissociates, it cannot get back
onto DNA without specialized helicase-loading factors, and
replication fork progression is halted. Therefore, even if a TLS
polymerase enters the replisome, the subsequent loss of Pol III
interaction with DnaB may result in DnaB dissociation, prevent-
ing further progression of the fork.
Fork breakdown upon takeover by Pol IV is supported by
recent studies that show that overexpression of Pol IV in the
absence of DNA damage inhibits cell growth in Bacillus subtilis
and£. coli (21, 22). The authors of the£. coli study (21) note that
induction of Pol IV stops replication abruptly and kills the cell,
suggesting that Pol IV entry into the replisome is catastrophic.
Interestingly, these observations did not require the clamp
I blocks
binding residues of Pol IV, suggesting that Pol IV
replication by a process distinct from TLS polymerase switching
u
on a clamp. However, Pol IV was overexpressed to abnormally
2
high levels in that study. Thus, the mechanism of the replication
block by Pol IV was suggested to be separate from the physio-
logical action of the enzyme. Lower levels of Pol IV expression
appeared to decrease replication without cell killing, suggesting
Pol IV may slow a replication fork, but this hypothesis could not
be confirmed and was not tested in vitro.
Author contributions: C.I., L.D.L., and M.O.D. designed research; C.I., LD.L, and O.Y.
performed research; M.F.G. contributed new reagents/analytic tools; C.I., LD.L, M.F.G.,
and M.O.D. analyzed data; and C.I., LD.L, and M.O.D. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
See Commentary on page 6027.
1To whom correspondence should be addressed. E-mail: odonnel@mail.rockefeller.edu.
This article contains supporting information online at www. pnas.org/cgi/content/fu 1 1/
0901 403 1 06/DCSupplemental.
PNAS | April 14,2009 | vol.106 | no. 15 | 6031-6038
Fig. 1. Pol II and Pol IV form alternative replisomes with DnaB helicase and the ß clamp. (A) Minicircle DNA was incubated with DnaB, then TLS polymerase
(5 pmol), clamp loader, clamp and dCTP, dGTP for 5 min. Replication was initiated by addition of SSB, DnaG primase, dATP, dTTP, [a32P]dTTP, and the 4 rNTPs.
Newly-synthesized leading strand DNA is in red, and lagging strand is in blue. As controls, either DnaB or clamp/clamp loader were excluded from the reaction.
(B and O Time courses of Pol II (B) or Pol IV (Q in the presence of DnaB, the j3-clamp, and the clamp loader (lanes 1-6), in the absence of clamp and clamp loader
(lanes 7-12), and in the absence of DnaB (lanes 13-18). Products were analyzed in alkaline agarose gels. (D) Pol II (15 pmol) and Pol IV [5 pmol (Center) or 7 pmol
(Right)] were added simultaneously to the reaction. (Left) The gel shows synthesis by Pol II in the absence of Pol IV for comparison.

In the current study we examine the effects of normal and absence of ssDNA-binding protein (SSB), then the ß-clamp was
induced levels of Pol II and Pol IV on moving replisomes loaded onto DNA by using the clamp loader followed by the
reconstituted in vitro. We find that these TLS polymerases can addition of Pol II or Pol IV in the presence of only dCTP and
gain access to the moving Pol III replisome despite the strong Pol dGTP to prevent fork movement. After 5 min, replication was
III-T-DnaB connection. Surprisingly, the fork does not break initiated upon adding dATP, dTTP, SSB, DnaG primase, and
down. Instead the TLS polymerases function with DnaB to form the 4 rNTPs (see Fig. L4). In these assays, dissociation of DnaB
replisomes that move more slowly than the intrinsic rate of from DNA will halt the fork because SSB is present and the assay
DnaB, with the rate of helicase unwinding adjusting to the speed does not contain DnaB-reloading factors. Replication fork pro-
of the polymerase at the fork. At the levels of Pol II and Pol IV gression is followed by analysis of timed aliquots in an alkaline
present in undamaged cells, the Pol III replisome is unaffected, agarose gel. The results are compared with similar reactions
whereas at levels of Pol II and Pol IV that correspond to their performed in the absence of DnaB helicase or the absence of the
concentrations in DNA damage-induced cells, the TLS poly- clamp and clamp loader.
merases dominate the replication fork and slow it down. Ex- Surprisingly, the analysis shows that Pol II and Pol IV function
pression of Pol II and Pol IV in vivo in the absence of DNA with DnaB and the ß-clamp, because the presence of both of
damage also slows DNA replication and depends on TLS these factors is required to synthesize long DNA chains (Fig. 1
polymerase interaction with the ß-clamp. Slow replication fork B and C, lanes 1-6). DNA synthesis continues for at least 20 min,
advance in the context of DNA damage makes biological sense indicating that the DnaB helicase acts processively and is main-
as it buys time for normal DNA repair, decreasing collision tained the entire time. The rates of Pol II and Pol IV fork
events of the fork with DNA lesions. progression are «40 nucleotides (ntd)/s and 1 ntd/s, respectively
(Fig. SI), significantly slower than Pol III replisomes under the
Results
same conditions and consistent with the slower synthetic rates of
Poland
Pol II and Pol IV Form Alternative Replisomes with the ß-Clamp II and Pol IV compared with Pol III (24, 25). The ability of
PolPol
DnaB Helicase. To investigate possible alternative roles for II and
II Pol IV to function with DnaB in the absence of Pol
and Pol IV beyond translesion synthesis, we examined III their
indicates that replisomes containing these TLS polymerases
ability to form a functional replisome with the ß-clamp and intact rather than undergo replication fork breakdown.
remain
DnaB helicase in the absence of Pol III. For these studies, we results also suggest that the speed of a replicative helicase
These
used a minicircle replication fork substrate consistingcan of bea limited by the rate of synthesis of the polymerase traveling
synthetic 100-mer circular duplex with a 5' T40 ssDNA tail along behind it. Lagging strand products are also observed by
(20,
using [a32P] dATP as the radiolabel as shown in Fig. S2. We
23). Each strand of the synthetic minicircle substrate contains
only 3 nt: dG, dC, dA on the leading strand template, and dG, that leading and lagging strand synthesis is uncoupled
presume
under these conditions, as further described in Discussion.
dC, dT on the lagging stand template. Hence, leading and lagging
Both Pol II and Pol IV are present in normal cells and are
strands can be specifically labeled in separate reactions using
either [a32?] dTTP or [a32F] dATP, respectively. greatly up-regulated during the SOS response. As shown in Fig.
The replication machinery at the fork was assembledID, bythe rate of DNA synthesis in the presence of Pol II and Pol
loading DnaB on the 5' dTw tail of the minicircle DNA in IV the
(Center and Right) was slower than that of Pol II alone (Left),

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 LU
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u

of unlabeled dNTPs (chase) was added to prevent incorporation

I
of additional [a32P] dTTP. Then, Pol III holoenzyme lacking the22<
jS-clamp (referred to as Pol IIP) was added. If Pol IIP takes
control of the replication fork, the radiolabel incorporated by Pol
II or Pol IV during the pulse period will quickly shift to a higher
molecular mass during the chase period because of the rapid rate>-
ce
of synthesis by Pol IIP-ß. As shown in Fig. IB, Pol II (Left) <
synthesized ^3 kb during the 7-min pulse of [a32P] dTTP, andZ LU

Pol IV (Right) synthesized ^1 kb during a 15-min pulse (lanes 1,

O
5, and 9), consistent with the results of Fig. 1. Addition of a large
excess of cold dNTPs had no effect on the rate of synthesis by LU

Pol II or Pol IV in the absence of added Pol IIP (Fig. IB, lanes
2, 6, and 10), but when Pol IIP was added immediately after the
chase, the pulse-labeled band shifted dramatically higher, in a
time-dependent manner (Fig. IB, lanes 3, 7, and 11). As a control
for the effectiveness of the chase, Pol IIP was added to
replication forks assembled without Pol II or Pol IV and showed
no detectable incorporation of [a32P] dTTP (Fig. IB, lanes 4, 8,
and 12).
This pulse -chase experiment confirms that the DnaB helicase
is fully functional within the alternative Pol II and Pol IV
replisomes and that the basic architecture of the machinery at
the replication fork is maintained by the specialized DNA
polymerases despite their slow speed compared with Pol IIP-ß.
Hence, DnaB is stable on DNA in a functional state, and its rate
Fig. 2. Pol III regains control from a moving Pol II- or Pol IV-based replisome. of DNA unwinding is determined by the speed of the DNA
(A) The Pol II- or Pol IV-based replisome was assembled as in Fig. 1 A After a polymerase that functions with it. We also conclude that Pol IIP
7-min pulse (Pol II) or 15-min pulse (Pol IV), a 100-fold excess of unlabeled takeover of Pol II and Pol IV replisomes is rapid and efficient.
dNTPs (chase) was added 10 s before Pol III (1 pmol), and reactions were
quenched at different times after the addition of Pol III. Radiolabeled DNA Pol II and Pol IV Recruit the Clamp and Helicase from a Moving Pol III
synthesized by the TLS polymerase during the pulse period is shown in red, and Replisome. Our previous studies with singly-primed ssDNA cir-
DNA synthesized by Pol III during the chase period is in blue. (B) DNA synthesis cles showed that when Pol III holoenzyme was stalled at a primed
by Pol II (Left) or Pol IV (Right) without Pol III and cold dNTPs (lanes 1, 5, and
site by nucleotide omission Pol IV takes control of the primer
9), with cold dNTPs (lanes 2, 6, and 10), and with Pol III and cold dNTPs (lanes
terminus even at low concentrations of Pol IV, but when Pol III
3, 7, and 11). Control experiments where Pol III was added to an unoccupied
fork after addition of cold dNTPs are shown in lanes 4, 8, and 12. is engaged in active DNA synthesis, much higher levels of Pol IV
are required to take over the primed site (14). However, at a
replication fork the Pol III holoenzyme has several additional
suggesting that both polymerases have access to the fork and connections that may anchor it to DNA and block entry of other
alternate in taking control of the growing DNA ends in a DNA polymerases. Specifically, at a replication fork Pol III
dynamic fashion. If the 2 polymerases did not repeatedly trade connects to 2 ß-clamps on both the leading and lagging strands
places at the fork, 2 distinct products would be observed that through its twin polymerase structure. Furthermore, Pol III
correspond to the rates of Pol II- and Pol I V-driven replicationholoenzyme also connects to the DnaB helicase via the T-subunit
forks. We confirmed that Pol II acts in a distributive manner at (26). These Pol Ill-specific connections may prevent takeover of
the replication fork by showing that the speed of Pol II repli- a Pol III replisome by Pol II or Pol IV.
somes depends on the Pol II concentration (Fig. S3). Based on the results of Figs. 1 and 2, one would predict that
if Pol II or Pol IV can recruit the ß-clamp from a Pol III
replisome, the result would be a functional, but slow, alternative
Pol III Switches with Pol II and Pol IV to Produce a Rapid Replisome. >■
ce
replisome. To examine the ability of TLS polymerases to take H-

The experiments of Fig. 1 indicate that DnaB is a component of V£>

over a moving replication fork, increasing concentrations of Pol

the Pol II and Pol IV replisomes and presumably stimulates LU

II and Pol IV were added to a moving Pol Ill-based replisome

X
u
synthesis by unwinding DNA. However, DnaB is not known to o
in the minicircle replication fork assay along with [a32P]dTTP CÛ
function with other DNA polymerases, nor is DnaB known to
(leading strand) or [o;32P]dATP (lagging strand), as illustrated in
unwind DNA as slowly as observed in these alternative repli-
Fig. 3A Timed aliquots were collected and analyzed in an
somes. Hence, it remained possible that DnaB helicase is acting
alkaline agarose gel. The first 4 lanes in Fig. 3B show the long
in some other capacity to stimulate synthesis on the minicircle
leading-strand products typically formed by the fast-moving Pol
replication fork substrate. If DnaB is truly functional and III replisome, as reported (20). Reactions were then analyzed in
encircles DNA in the alternative replisomes, then Pol III should
the presence of increasing amounts of Pol II (Fig. 3B Left, lanes
be capable of displacing the TLS polymerase with resumption of or Pol IV (Fig. 3B Right, lanes 5-20). The results indicate
5-20)
the rapid unwinding characteristic of the coupled DnaB-Polthat III both Pol II and Pol IV interfere with the accumulation of
replisome. To examine this issue and provide further evidence long leading-strand products generated by Pol III in a dose-
that replisomes containing Pol II or Pol IV are fully functional
dependent manner. Inspection of the gel clearly shows that Pol
and have not broken down, we performed a series of pulse-chase IV is more efficient at inhibiting the replisome than Pol II;
experiments by adding Pol III to a prelabeled replication fork rolling circle replication was reduced ^50% upon addition of 0.5
containing a moving Pol II or Pol IV (see Fig. 24). Pol II or Pol
pmol of Pol IV (Fig. 3B Right, lanes 9-12), whereas 2 pmol of Pol
IV was first assembled with DnaB, the sliding clamp, and clamp II was required for a 40% decrease of DNA synthesis (Fig. 3B
loader in a 5-min preincubation, and then DNA synthesis was Left, lanes 13-16). Lagging-strand replication was reduced to the
initiated for 7 min (Pol II) or 15 min (Pol IV) in the presencesame
of extent as leading-strand synthesis (Fig. S4).
[o!32P] dTTP (pulse). After this labeling reaction, a large excessAll 5.E. coli polymerases function with the clamp and bind ß

Indiani et al. PNAS | April 14, 2009 | vol.106 | no. 15 | 6033

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 Fig. 4. Pol II and Pol IV slow down the replication fork. (A) The Pol Ill-based
replisome was assembled as described in Fig. 3A DNA synthesized by Pol III is
in blue, and DNA synthesized by the TLS polymerase is in red. (B) After 25 s (0
time point, lanes 1 and 9) a high concentration of Pol II (27 pmol; Left) or Pol
IV (35 pmol; Right) was added, and aliquots were collected (lanes 2-8). Time
courses of Pol III alone (lanes 9-1 6) are shown for comparison. The dotted red
line highlights the difference in DNA chain length at 120 s with and without
added Pol II.

Fig. 3. Pol II and Pol IV takeover of Pol Ill-based replisomes. (^)The replisome
is assembled on DNA as in Fig. 1, except Pol III* is used instead of Pol II or Pol
IV. After 10 s, different amounts of TLS polymerase (pink) are added together
with [a32P]dTTPto label leading-strand DNA. Newly synthesized DNA is in blue
(lagging) and red (leading). (B) Time courses of Pol III replication in the
presence of 0 pmol (lanes 1-4), 0.1 pmol (lanes 5-8), 0.5 pmol (lanes 9-12), 2
pmol (lanes 13-16) and 5 pmol (lanes 17-20) of Pol II (Left) or Pol IV (Right).
Timed aliquots of leading strands were analyzed in alkaline agarose gels
(lagging strand analysis is in Fig. S4). (Q Five picomoles of WT Pol II or Pol IV
(lanes 7-12) or Pol II/IV AC-term (lanes 13-18) were added to a moving Pol III
on a minicircle substrate. Leading-strand synthesis is shown (Pol III only is in
lanes 1-6).
fork by the T-subunit of the clamp loader, and this connection is
essential for rapid, processive rolling circle replication by Pol
III*. Because Pol III is poorly processive in the absence of t, we
were not able to determine whether t plays a role in the takeover
of a Pol III replisome by Pol II or Pol IV. We did, however,
determine that r is not required for rolling circle replication by
Pol II (Fig. S5), suggesting that the role played by rin stimulating
DnaB-dependent replication might be unique to Pol III, perhaps
by making Pol III more processive in the context of the repli-
cation fork rather than by directly stimulating unwinding by
DnaB.

through a conserved consensus sequence (27). Pol II and Pol IV Pol II and Pol IV Remodel the Pol Ill-Based Replisome and Slow
bind to ß via the C-terminal 5 or 6 amino acid residues, Replication Fork Advance. The results of Fig. 3 indicate that when
respectively (24, 28). To determine whether Pol II and Pol IV either Pol II or Pol IV is added to a Pol Ill-based replisome, the
function with ß in takeover of Pol Ill-based replisomes, we replisome is remodeled to include the TLS polymerase, leading
expressed and purified mutant versions of Pol II and Pol IV that to a slower moving fork that does not break down but remains
lack their respective C-terminal ß-binding residues and tested intact. Nonetheless, it remains possible that Pol II and Pol IV
them in the minicircle replication assay outlined in Fig. 3A. For simply inhibit the replication reaction without taking control of
these experiments, 5 pmol of either WT or mutant Pol II or Pol the replication fork or stop the fork and cause it to break down.
IV was added 10 s after initiation of replication by Pol III. As Hence, we designed the experiments illustrated in the scheme of
expected for ß-dependent function, the C-terminal mutant Pol Fig. 44 to determine whether the replication fork continues to
II and Pol IV were no longer capable of inhibiting Pol Ill-based slowly advance after the addition of Pol II or Pol IV. Because Pol
replisomes in the minicircle replication assay (compare WT Ill-based replisomes quickly produce replication products that
lanes 7-12 vs. AC-term lanes 13-18 for Pol II, Fig. 3C Left, and approach the resolution limit of alkaline gels, we examined very
Pol IV, Fig. 3C Right). These results support the conclusion that short time points after addition of the TLS polymerase. In the
Pol II and Pol IV take control of the replisome from Pol III by case of Pol II, it is clear that the fork does not break down; fork
competitive binding to the clamp and slow the fork. As a control, progression continues but at a slower rate (Fig. AB Left). Pol IV
we determined that the mutant Pol II and Pol IV retained >70% appears to stop the replication fork (Fig. AB Right), but consid-
ering that the Pol IV fork only moves 1 ntd/s it would only extend
of the activity of the corresponding WT polymerase in gap-filling
assays that do not require ß. DNA by 120 nt at the final time point of this experiment. This
short extension cannot be detected considering that the Pol III
Pol III is connected to the DnaB helicase at the replication

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 |
<
eg

To test whether the replication inhibition observed upon

expression of Pol II and Pol IV in vivo depends on interaction
of the 2 specialized polymerases with the ß-clamp, genes en-
I
coding Pol II and Pol IV mutants that lack the C-terminal
ß-interacting residues were cloned into the arabinose inducible
plasmid and tested for [3H] thymidine incorporation in the
presence and absence of arabinose. The mutant polymerases
were found to have no measurable effect on DNA synthesis. The |
results in Fig. 5 D and E indicate that Pol II and Pol IV interact
with ß to reduce intracellular replication, consistent with the in
vitro results of Fig. 3C.
Although we were unable to distinguish whether DNA syn-
thesis in vivo was slowed or stopped by overexpression of Pol II
and Pol IV, the results of Fig. 5 are remarkably consistent with
the in vitro experiments of Fig. 3, suggesting that the reconsti-
tuted rolling circle replication system can be mimicked by Pol II
and Pol IV overexpression in living cells. Despite the good
agreement of the in vivo and in vitro data, we cannot eliminate
the possibility that differences in sulA inhibition of cell growth
could reflect a differential induction of the SOS response when
WT Pol II or Pol IV are overexpressed, and thereby might
account, at least in part, for the differences in DNA synthesis
(Fig. 5 B and C). It is interesting to note that UV irradiation of
Pol II mutant cells delays recovery of replication restart (29),
which may be caused, in part, by more efficient takeover by Pol
IV in the absence of Pol II (i.e., Pol IV is the more potent of the
2 polymerases in slowing the fork). However, a subsequent study
did not observe a defect in the rate of recovery after UV
irradiation of either Pol II or Pol IV mutant cells (30), suggesting
that the rate of recovery depends on the kinetics of the repair
Fig. 5. Expression of Pol II and Pol IV in vivo reduces chromosomal DNA processes themselves and not on whether replication proceeds
synthesis. The rate of DNA synthesis is expressed as incorporation of [3H]thy-
slowly while repair is occurring behind the fork.
midine over a 3-min interval starting 45 min after overexpression of the TLS
polymerase by the addition of arabinose. (A) Scheme of the experiment as Discussion
detailed in Experimental Procedures. (B) Arabinose-induced expression of Pol
This article demonstrates that E. coli translesion DNA poly-
II WT. (O Pol IV WT. (D) Pol II AC-term. (£) Pol IV AC-term. Pol II AC-term and
merases Pol II and Pol IV form alternative replisomes with
Pol IV AC-term are impaired in their ability to bind the clamp. For B-E, 3H
DnaB helicase (Fig. 1) and maintain the architecture of the
incorporation was adjusted to account for different cell densities of unin-
duced and induced cultures. replication machinery for eventual resumption of synthesis by
Pol III (Fig. 2). Whether overexpressed in the cell or studied in
vitro, high levels of Pol II and Pol IV clearly interfere with
replisome has already moved nearly 10 kb by the time Pol IV is processive synthesis by Pol III (Figs. 3 and 5), by slowing
ongoing
added. Based on the results of Fig. 1C, showing that Polrather IV than stopping the replisome (Fig. 4), and this inhibition of
functions with DnaB, and Fig. 2B, showing that Pol IV Pol repli-
III replication depends on the interaction of Pol II and Pol
somes are competent for resumption of synthesis by Pol III*, we the ß-clamp both in vitro (Fig. 3) and in vivo (Fig. 5).
IV with
presume that takeover of the fork by Pol IV causes the fork On theto basis of these results, we conclude that high levels of Pol
slow dramatically rather than break down. II and Pol IV recruit both the ß-clamp and DnaB helicase from
Pol III at a moving replication fork during the SOS response.
Induction of Pol II and Pol IV in the Cell Reduces Chromosome
Surprisingly, these TLS polymerases modulate the rate of the
Replication in the Absence of DNA Damage. The in vitro reactions
DnaB helicase and slow the replication fork without causing it to LU
I
of Figs. 3 and 4 predict that high levels of Pol II and/orbreakPoldown.
IV, X
u
O
as found in the early stages of the SOS response, take over the 00

replisome and slow down the rate of chromosome replication DnaB as ainRegulated Motor. DnaB is not known to function with
vivo. To test this prediction, the genes encoding Pol IIother and polymerases
Pol besides Pol III, yet Pol II and Pol IV slow the
IV were cloned into a plasmid under control of the inducible
rate of DnaB unwinding, suggesting they may connect to DnaB
arabinose promoter, and the incorporation of [3H] thymidineand regulate helicase speed. A reinterpret ation of previous
during replication was measured before and after induction studies(see
suggests another explanation. Specifically, the rate of
Fig. 5/4). The amounts of arabinose used for induction yielded
DnaB unwinding may simply be determined by the rate of any
1,200 molecules of Pol II per cell and 2,500 molecules of DNA Pol IV
polymerase that acts on the unwound ssDNA product as
per cell, within 2- to 3-fold the levels present in the cell during in bacteriophage T4 and T7 systems (43, 44). The 35
illustrated
the SOS response (9, 10). The results of Fig. 5B indicate bp/sthat
rate of DnaB unwinding observed in an earlier study was
induction of Pol II is accompanied by a 55% decrease determined
in DNA by monitoring chain extension by Pol III in the
replication, and induction of Pol IV decreases DNA synthesis
absenceby of the T-subunit (3). Because t, which binds DnaB, was
^99% (Fig. 5C). The more severe decrease in DNA synthesis not present it was thought that the observed rate of unwinding
upon overexpression of Pol IV compared with Pol II correlates
was the intrinsic rate of DnaB. Instead, the observed unwinding
well with the results of the in vitro assays of Fig. 3. rate
Control
may represent the rate of DnaB unwinding when function-
experiments using the same plasmid, but without the Poling with
II or PolPol III core. The same study also observed a 35 bp/s rate
IV genes, showed that the vector does not interfere by thewithprimosome, which contains DnaB and other proteins,
chromosomal replication. including DnA helicase (3). The current study shows that Pol II

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Fig. 6. TLS polymerases function with DnaB and ß clamps. (Left) Scheme of
Pol Ill-based replisome. The r-subunit of Pol III binds the DnaB helicase and
enables rapid unwinding. Two x-subunits are contained within a clamp loader
assembly (the clamp loader is not illustrated) and thereby dimerize 2 Pol III
molecules for coupled leading- and lagging-strand synthesis. (Right) TLS
polymerases trade places with one another on the /3-clamp with Pol III to form
a TLS replisome. The TLS polymerases function with DnaB helicase, which
slows to match their slow rate of synthesis. Leading and lagging strands may
be uncoupled in the TLS replisome.
and Pol IV, which are slower than Pol III, function with DnaB
to move replication forks at jrates of 10 and 1 bp/s, respectively.
If the intrinsic rate of DnaB unwinding is truly 35 bp/s, the
current study indicates that Pol II and Pol IV slow DnaB. The
highest rate of DnaB unwinding is observed by using Pol III
holoenzyme containing t (>500 bp/s).
In light of these 4 different rates of unwinding, ranging
>500-fold, we propose that the DnaB unwinding rate is coupled
to DNA synthetic rate, even in the absence of a direct coupling
factor like t. This observation suggests that DnaB acts as a
"regulated motor" that must be coupled to some other activity,
like DNA polymerization, to promote efficient unwinding (43,
44). A replicative helicase acting as a regulated motor will ensure
that unwinding is tightly coordinated with DNA synthesis, a
favorable property for replisome action. Otherwise, ssDNA will
be generated that, besides being vulnerable to nucleases and
strand breaks, leads to the SOS response.
Logic of Slower Alternative Replisomes in the Face of DNA Damage.
The concept of switching between Pol III and TLS polymerases
has been considered an ephemeral phenomenon, where a stalled
replicative polymerase briefly yields to the TLS polymerase only
for bypass of the lesion, and Pol III immediately retakes control
of the growing DNA end to resume processive replication. The
results shown here compel a reconsideration of this notion,
because high levels of Pol II and Pol IV, as found in the early
stages of the SOS response, clearly take over replication by Pol
III to form slower replisomes that function not only with the
clamp but also with the replicative DnaB helicase. Unlike Pol III
replicase that contains 2 Pol III cores connected to a clamp
loader, Pol II and Pol IV are not known to dimerize or connect
to other proteins besides ß. Hence, we presume that leading- and
lagging-strand synthesis in TLS replisomes are constitutively
uncoupled, with no need to hypothesize a DNA trombone loop
(illustrated in Fig. 6). We propose that ß-clamps are assembled
on lagging strand RNA primers by the y-complex, and perhaps
it may be the reason for production of both y and t in cells.
Nevertheless, it remains possible that TLS polymerases dimerize
or connect to other proteins in the context of a replication fork.
The Pol II and Pol IV replisomes are highly stable and
therefore are probably the active form during the entire SOS
response. These replisomes are also highly dynamic. We dem-
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onstrate here that all 3 DNA polymerases (Pols II, III, and IV)
freely exchange with one another while preserving the DNA-
bound helicase and sliding clamps. We presume the average
residence time of any particular polymerase at the fork is defined
by the relative amounts of the different DNA polymerases in the
cell, their processivity, and their affinity for the sliding clamp and
helicase. Likewise, the speed of the replication fork will be a
function of the residence time of each DNA polymerase and its
intrinsic rate of DNA polymerization. As the intracellular con-
centration of TLS polymerases decreases, the population time of
Pol III at the replication fork will increase, with a corresponding
increase in replication fork speed.
What would be the purpose of establishing slower replication
forks in the face of DNA damage? We propose an additional role
for SOS-induced Pol II and Pol IV besides their ability to bypass
lesions. Specifically that TLS polymerases slow the fork, which
may give more time for repair of DNA damage by the excision
repair system, which can only function on dsDNA before a
replication fork collides with a lesion. Of course, sometimes the
replisome will encounter a lesion, and TLS synthesis will bypass
it, producing a mutation. Nevertheless, slower moving forks
should lower the frequency of these events. Pol II and Pol IV are
induced early in the SOS damage response and therefore
replication fork control by these polymerases is expected to be
specific to the first phase (<45 min) of SOS induction before the
highly mutagenic Pol V is produced (4). If the burden of DNA
damage is high, prolonging the SOS response, the cell eventually
up-regulates production of Pol V to effectuate a salvage program
to deal with unrepaired lesions.
SOS Response Can Be Activated by ssDNA Gaps Without Fork Break-
down. It is widely accepted that the presence of ssDNA is the
trigger for activation of the SOS response by RecA (31).
Research showing that replication is necessary for SOS induction
has been interpreted to suggest that one fork must stall or break
down, uncoupling helicase unwinding from DNA synthesis, to
generate ssDNA (19). However, cellular studies have shown that
DNA damage leads to single-strand gaps in both leading and
lagging strands, and therefore replication forks skip over lesions,
leaving ssDNA gaps behind them (32-35). These gaps may then
serve as substrate for RecA binding and consequent induction of
the SOS response.
Gaps in the lagging strand are made possible by the numerous
priming events that occur during discontinuous synthesis. Spe-
cifically, when a polymerase extending an Okazaki fragment
stalls at a lesion, it can transfer from the incomplete fragment to
a new RNA primer, leaving behind a ssDNA gap. Recent studies
demonstrate repriming of DNA synthesis on the leading strand
as well, and therefore the same process can explain leading
strand gaps (32, 36). Formation of ssDNA gaps behind the
replication fork offers an alternative means to generate ssDNA
for SOS induction that does not require fork stalling or break-
down. The ability to skip over damage implies that replication
forks do not necessarily stall at every leading-strand lesion, but
if Pol III were to continue rapid synthesis in the presence of
significant DNA damage, encounters with additional lesions,
and the risk of stalling or collapse would increase. By slowing the
fork, TLS polymerases diminish this risk while also assuring that
the fork does not break down. It is important to note that when
the lesion is a nick, replication forks will collapse, explaining the
need for primosomal replication restart proteins.
How Are Mutations Prevented If Error-Prone TLS Replisomes Are
Long-Lived? Pol II and Pol IV replicate DNA with lower fidelity
than Pol III, and therefore an obvious concern about alternative
TLS replisomes is the increased risk of introducing heritable
mutations, undermining rather than enhancing genome integ-
rity. However, the cell contains several safeguards that prevent
Indiani et al.
 production of heritable mutations under these circumstances.
First, errors produced by a TLS enzyme operating on undam-
aged DNA result in simple mismatches that can be corrected by
the postreplication mismatch repair system. Second, TLS DNA
polymerases are very slow, thereby ensuring that mismatches
caused by an inherently low-fidelity polymerase are not pro-
duced often. For example Pol IV, which makes a mistake
approximately every 1,000 nt, moving at a speed of 1 ntd/s will
generous gift from Jay Keasling (University of California, Berkeley). Buffer A
is 20 mM Tris-Cl (pH 7.5), 0.1 mM EDTA, 5 mM DTT, 5% glycerol, 40 /ig/ml BSA,
0.5 mM ATP, and 8 mM MgCI2.
Rolling Circle Replication Reactions. DnaB was assembled on the minicircle
DNA by incubating 100 fmol of DNA with 4 pmol of DnaB (as hexamer) in 15
yX. of buffer A for 30 s at 37 °C, at which time Pol III* (100 fmol), ß (350 fmol), >
CC
and 60 /iM each of dGTP and dCTP were added for 5 min. DNA synthesis was
initiated upon adding 1 /¿g of SSB, 50 /iM of the 4 rNTPs, 60 /u,M DnaG primase, in

make only about 4 mismatches in 1 h. Correcting these errors is 60 /xM dATP, and 20 /liM dTTP (leading strand) or 60 /u,M dTTP and 20 /liM dATP
a relatively small task for the mismatch repair system. Third, Pol (lagging strand) in 25 /ulL DNA synthesis was monitored by using [a32P]dTTP for u

II is a high-fidelity enzyme because it contains a proofreading the leading strand and [c*32P] dATP for the lagging strand (3,000-5,000 UJ
LU
CO

exonuclease. Finally, Pol IV is held in a high-fidelity state in the
early phase of the SOS response by interaction with full-length
UmuD2 and RecA (6, 37). When the SOS response is prolonged,
indicating the presence of significant or unrepairable damage,
UmuD2 is cleaved to a form that no longer modulates the fidelity
of Pol IV interacts with UmuC to form the low-fidelity Pol V
TLS polymerase, thereby defining the later, mutagenic phase of
the SOS response. Regulation of the fidelity of Pol IV by UmuD
also explains why overexpression of Pol IV alone was found to
be mutagenic whereas co-overexpression of Pol IV along with an
uncleavable form of UmuD, UmuD(S60A), was not (37, 38).
One may presume that during a catastrophic and prolonged SOS
response, bypass of lesions by Pol V is of utmost importance,
even at the cost of forming Heritable mutations.
Experimental Procedures
Materials. Subunits of Pol III holoenzyme (a, e, 0, y, t, 8, 8', x. ty. and ß) were
purified as described (20). DnaB, DnaG, and SSB were purified as described
(39). Pol III* (all holoenzyme subunits except j3) was reconstituted as described
(14). The C-terminal 5-residue deletion of Pol II (polB A779-783) and 6-residue
deletion of Pol IV (dinB A346-351) containing a His6tag were cloned into the
pET1 6 expression vector and purified by using Ni2+ affinity chromatography;
their activity was nearly that of WT Pol II and Pol IV (>70%). The tailed form
II duplex minicircle DNA was as described (20). The BW27786 strain was a
cpm/pmol). Pol II or Pol IV were added as indicated in the figure legends.
Reactions lacking Pol III* contained 5 pmol of Pol IV (or Pol II), 50 fmol of
T-complex, and 350 fmol of ß (Fig. 1). All reactions were quenched upon the
addition of 0.5% SDS and 20 mM EDTA, and DNA synthesis was quantitated as
described (40). Products were analyzed on 0.6% alkaline agarose gels.
Measurement of DNA Synthesis After Protein Overexpression. The protocol
used [3H]thymidine to monitor DNA synthesis after overexpression of differ-
ent DNA polymerases as described (41). Dose-dependent expression of Pol II or
Pol IV was obtained by using the araC-pBAD system (Invitrogen). The plasmid
was transformed into E. coli strain BW27786, which overexpresses the arabi-
nose permease, araE, allowing consistent and highly regulated expression of
pBAD in a population of cells (42): A single colony was inoculated in 5 ml_ of
LB, overnight cultures were diluted 1 :100 in 100 m Lof LB, then grown to OD6oo
= 0.1 . 45 min after adding arabinose (0.1 % for Pol II and 0.01 % for Pol IV), and
0.5-ml aliquots were removed and added to a tube containing pulse-label
solution {30 /ml of [methyl-3H] thymidine (1mCi/mL) in 2.97 mL of LB}. After
exactly 3 min at 37 °C, ice-cold 5% trichloroacetic acid was added to lyse cells
and precipitate DNA. Pol II and Pol IV were quantitated by Western blot
analysis. Control samples (-Ara) were from parallel cultures not treated with
arabinose.
ACKNOWLEDGMENTS. We thank Jay Keasling for the AraE overexpression
strain. This work was supported by National Institutes of Health Grants
GM38839 (to M.O.), AI065508 (to M.O.), R37GM21422 (to M.F.G.), and ESO
12259 (to M.F.G.).

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