The Discovery of Error-prone DNA Polymerase V and Its Unique Regulation by RecA and ATP
The Discovery of Error-prone
DNA Polymerase V and Its
Unique Regulation by RecA
and ATP
Published, JBC Papers in Press, August 26, 2014, DOI 10.1074/jbc.X114.607374
Myron F. Goodman
From the Molecular and Computational Section, Departments of Biological Sciences and
Chemistry, University of Southern California, Los Angeles, California 90089
My career pathway has taken a circuitous route, beginning with a Ph.D. degree in
electrical engineering from The Johns Hopkins University, followed by five postdoc-
toral years in biology at Hopkins and culminating in a faculty position in biological
sciences at the University of Southern California. My startup package in 1973 con-
sisted of $2,500, not to be spent all at once, plus an ancient Packard scintillation
counter that had a series of rapidly flashing light bulbs to indicate a radioactive read-
out in counts/minute. My research pathway has been similarly circuitous. The discov-
ery of Escherichia coli DNA polymerase V (pol V) began with an attempt to identify the
mutagenic DNA polymerase responsible for copying damaged DNA as part of the well
known SOS regulon. Although we succeeded in identifying a DNA polymerase, one
that was induced as part of the SOS response, we actually rediscovered DNA poly-
merase II, albeit in a new role. A decade later, we discovered a new polymerase, pol V,
whose activity turned out to be regulated by bound molecules of RecA protein and
ATP. This Reflections article describes our research trajectory, includes a review of
key features of DNA damage-induced SOS mutagenesis leading us to pol V, and
reflects on some of the principal researchers who have made indispensable contribu-
tions to our efforts.
N
ever having taken a college-level course in biology, I was nonetheless appointed as an
assistant professor in biological sciences at the University of Southern California in
1973. My undergraduate training at Queens College and Columbia University was
primarily in physics and electrical engineering. My Ph.D. thesis in electrical engineer-
ing from The Johns Hopkins University was entitled Selective Hydrolysis of Adenosine Triphos-
phate Resulting from the Absorption of Laser Light in a Stretching Mode of the Terminal Phosphate
Group, which meant that, at the very least, I needed to learn how to measure the hydrolysis of ATP
3 ADP � Pi. Maurice (Moishe) J. Bessman (Fig. 1), a professor of biology at Hopkins, provided a
lab bench and personalized instruction for measuring inorganic phosphate release using the
Lowry-Lopez colorimetric assay. The intellectual interest in Moishe’s lab focused on understand-
ing the biochemical basis of mutagenesis. Their exciting studies on DNA synthesis fidelity using
bacteriophage T4 mutator and antimutator DNA polymerases proved infectious, so much so that
when I received a Ph.D. degree in electrical engineering in 1968 from Hopkins, in lieu of a $30,000
salary offer from Bell Labs (Murray Hill, New Jersey), I chose instead to pursue the opportunity to
learn and, more importantly, appreciate biochemical enzymology as a postdoctoral fellow with
Moishe at a National Institutes of Health fellowship stipend of $6,500. From 1968 onward, the
study of DNA polymerase fidelity became an enduring interest.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 39, pp. 26772–26782, September 26, 2014
© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
26772 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 39 • SEPTEMBER 26, 2014
REFLECTIONS
This is an open access article under the CC BY license.
http://creativecommons.org/licenses/by/4.0/
Mutations for Worse or Better
Mutations are almost always bad, typically with nega-
tive consequences, in all forms of life. However, mutations
can also be good, in fact essential, by serving as the engine
that drives evolution. A specialized DNA polymerase fam-
ily, the Y-family, has been studied for over a decade. With
examples spanning bacteria to humans, the central role of
Y-family polymerases appears to be copying past damaged
DNA template bases that block replication. There is no
free lunch, however, because the costs of fully replicating
the genome are often mutations frequently targeted at
lesion sites, but occasionally occurring within undamaged
template regions. These Y-family enzymes are referred to
as translesion synthesis (TLS)1 polymerases. This Reflec-
tions article describes the path leading to our discovery of
the Y-family Escherichia coli DNA polymerase V (pol V)
and to the further discovery of pol V properties that set it
apart even from other Y-family polymerases.
The path to the discovery of pol V is closely linked to
more than sixty years of experiments and concepts in the
area referred to as “SOS error-prone DNA repair.” The
logical place to begin is by reflecting on the insights that
revealed the transcriptionally regulated error-prone DNA
repair pathway in E. coli, the SOS regulon, which led us to
pol V.
The Origin of SOS Error-prone DNA Repair
The year of the discovery of the structure of DNA, 1953,
is coincidentally the year that Jean Weigle made a seminal
observation showing that bacteriophage � can be revital-
ized after killing it with UV light (1). Although the word
“seminal” tends to be overused, especially when viewed
alongside Watson and Crick, Weigle’s experiment was
nevertheless remarkable. It showed that UV light-irradi-
ated bacteriophage �, which were unable to generate prog-
eny phage (i.e. form plaques when infecting E. coli), did in
fact form plaques when the infected E. coli had also been
exposed to UV light. Weigle further found that reactiva-
tion of bacteriophage � was accompanied by a large
increase in phage mutagenesis (1).
During the late 1960s to early 1970s, genetic studies by
Evelyn Witkin (Fig. 2) were instrumental in showing that
phage reactivation could be attributed to the presence of a
DNA damage-inducible bacterial pathway that regulated
the transcription of genes designed to repair the E. coli
chromosome (2, 3). While eliminating DNA damage in the
bacterial chromosome, newly induced bacterial repair and
recombination proteins also repaired the phage DNA,
enabling bacteriophage � to generate progeny and subse-
quently lyse the cell. Miroslav Radman (Fig. 2) proposed in
1974 that this was a repair pathway of last resort, an “SOS”
mutagenic pathway that could repair DNA in an error-free
manner but could also allow unrepaired DNA to be copied
by an error-prone process (4).
DNA template lesions that would normally block repli-
cation fork progression could be copied via TLS, but at the
cost of causing mutations targeted at DNA template
lesions and elsewhere as well. A genetic locus was identi-
fied in the late 1970s that fit the error-prone role envi-
sioned by Radman, the UV mutagenesis (umu) locus (5, 6).
The locus was so named because when the umu genes
were mutated, UV radiation did not produce chromo-
somal mutations in excess of spontaneous background
levels. This locus was subsequently shown to encode two
1 The abbreviations used are: TLS, translesion synthesis; pol V; DNA
polymerase V; ssDNA, single-stranded DNA; pol III HE, pol III holoen-
zyme complex; ATP�S, adenosine 5�-O-(thiotriphosphate); MALS,
multiangle light scattering.
FIGURE 1. Maurice J. Bessman (1973) shown brewing a cup of joe
bearing his initials, “MJB” (see coffee label at rear).
FIGURE 2. Miroslav Radman (left) with Evelyn Witkin (right), Paris
(2012).
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SOS-regulated genes, umuC and umuD (7, 8), which are
used to form pol V (9).
Two proteins regulate the SOS response, LexA and
RecA. LexA is a repressor that binds to more than 40 oper-
ators in the DNA damage-inducible regulon. Each opera-
tor has a consensus LexA-binding sequence, but with
different neighboring sequences that determine repres-
sor-operator binding affinities. Early protein induction,
e.g. RecA (�1 min post-UV), accompanies weak LexA
binding, whereas late protein induction, e.g. pol V (�45
min), results from strong LexA binding to the umuDC
operon. The late appearance of pol V provides the cell an
early temporal window to feature error-free ways to excise
DNA damage involving base excision, nucleotide excision,
and homologous recombination. At later times, chromo-
somal damage that remains unrepaired is then subjected
to pol V-catalyzed TLS in a last-ditch effort to restart
replication.
RecA protein is induced rapidly following the exposure
of E. coli to UV radiation or to chemicals that damage
DNA. RecA assembles cooperatively on regions of single-
stranded DNA (ssDNA) in the presence of ATP to form a
nucleoprotein filament, often referred to as RecA*. RecA*
plays so many different roles and is so important to the cell
that I sometimes present a tongue-in-cheek seminar slide
listing the three most salient aspects of life: death, taxes,
and RecA nucleoprotein filaments. Roles for RecA*
include initiating DNA strand invasion as a first step in
homologous recombination (10) and serving as a copro-
tease facilitating the autocatalytic cleavage of the LexA
repressor to induce the SOS response and, similarly, facil-
itating cleavage of the UmuD2 protein to its shorter muta-
genically active form, UmuD�2 (11–13). UmuD�2 interacts
with UmuC to form pol V (UmuD�2C) (9).
pol V copies undamaged DNA and performs TLS in the
absence of any other E. coli DNA polymerase (9) but only
when RecA* is present in the reaction. pol V (9) or UmuC
(14) has minimal activity in the absence of RecA*. In 2009,
we determined that the role of RecA* was to transfer a
RecA monomer from the 3�-end of the nucleoprotein fil-
ament, which, along with a molecule of ATP, “activates”
pol V to a mutasome complex, designated as pol V Mut
(UmuD�2C-RecA-ATP) (15).
A Separate Role for RecA in SOS Mutagenesis
In 1989, Raymond Devoret (Fig. 3) and colleagues iden-
tified a recA mutant (originally named recA1730, which we
now know to be recA(S117F)) that abolished SOS
mutagenesis entirely (16). UV light-induced mutations,
which typically increased by �100-fold in wild-type recA
cells, remained at spontaneous background levels in
recA1730 cells. The other RecA functions, including
homologous recombination, conversion of UmuD to
UmuD�, cleavage of the LexA protein, and induction of
SOS, all occurred in cells that overexpressed RecA(S117F)
(16), but SOS mutagenesis did not. Therefore, the absence
of UV light-induced mutagenesis in this recA mutant
background could not be attributed to the seemingly obvi-
ous reason that SOS could not be turned on. Instead, RecA
must possess a separate mutagenic function. The identifi-
cation of this new RecA function, especially in its connec-
tion to pol V, became the overriding research goal for my
laboratory.
Damaged Goods
We entered the SOS mutagenesis field serendipitously
as a result of an “out of the blue” phone call in 1986 from
Bruce Kaplan and Ramon Eritja at City of Hope in nearby
Duarte, California. They asked if I could think of a biolog-
ical use for a new reduced deoxyribose compound that
they had recently synthesized in the form of a phosphora-
midite incorporated in a DNA strand using their home-
made DNA synthesizer. This was the first type of abasic
site to be synthesized. Reduction at the C-1 position of the
sugar rendered the potentially labile sugar stable as a rock,
so the abasic lesion could be incorporated on a DNA tem-
plate strand site-specifically and with high yield. The
lesion-containing template annealed to an oligonucleotide
5�-32P-labeled primer strand could then be copied by any
DNA polymerase. We could determine the fraction of
extended primers that were blocked one base before the
lesion, stopped opposite the lesion, and, most importantly,
extended beyond the lesion, indicating successful TLS
(17).
FIGURE 3. Raymond Devoret a pris dîner à Sage American Grill and
Oyster Bar, New Haven, Connecticut (2013).
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Parenthetically, we analyzed the polymerase-catalyzed
32P-labeled primer extension data using a brand-new tech-
nological marvel. Our lab had recently purchased a first
edition PhosphorImager, which was developed by a
startup company called Molecular Dynamics. This new
technology allowed us to simultaneously visualize about
twenty lanes of a polyacrylamide gel on which primers
were separated electrophoretically according to length, so
primers differing by just single-nucleotide additions were
easily observed. The imaging provided high resolution and
unheard-of sensitivity. A barely visible gel band, corre-
sponding to polymerase extension directly opposite a
lesion, and faint TLS extension bands continuing beyond
the lesion could be resolved from the much darker gel
band, which corresponded to a polymerase blocked at the
lesion. The faint-to-dark band detection sensitivity was
about �1000-fold, far exceeding the 30-fold range previ-
ously available using x-ray film scanning densitometry. A
gel fidelity assay developed concurrently in our lab by my
graduate students Michael Boosalis and Sandra Randall
and my next-door faculty colleague John Petruska
required the ratio of adjacent integrated gel band intensi-
ties as input to quantify nucleotide incorporation veloci-
ties. We showed that by determining the ratio of right and
wrong incorporations, we could obtain the fidelity for any
polymerase, including those with 3� 3 5�-exonuclease
proofreading (18).
A Digression into DNA Polymerase II
RecA had yet to appear on our radar screen when we
began looking for a biochemical basis for SOS mutagenesis
in 1986. Devoret’s paper demonstrating a separate role for
RecA in SOS mutagenesis would be published three years
later (16). Our initial foray into SOS was to take a bot-
tom-up approach by incubating crude lysates prepared
from untreated and nalidixic acid-treated E. coli with
primer-template DNA containing an abasic template
lesion. We observed a 7-fold increase in an unknown
polymerase activity in DNA-damaged cell lysates exposed
to nalidixic acid compared with undamaged cell lysates
(19). DNA polymerase II (pol II) emerged as the enzyme
activity responsible for the DNA damage-induced TLS
activity based on its similar molecular mass and enzymatic
properties to those of pol II reported by Jerry Hurwitz in
1972 (20).
However, the 7-fold activity induction number turned
out to be especially important. By inserting a lacZ reporter
gene at various locations on the E. coli chromosome, Gra-
ham Walker (Fig. 4) had previously identified a series of
individual loci distributed along the chromosome that
were induced in response to DNA damage, which he called
damage-inducible (din) genes (21). This important study
provided a salient piece of information: one of the induced
genes, dinA, showed a 7-fold increase in transcription. The
close correspondence of transcriptional induction to the
polymerase activity induction suggested to us that dinA
could be the structural gene for pol II, which was con-
firmed by showing that the nucleotide sequences for dinA
and our structural gene for pol II were the same (22). A
concurrent paper identifying dinA as the gene for pol II
was published by Shinagawa and colleagues (23).
Trials and Tribulations of UmuC and the
Identification of a UmuD�2C Complex
The power of E. coli genetics to investigate complex bio-
chemical processes was clearly evident in the reconstitu-
tion of a minimal system that was able to catalyze TLS in
vitro. The complement of genetic requirements included
the UV mutagenesis proteins UmuC and UmuD� and the
RecA protein. A primer-template DNA, including a dam-
aged base on the template strand, could be used as a sub-
strate. Based on genetic evidence available in the mid-
1980s, the first TLS model was formulated by Bryn Bridges
and his graduate student Roger Woodgate (Fig. 5). The
Bridges-Woodgate two-step model (Fig. 6A) proposed
that the role of the UmuDC proteins was to shepherd
DNA polymerase III (pol III) past replication-blocking
lesions by reducing polymerase fidelity, e.g. possibly by
inhibiting exonucleolytic proofreading (24). It was sug-
gested that in the first step, pol III could insert a nucleotide
FIGURE 4. Graham Walker, Cambridge, Massachusetts (2013).
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opposite a template lesion but could go no further. It
required the Umu proteins and RecA* to copy past the
lesion, after which there would be clear sailing on undam-
aged template DNA until further damage was encoun-
tered. Whatever the mechanism for TLS might turn out to
be, it seemed clear that the Umu proteins working along
with RecA* were required for pol III-catalyzed TLS.
Following the discovery in 1988 that SOS mutagenesis
required cleavage of UmuD to UmuD� (11–13), an
updated TLS model was proposed by Harrison (Hatch)
Echols (Fig. 7) and me (Fig. 5). This next generation model
included UmuD� in place of UmuD and the pol III holoen-
zyme complex (pol III HE) in place of pol III (Fig. 6B) (25).
The stage was almost, but not quite, set to reconstitute
TLS in vitro. pol III HE, RecA*, and UmuD� were available
as purified proteins. However, UmuC was lacking. In 1989,
Hatch and his postdoctoral student Roger Woodgate, who
was to become a longtime and current collaborator of ours,
reported the purification of UmuC and its interaction with
UmuD and UmuD� (26). UmuD and UmuD� were soluble in
aqueous solution. In contrast, UmuC was insoluble and con-
fined to inclusion bodies, prompting Roger to denature the
protein in urea and renature it by slowly dialyzing out the
urea. The result was a refolded form of UmuC that was solu-
ble in aqueous solution. A similar denaturation-renaturation
strategy had proved successful for purifying the insoluble
�-proofreading subunit of E. coli pol III, resulting in a water-
soluble active 3�-exonuclease (27). However, extension of a
primer past a template lesion (i.e. TLS) was marginal when
renatured UmuC was used along with pol III HE, UmuD�,
and RecA* (28).
FIGURE 5. Roger Woodgate, Bryn Bridges, Myron F. Goodman, and Mengjia Tang (left to right): four SOS error-prone generations, Hilton
Head, South Carolina (1999).
FIGURE 6. Evolving models for SOS error-prone TLS involving
UmuD�2C (pol V) and RecA*. A, pol III core (composed of �-polymerase,
�-proofreading exonuclease, and �-subunits) copies past a damaged
template site X, requiring the presence of UmuDC and RecA* situated in
cis on the damaged template strand being copied. B, pol III HE (com-
posed of pol III core � �-sliding clamp � �-clamp-loading complex)
catalyzes TLS, requiring the presence of UmuD�C and cis-RecA*. C, pol V
(composed of UmuD�2C) catalyzes TLS, requiring the presence of cis-
RecA*. D, pol V activated by a RecA* located in trans to the primer-
template DNA catalyzes TLS. E, pol V Mut (composed of pol V � RecA
(red circle) � ATP (inverted blue triangle)) catalyzes TLS in the absence of
RecA*.
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Sadly, my close friend Hatch passed away on April 11,
1993. Several days earlier, I was invited by his wife, Carol
Gross, to visit Hatch in their Berkeley home. During this
very touching and brief visit, Hatch asked that I continue
trying to reconstitute a biochemical system for TLS.
One could study the renatured form of � with confi-
dence because it was known that the native � had a clearly
defined 3�-exonuclease activity when present as a compo-
nent of the soluble pol III core. However, in the case of
UmuC, an alternative approach might be better suited to
investigate a protein with no known biochemical function.
Irina Bruck (a graduate student at the University of South-
ern California), Roger and I rebooted the study on UmuC,
taking an alternative approach that opened the way to
identify UmuC, not by itself but rather as part of soluble
heterotrimeric complex, UmuD�2C (29).
By coexpressing the UmuD�C proteins, overproduced
from a high-expression promoter, and purifying the
UmuC protein from a strain carrying a deletion of the
entire umuDC operon, we obtained a sizable amount of a
soluble 46-kDa protein that we identified as UmuC by
Western blotting and microsequencing. At the end of a
multistep purification process, UmuC remained soluble
but, as we soon discovered, only because it was accompa-
nied throughout each step in the purification by UmuD�.
During the purification, UmuC remained bound to
UmuD� as a stable 70-kDa heterotrimeric complex,
UmuD�2C (29). Having UmuD�2C in hand, we then
embarked on reconstituting an SOS TLS system in vitro
that included the minimal requirements determined
genetically, the UmuC and UmuD� proteins, RecA*, prim-
er-template DNA, and, of course, a DNA polymerase that
was almost surely pol III HE (25). However, that turned
out not to be the case as described next.
TLS Requires the Umu Proteins and RecA, But Not
pol III
The approach to reconstitute TLS with purified pro-
teins dictated by the genetics was straightforward. Simply
take a primer-template DNA having a 5�-32P-labeled
primer and a template containing an abasic lesion and
incubate it with purified RecA, UmuD�2C, and pol III HE.
The expectation was that pol III HE would extend the
32P-labeled primer up to the template lesion, but not
beyond, in the absence of either RecA or UmuD�2C. The
hope was that the addition of both RecA and UmuD�2C
would allow pol III to insert a deoxynucleotide opposite
the noncoding lesion and then continue primer extension
to reach the end of the template strand. The hoped-for result
was attained except for one unforeseen observation. Exten-
sion of the primer, copying undamaged template DNA to
reach the lesion and then copying past the lesion (TLS),
occurred in the absence of pol III core (30)! The wholly sur-
prising result was that something other than pol III was able
to avidly synthesize DNA. TLS was not the only unusual
activity observed. Experiments leaving out one of the dNTP
substrates made it pretty obvious that deoxynucleotide mis-
incorporations were also occurring to an unprecedented
extent opposite normal template bases.
It had occurred to us that UmuD�2C might be a new
type of low-fidelity DNA polymerase. I had mentioned at a
1998 Taos conference that “DNA processing takes place in
accordance with the ‘four Rs’: Replication, Recombina-
tion, Repair, REDUNDANCY,” and added that “if
UmuD�2C walks like a duck and quacks like a duck, then
perhaps it is a duck, disguised as a new type of DNA
polymerase.” We titled the 1998 PNAS paper describing
TLS “Reconstitution of in vitro lesion bypass dependent
on the UmuD�2C mutagenic complex and RecA protein”
(30). Bob Lehman, who communicated the paper to
PNAS, suggested that we were being overly conservative
by not stating flat out in the title that UmuD�2C was a new
SOS-induced error-prone DNA polymerase. In hindsight,
I obviously should have taken Bob’s advice, but as Dick
Vermeil, the superb head football coach for our “hated”
rival school UCLA, had aptly said in a quote paraphrased
from the poet Henry David Thoreau, “Never look back
unless you are planning to go that way.”
There was, however, a mitigating factor in my chicken-
ing out. The specific activity of the low-fidelity TLS activ-
ity was miniscule, nowhere near that of pol I, II, or III, and
perhaps a small contaminant from one or more of the pols
FIGURE 7. Hatch Echols, Berkeley, California (circa 1980).
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could be present. Thanks to an exquisitely sensitive neu-
tralizing antibody against pol I provided to us by Larry
Loeb at the University of Washington, we could confi-
dently rule out pol I as a contaminant. The deliberate addi-
tion of pol III inhibited TLS, suggesting that it was unlikely
to be present in the preparation, but we could not entirely
eliminate an adventitious presence of pol II. We had no
antibody against pol II and no pol II deletion strain to
exploit. Notably, as recounted above under “A Digression
into DNA Polymerase II,” pol II was itself pretty adept at
TLS. pol II was also induced by the SOS regulon in vivo in
response to DNA damage (19, 22). Here, I reflect on that,
back in the day, circa 1998 –2004, we were fortunate to
recover �2– 4 mg of purified UmuD�2C from 30 liters of
cells, even when overproduced. To obtain more workable
amounts of UmuD�2C, Roger Woodgate fired up the 200-
liter fermenter at the National Institutes of Health, ship-
ping many grams of spun-down cell pellets to us on dry ice.
This ensured a quarterly profit for FedEx lasting for several
years. It also gave rise to heavy-duty KP (kold room patrol)
by graduate students Irina Bruck, Mengjia Tang (Fig. 5),
and Xuan Shen. Under such trying circumstances, a minor
polymerase contaminant would not be too surprising.
A concurrent paper by Zvi Livneh and colleagues
showed that when UmuC had a maltose-binding protein
attached to its N terminus, it was soluble in aqueous solu-
tion; maltose-binding protein-UmuC along with UmuD�,
RecA*, and pol III HE performed TLS. However, in con-
trast with our data, pol III HE was a necessary component
in their reconstituted system (31). Absent pol III, there was
no detectable TLS in Livneh’s system. A PNAS Commen-
tary discussing the two papers was written by Graham
Walker and entitled “Skiing the black diamond slope: pro-
gress on the biochemistry of translesion DNA synthesis”
(32). Graham’s title referred specifically to a statement
made by Hatch to a packed audience during a replication
and repair “skiing” meeting held in Taos, New Mexico, in
1992, where he referred to UmuC as the “black diamond
slope of DNA biochemistry.”
Black double diamond slope is what I remember Hatch
having said. Either way, one or two diamonds, by mid-
1998, the difficulties with UmuC and, more generally, with
the biochemical reconstitution of TLS were at long last
being bypassed (pun intended). In 1999, the heterotri-
meric UmuD�2C complex was unequivocally shown to be
a bona fide DNA polymerase (9), with the polymerase
activity residing in the UmuC subunit (9, 14). The year
2001 witnessed the identification of a new polymerase
family, the Y-family polymerases (33), that grew rapidly. A
recent count identified 13 family members spanning from
microorganisms to humans (34, 35).
A Fly in the cis-RecA* Filament Ointment
The model for TLS that Hatch and I proposed in 1990
(Fig. 6B) had not strayed far from the 1985 Bridges-
Woodgate model (Fig. 6A), although the proteins involved
were modified according to new biochemical data, includ-
ing dispensing with pol III HE and replacing it with pol V
(UmuD�2C) (Fig. 6C) (9). However, the core feature of the
model remained: TLS was assumed to require the pres-
ence of a RecA* nucleoprotein filament assembled on the
region of ssDNA located in cis at the 5�-side of the dam-
aged template base (Fig. 6, A–C). Positioning of RecA* on
the template strand proximal to the lesion surely made
sense. When asked why he robbed banks, Willie Sutton
famously replied, “Because that’s where the money is.” The
same localization principle logically accounted for posi-
tioning of RecA*. Although assembly of RecA* in cis on the
template strand being copied was a reasonable supposi-
tion, there was no direct evidence to support it.
We were soon led to rethinking where and how RecA*
functioned during TLS because of a talk that I gave in
November 2000 at the National Academy of Sciences Col-
loquium on “Links Between Recombination and Replica-
tion: Vital Roles of Recombination” at University of Cali-
fornia, Irvine. Projected on the screen were PAGE data
depicting 32P-labeled primer elongation bands visualized
at single-nucleotide resolution with remarkable clarity
using the aforementioned PhosphorImager. Although the
data showed that TLS was greatest with a RecA/ssDNA
ratio of �1 RecA monomer/3–5 nucleotides, which is
consistent with the optimal ratio for RecA* filament for-
mation, TLS was also clearly observed at �1 RecA mono-
mer/50 nucleotides. Following my talk, Mike Cox (Fig. 8)
mentioned to me privately that RecA* would be unlikely to
assemble on the region of ssDNA downstream of the tem-
plate lesion at 1 RecA monomer/50 nucleotides.
I was grateful that Mike spared me a public “gotcha,” all
the more so because his point proved to be spot-on. When
one makes primer-template DNA by annealing a relatively
short primer to a longer template strand, thermodynamics
dictates that a portion of the DNA remains single-
stranded. Furthermore, because we typically used an
excess of primers over template to optimize the yield of the
primer-template DNA duplex, there was always an excess
of primer strands in solution to bind with RecA. Although
the ratio of �1 RecA monomer/50 nucleotides was not
sufficiently high for RecA nucleoprotein filament assem-
bly on the primer-template DNA duplex, the ability to
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observe pol V-catalyzed TLS (36) suggested that perhaps
RecA* forming on the shorter free primer strands was
somehow able to facilitate lesion bypass by pol V by acting
in trans (Fig. 6D) (37). We viewed these data as a potential
“fly in the cis-RecA* filament ointment.” Perhaps RecA*
assembled in trans on a ssDNA strand that was not being
copied by pol V.
Transactivation of pol V by RecA*
Compelling evidence for transactivation of pol V-cata-
lyzed DNA synthesis, including TLS, was obtained by
Katharina (Kathi) Schlacher, a graduate student who came
to our lab initially with the limited objective of performing
a Master’s thesis project for a year and defending in Aus-
tria, which she did. To our good fortune, Kathi then
decided to return to the lab to initiate the transactivation
studies, which I am pleased to say won her the “Best Ph.D.
Dissertation of 2006” award from the University of South-
ern California. The key to proving that pol V can be acti-
vated via the assembly of RecA* on ssDNA that is not
being copied was to design a primer-template DNA that
could not support template filament formation. The prim-
er-template was formed as a stable DNA hairpin with just
a 3-nucleotide overhang region at its 5�-end to serve the
template strand (see e.g. Fig. 9B, left) (37). A RecA mono-
mer has a 3-nucleotide footprint on ssDNA, leaving no
space on the template strand to form a cis-RecA* filament
downstream of UmuD�2C. Instead, RecA* filaments were
assembled on separate ssDNA molecules, which acted in
trans on the primer-template DNA duplex hairpins. The
3-nucleotide template overhangs were copied but only
with trans-RecA* present (37). The reaction of pol V with
the primer-template DNA hairpins was second-order, i.e.
linearly proportional to the concentration of trans-RecA*,
leaving little (if any) room for doubt that RecA* assembled
on a DNA different from the one being copied was respon-
sible for pol V activity (Fig. 6D).
The historical positioning of RecA* downstream of pol
V on the template strand being copied has always been
based on an Occam’s razor-like rationale that because
RecA* was required for TLS, it had to act at a blocked
replication fork (38). What had not been contemplated is
that the role of RecA* might actually be indirect: that it is
not interacting with a RecA* filament, be it located either
in cis or in trans, that activates pol V, but instead, RecA* is
FIGURE 8. Mike Cox, Madison, Wisconsin (circa 2009). This is how he
looks today.
FIGURE 9. pol V Mut (UmuD�2C-RecA-ATP) is a stand-alone DNA polymerase able to synthesize DNA in the absence of RecA*. A, pol V Mut is
formed by incubating pol V with RecA* bound to resin and removing the resin-bound RecA*, leaving pol V Mut in solution. B, pol V Mut formed with
wild-type RecA copies DNA, whereas pol V Mut formed with Devoret’s SOS nonmutagenic RecA1730(S117F) cannot copy DNA. Red circle, RecA;
inverted blue triangle, ATP or ATP�S. nt, nucleotides.
REFLECTIONS: Discovery of Error-prone DNA Polymerase V
SEPTEMBER 26, 2014 • VOLUME 289 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 26779
needed to transfer a RecA monomer from its 3�-proximal
tip to pol V and thereby activate it for DNA synthesis (15).
Captured Alive, the Active Form of pol V Is
UmuD�2C-RecA-ATP
Deciphering the role of RecA* remained the key bio-
chemical challenge, a mystery that had persisted since the
mid-1980s with the Bridges-Woodgate model (Fig. 6A)
(24) and crystallized by Devoret’s discovery of an essential
role for RecA* in damage-induced mutagenesis, distinct
from its roles in recombination and the coproteolytic
cleavage of the LexA repressor and UmuD (16). Mixing
pol V with trans-RecA* resulted in an active enzyme capa-
ble of copying undamaged and damaged templates (37),
but mixing pol V with RecA did not (39). An experiment
was needed to nail down unambiguously how and why
RecA* acts during pol V-catalyzed TLS.
We addressed the “how and why” by incubating pol V
with RecA* in the absence of primer-template DNA and
then isolating a possibly modified form of the polymerase
with RecA* no longer present (Fig. 9A). The strategy
entailed assembling RecA* filaments on ssDNA oligonu-
cleotides that were stably attached to resins or beads (15).
Following pol V incubation with immobilized RecA* and
removal of the RecA* by centrifugation, we found that pol
V had undergone a major modification. A single RecA
molecule had been transferred from the 3�-proximal tip of
RecA* to UmuD�2C, which, along with the binding of a
molecule of ATP (or ATP�S), converted the virtually inac-
tive pol V to the stand-alone activated pol V Mut
(UmuD�2C-RecA-ATP) (Fig. 9A). This was the first time
that pol V was observed to synthesize DNA without RecA*
(Fig. 9B) (15). A new model for TLS involved pol V Mut
with RecA* no longer directly in the picture (Fig. 6E).
pol V Mut was active with a bound wild-type RecA
monomer, whereas pol V Mut was dead when formed with
Devoret’s UV light-nonmutable RecA(S117F) mutant (Fig.
9B) (15). pol V Mut(S117F) had no measurable DNA synthe-
sis activity in vitro even though it was also transferred to
UmuD�2C from the 3�-RecA* filament tip to form a stable, yet
inactive, pol V Mut complex (15). It was gratifying to give a
biochemical face to Devoret’s critically important genetic
identification of an independent mutagenic function for
RecA*, which had provided the impetus for our work.
After submitting a manuscript to Nature, a multiangle
light scattering (MALS) instrument arrived at the Univer-
sity of Southern California in the two weeks intervening
between receipt of two referees’ reports and the third
report, which requested (i.e. demanded) more convincing
evidence for the existence of a stable complex composed
of a RecA monomer bound to UmuD�2C. MALS measures
the absolute molecular mass of a pure protein in aqueous
solution, subject only to a few nonrestrictive assumptions
regarding protein globular shape (40). A mass standard for
either calibration or comparison is not even required.
Using MALS, we identified a light scattering peak centered
at 113 kDa corresponding to a complex of UmuD�2C-
RecA. Using SDS-PAGE, we showed that the protein com-
position in the 113-kDa peak contained UmuC, UmuD�,
and RecA.
The Unexpected Identification of pol V Mut as
DNA-dependent ATPase
Thanks to MALS and the third reviewer, pol V Mut was
captured alive as UmuD�2C-RecA (15). However, a ques-
tion remained: what about ATP? Was ATP required for
pol V Mut activity? The short answer is that when present
as UmuD�2C-RecA, pol V Mut lacking ATP cannot syn-
thesize DNA. To be active, it must have a molecule of ATP
(or ATP�S) bound to form UmuD�2C-RecA-ATP (15).
FIGURE 10. Model for pol V Mut regulation by RecA and ATP. Transfer
of a RecA molecule (red circle) from RecA* and the subsequent binding
of an ATP molecule (inverted blue triangle) to UmuD�2C form activated
pol V Mut (UmuD�2C-RecA-ATP). A bound ATP molecule is required for
polymerase binding to primer-template DNA. pol V copies an undam-
aged region of DNA, inserts a deoxynucleotide opposite a lesion (X), and
continues DNA synthesis beyond the lesion (TLS). pol V Mut is deacti-
vated after dissociating from the primer-template DNA. Dissociation is
dependent on pol V Mut-dependent ATP hydrolysis.
REFLECTIONS: Discovery of Error-prone DNA Polymerase V
26780 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 39 • SEPTEMBER 26, 2014
The unresolved question was why. The answer that we
arrived at while I was writing these Reflections is that pol V
Mut function is regulated by an intrinsic DNA-dependent
ATPase activity (41). The binding of a single ATP mole-
cule to pol V Mut is required to bind the polymerase to
primer-template DNA. DNA synthesis, including TLS,
then takes place. ATP hydrolysis triggers the dissociation
of pol V Mut from the DNA. pol V Mut appears to func-
tion as an on-off toggle switch. Once activated, it performs
a single round of DNA synthesis. Following ATP hydroly-
sis, the enzyme dissociates from primer-template DNA in
a deactivated form (Fig. 10). No such ATPase activity or
autoregulatory mechanism has previously been found for
a DNA polymerase.
What is the biological basis for the complex regulation
of pol V by ATP? Mutagenic DNA synthesis during the
SOS response is seemingly an act of cellular desperation
that would best be limited as to when and where it is used.
The regulation of pol V Mut is needed to limit mutations,
especially in rapidly dividing cells (42). Mutational lethal-
ity can be avoided by restricting low-fidelity DNA synthe-
sis to short DNA segments confined to replication forks
blocked by DNA damage and to replication restart at
stalled replication forks on undamaged DNA. Keeping pol
V Mut processivity in check appears to be role of the inter-
nal ATPase that triggers polymerase dissociation from
primer-template DNA (41). In essence, the enzyme has
evolved to do the absolute minimum to get cellular DNA
synthesis restarted.
Acknowledgments—I thank Matty Scharff and Phuong Pham for provid-
ing valuable and constructive criticisms on the manuscript and Jeff Ber-
tram and Phuong for aid with the illustrations. I thank Evelyn Witkin,
Michael Devoret, Mike Cox, Maurice Bessman, Graham Walker, and
Stuart Linn for generously sending photographs. I am indebted to past
and present students and postdoctoral fellows who provided invaluable
intellectual support for the SOS-pol V studies while having interests that
differed from pol V. These other projects investigated the fidelity of DNA
polymerases and, more recently, the mutator mechanisms of APOBEC
dC deaminases. The NIGMS, NIEHS, and NCI have provided generous
sustenance. I especially thank Dan Shaughnessy (NIEHS), Dick Pelroy
(NCI), and the late Paul B. Wolfe (NIGMS) for their gracious support and
advice throughout my forty years and counting in the business.
Address correspondence to: mgoodman@usc.edu.
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REFLECTIONS: Discovery of Error-prone DNA Polymerase V
26782 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 39 • SEPTEMBER 26, 2014
The Discovery of Error-prone DNA Polymerase V and Its Unique Regulation by RecA and ATP
The Origin of SOS Error-prone DNA Repair
A Separate Role for RecA in SOS Mutagenesis
Damaged Goods
A Digression into DNA Polymerase II
Trials and Tribulations of UmuC and the Identification of a UmuD'2C Complex
TLS Requires the Umu Proteins and RecA, But Not pol III
A Fly in the cis-RecA* Filament Ointment
Transactivation of pol V by RecA*
Captured Alive, the Active Form of pol V Is UmuD'2C-RecA-ATP
The Unexpected Identification of pol V Mut as DNA-dependent ATPase
Acknowledgments
REFERENCES