Structure of a small-molecule inhibitor of a DNA polymerase sliding clamp.
ABSTRACT DNA polymerases attach to the DNA sliding clamp through a common overlapping binding site. We identify a small-molecule compound that binds the protein-binding site in the Escherichia coli beta-clamp and differentially affects the activity of DNA polymerases II, III, and IV. To understand the molecular basis of this discrimination, the cocrystal structure of the chemical inhibitor is solved in complex with beta and is compared with the structures of Pol II, Pol III, and Pol IV peptides bound to beta. The analysis reveals that the small molecule localizes in a region of the clamp to which the DNA polymerases attach in different ways. The results suggest that the small molecule may be useful in the future to probe polymerase function with beta, and that the beta-clamp may represent an antibiotic target.
- SourceAvailable from: Markus B. Cieslik[Show abstract] [Hide abstract]
ABSTRACT: Halogen bonding has recently experienced a renaissance, gaining increased recognition as a useful molecular interaction in the life sciences. Halogen bonds are favorable, fairly directional interactions between an electropositive region on the halogen (the σ-hole) and a number of different nucleophilic interaction partners. Some aspects of halogen bonding are not yet understood well enough to take full advantage of its potential in drug discovery. We describe and present the concept of halogen-enriched fragment libraries. These libraries consist of unique chemical probes, facilitating the identification of favorable halogen bonds by sharing the advantages of classical fragment-based screening. Besides providing insights into the nature and applicability of halogen bonding, halogen-enriched fragment libraries provide smart starting points for hit-to-lead evolution.Future medicinal chemistry 04/2014; 6(6):617-639. · 3.31 Impact Factor
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ABSTRACT: Evidence suggests that some nonsteroidal anti-inflammatory drugs (NSAIDs) possess antibacterial properties with an unknown mechanism. We describe the in vitro antibacterial properties of the NSAIDs carprofen, bromfenac, and vedaprofen, and show that these NSAIDs inhibit the Escherichia coli DNA polymerase III β subunit, an essential interaction hub that acts as a mobile tether on DNA for many essential partner proteins in DNA replication and repair. Crystal structures show that the three NSAIDs bind to the sliding clamp at a common binding site required for partner binding. Inhibition of interaction of the clamp loader and/or the replicative polymerase α subunit with the sliding clamp is demonstrated using an in vitro DNA replication assay. NSAIDs thus present promising lead scaffolds for novel antibacterial agents targeting the sliding clamp.Chemistry & biology 03/2014; · 6.52 Impact Factor
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ABSTRACT: Bacterial sliding clamps are molecular hubs that interact with many proteins involved in DNA metabolism through their binding, via a conserved peptidic sequence, into a universally conserved pocket. This interacting pocket is acknowledged as a potential molecular target for the development of new antibiotics. We previously designed short peptides with an improved affinity for the Escherichia coli binding pocket. Here we show that these peptides differentially interact with other bacterial clamps, despite the fact that all pockets are structurally similar. Thermodynamic and modeling analyses of the interactions differentiate between two categories of clamps: group I clamps interacts efficiently with our designed peptides and assembles the Escherichia coli and related orthologs clamps, while group II poorly interact with the same peptides and includes Bacillus subtilis and other Gram+ clamps. These studies also suggest that the peptide binding process could occur via different mechanisms depending on which type of clamp it binds to.Journal of medicinal chemistry. 08/2014;
Structure of a small-molecule inhibitor of a DNA
polymerase sliding clamp
Roxana E. Georgescu†‡, Olga Yurieva†‡, Seung-Sup Kim§, John Kuriyan¶?††, Xiang-Peng Kong§, and Mike O’Donnell†‡‡
†The Rockefeller University and‡Howard Hughes Medical Institute, 1230 York Avenue, P. O. Box 228, New York, NY 10065;¶Department of Molecular and
Cell Biology and††Howard Hughes Medical Institute, University of California, 401 Barker MC3202, Berkeley, CA 94720;?Lawrence Livermore National
Laboratory, 7000 East Avenue, Livermore, CA 94550; and§Department of Biochemistry, New York University School of Medicine, 550 First Avenue,
New York, NY 10016
Contributed by Mike O’Donnell, May 25, 2008 (sent for review April 3, 2008)
DNA polymerases attach to the DNA sliding clamp through a
common overlapping binding site. We identify a small-molecule
?-clamp and differentially affects the activity of DNA polymerases
II, III, and IV. To understand the molecular basis of this discrimina-
tion, the cocrystal structure of the chemical inhibitor is solved in
complex with ? and is compared with the structures of Pol II, Pol
III, and Pol IV peptides bound to ?. The analysis reveals that the
small molecule localizes in a region of the clamp to which the DNA
polymerases attach in different ways. The results suggest that the
small molecule may be useful in the future to probe polymerase
function with ?, and that the ?-clamp may represent an antibiotic
antibiotic target ? rational drug design ? fluorescence anisotropy ?
the duplex, thus acting as a mobile tether to hold the chromo-
somal replicase to DNA for high processivity (1–3). Sliding
clamps from the three domains of life are remarkably similar in
architecture (4–6). They consist of six domains of similar chain
fold. The bacterial ?-clamp is a homodimer, and each protomer
consists of three domains, whereas eukaryotic and archaeal
proliferating cell nuclear antigen (PCNA) clamps are homotri-
mers formed from protomers containing two domains each.
Initially, ? and PCNA were identified as processivity factors
for chromosomal replicases, but sliding clamps are now known
to function with diverse DNA polymerases, repair factors, and
cell cycle-control proteins (reviewed in ref. 1). Proteins typically
bind PCNA through a conserved sequence, referred to as a
PCNA interaction peptide (PIP) (7). The detailed interaction of
a PIP sequence with PCNA was originally observed for human
PCNA bound to a C-terminal peptide of the p21CIP1/WAF1cyclin
kinase inhibitor (8). Proteins that bind to the bacterial ?-clamp
contain a five- or six-residue consensus sequence, QL[S/D]LF
and QLxLx[L/F] (9).
The peptide-binding pocket of sliding clamps is located be-
of the bacterial ?-clamp is located between domains II and III,
as demonstrated by structures of ? bound to the ?-clamp loading
subunit (11) and the ?-Pol IV complex (10, 12). The protein-
binding pocket of ? consists of two subsites (10). Subsite 1 is 8
Å ? 10 Å and 8.5 Å deep, whereas subsite 2 is 14 ? 7.5 Å and
4.5 Å deep. Clamp-binding proteins can have additional points
of contact with the clamp, as exemplified by Escherichia coli Pol
IV, which also interacts with the edge of the ?-ring (12).
E. coli harbors five DNA polymerases. Pols II, III, IV, and V
are greatly stimulated by interaction with the ?-clamp (12). Pol
III is the chromosomal replicase, whereas Pols II, IV, and V are
induced upon DNA damage and function in repair and chro-
mosome maintenance (13). Pol IV and Pol V are Y-family,
error-prone DNA polymerases that lack 3?–5? proofreading
exonuclease activity and are thought to advance replication forks
he replication machinery of all cells utilizes a ring-shaped,
sliding-clamp protein that encircles DNA and slides along
over template lesions that block the Pol III replicase. Pol V is
detectable only after DNA damage and is the main DNA
polymerase responsible for mutagenic lesion bypass. Interest-
ingly, whereas Pol II and Pol IV are induced 7- to 10-fold upon
DNA damage, they are also present in undamaged cells (50 and
250 copies per cell, respectively) and may play roles during
normal cell growth as well as during the DNA damage response.
The roles of Pol II and Pol IV are relatively obscure.
The fact that the ?-clamp is an essential protein and uses the
same peptide-binding pocket for all of the DNA polymerases
diverse polymerases function with ?. Thus, a chemical may be
used in the future to probe and better define the function of Pol
II and Pol IV with ? and their interplay with Pol III. To further
this endeavor, the current report identifies a small-molecule
compound that binds to the peptide-binding pocket of the
Pol IV. To determine the molecular basis by which the com-
pound selectively alters the function of ? with these different
DNA polymerases, we solve the structures of ? bound to the
compound as well as the related peptides of Pol II and Pol III
with ?, and compare them with the Pol IV-? structure. The
analysis indicates how the chemical compound may discriminate
among these different DNA polymerase-?-clamp interactions.
Interestingly, the compound inhibits the bacterial Pol III repli-
case without disrupting the eukaryotic replicase. Hence, the
?-clamp may represent a target for antibiotic compounds.
Identification of a Small-Molecule Compound That Binds the Peptide-
Binding Pocket of the ?-Clamp. To identify small-molecule com-
pounds that bind the peptide-binding pocket of ?, we developed
a fluorescence anisotropy assay that is easily adapted to a
high-throughput approach. The assay uses a TAMN-labeled
20-mer peptide derived from the Pol III C terminus. Titration of
? into the TAMN-peptide yields an apparent Kdof 2.7 ? 0.4 ?M
(Fig. 1A). Compounds that disrupt this interaction should dis-
place the TAMN-peptide, resulting in a decrease in anisotropy.
The peptide displacement assay was used to screen the Rock-
efeller University chemical library consisting of ?30,600 polar
organic compounds. An example result from one 386-well plate
is shown in supporting information (SI) Fig. S1. The screen gave
Author contributions: R.E.G. and M.O. designed research; R.E.G. and O.Y. performed
research; R.E.G., O.Y., S.-S.K., J.K., X.-P.K., and M.O. analyzed data; and R.E.G. and M.O.
wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
Data deposition: The atomic coordinates have been deposited in the Protein Data Bank,
www.pdb.org (PDB ID codes 3D1E, 3D1F, and 3DIG).
‡‡To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
August 12, 2008 ?
vol. 105 ?
no. 32 www.pnas.org?cgi?doi?10.1073?pnas.0804754105
baseline dispersion values that grouped within 5%, with a
Z-score of 0.901 ? 0.032 (14). Using a threshold value of 30%
displacement, the screen yielded 91 different compounds. The
screen was repeated, and chemicals that reproducibly displayed
30% displacement of Pol III peptide from ? were screened
further in Pol III ?-dependent DNA synthesis assays on a singly
primed M13mp18 ssDNA substrate (Fig. 1B). Compounds were
also tested for nonspecific inhibition in ?-independent assays by
using Pol I Klenow fragment (Fig. 1B). Inhibition of Pol I
Klenow fragment is indicative of nonspecific inhibition, for
example, by binding DNA or protein denaturation, because Pol
I, in contrast to the other Pols, is only stimulated 2-fold by the
Compounds that inhibit Pol III substantially greater than Pol
I (red circles in Fig. 1B) were tested in a TAMN-peptide
displacement assay by using Streptococcus pyogenes ? and a
TAMN-20-mer peptide derived from the C terminus of S.
pyogenes Pol C replicase (Kd ? 1.5 ?M; data not shown).
Residues lining the peptide-binding pocket of E. coli ? are
conserved across diverse bacterial species, and accordingly, a
subset of the compounds that disrupt the Gram-negative E. coli
Pol III-? interaction also score positive in the Gram-positive
S. pyogenes peptide displacement assay (Fig. 1C).
A Small-Molecule Compound Differentiates Between Function of Pols
II, III, and IV with ?. With a long-term goal of understanding the
roles of Pol II and Pol IV with ?, and their interplay with Pol III,
we wished to identify a compound that differentiates between
the actions of these DNA polymerases with ?. Pols II, III, and
IV display ?-dependent activity with a 7.2-Kb M13mp18 ssDNA
primed with a single oligonucleotide, and this assay revealed a
compound, RU7, that differentially inhibits these DNA poly-
merases (Fig. 2A). At high concentrations of RU7, only Pol IV
remains functional with ?; the RU7 compound is at least a
50-fold less effective inhibitor of Pol IV than Pol III. At
intermediate concentrations of RU7, the function of Pol III with
? is inhibited to a greater extent than Pol II.
Despite the similar structures of bacterial ? and eukaryotic
PCNA, the amino acid sequence of these clamps is highly
divergent, and sequence comparison algorithms do not detect
similarity between them (4, 5). Hence, a small-molecule com-
pound that binds ? and inhibits Pol III would not be expected to
bind PCNA and inhibit the eukaryotic replicase. In Fig. 2B, the
RU7 compound is titrated into replication reactions by using the
E. coli Pol III replicase or the PCNA-dependent yeast Pol ?
replicase. The results show that RU7 inhibits the E. coli system
with a Kiof ?10 ?M but does not inhibit the PCNA-dependent
Saccharomyces cereviase Pol ?. The assays in Fig. 2A contain an
additional 50 mM NaCl, compared with the assays of Fig. 2B,
which results in slightly different Kivalues.
The RU7 compound is shown in Fig. 3A. RU7 presumably
by using a peptide-displacement assay. However, the peptide-
displacement assay is indirect and could be explained in other
ways. For example, RU7 could bind the Pol III peptide or could
bind another position on ? and displace the TAMN-peptide by
an allosteric mechanism. To obtain direct evidence that the RU7
compound binds to the peptide-binding pocket in ?, we solved
the structure of a RU7-? complex.
Cocrystal Structure of ? Bound to a Chemical Inhibitor. The RU7-?
the apoenzyme structure (4). The region containing the com-
peptide from the ?-clamp. (A) Titration of E. coli ? into TAMN-labeled Pol III
C-terminal 20-mer peptide is monitored by fluorescence anisotropy. (B) Inhi-
bition of DNA replication by compounds identified in the peptide-
Pol I Klenow versus ?-dependent synthesis by Pol III* in the presence of 20 ?M
compound. (C) Chemicals (i.e., at 50 ?M) that displace E. coli Pol III ?-peptide
S. pyogenes ?.
High-throughput screen for chemical inhibitors that displace a Pol III
compound is titrated into ?-dependent replication reactions by using Pol II
(blue triangles), Pol III (red circles), or Pol IV (green diamonds). (B) The effect
of RU7 on replication assays by using either the E. coli replication system (i.e.,
yeast Pol ?, RFC, and PCNA; green circles).
A small-molecule compound selectively inhibits Pol III. (A) The RU7
RU7 compound. (B) Distances between RU7 and the ?-clamp. Side-chain
? in the absence or presence of RU7, respectively. Distances between RU7 and
protein residues are marked in black; the distances 3.73, 3.41, and 3.03 Å are
marked a, b, and c, respectively.
Georgescu et al.
August 12, 2008 ?
vol. 105 ?
no. 32 ?
pound was particularly well ordered (Fig. S2). The structure,
refined to 1.64 Å resolution, reveals that RU7 occupies subsite
1 of the peptide-binding pocket on both subunits (Fig. 3B).
Subsite 1 is the deeper of the two subsites and is shaped by
residues R152, L155, L177, V247, V347, and V360. The chemical
compound interacts with V247, P242, R152, R246, M362, and T172
(Fig. 3B). With the exception of the R152side chain that swings
5 Å to form a H-bond with the carbonyl oxygen 4 of RU7 (2.7
Å), the peptide-binding pocket residues do not undergo a
significant conformational change upon complex formation
To understand how RU7 derives specificity for Pol III over Pol
II and Pol IV, we solved structures of ? with peptides of Pol II
and Pol III and compared them with the previously determined
Pol IV-? structure and the structure of RU7 bound to ?.
Cocrystal Structure of ? Bound to a Pol III Peptide. Studies have
identified two sequences within the Pol III ? subunit that bind
to ?; one is located at the extreme C terminus of ? (15), and the
other is an internal site 220 residues upstream of the ? C
terminus (16). The two sequences differ to some extent, but both
ing peptides (9). Two 9-mer peptides, corresponding to the
internal and C-terminal clamp-binding sequences of Pol III ?,
were studied for ?-binding affinity by using isothermal titration
calorimetry (ITC) (Fig. S3). The analysis shows that the
C-terminal peptide of Pol III ? binds ? ?10-fold tighter than the
internal ?-binding sequence. We also examined ?-binding to
C-terminal Pol III peptide sequences of different lengths (9-mer,
20-mer, and 30-mer) to determine whether longer peptides bind
? tighter than the 9-mer (Fig. S3). Overall, the analysis indicates
that the major contribution of binding enthalpy is contained in
the 9-mer ?-clamp interactions (Fig. S3). We also used phage
display to determine whether there exists a peptide that binds to
? tighter than Pol III and other enzymes. The predominant
?-binding peptide sequence selected by phage display belonged
to the six-residue ?-binding motif (Fig. S4). ITC analysis of this
sequence, LQLELDF, shows that it binds to ? with similar
affinity as the Pol III 9-mer peptide (Kd? 1.8 ?M; data not
We tried to form cocrystals of ? with both the C-terminal and
the internal Pol III ?-binding peptides, but only cocrystals using
the Pol III ? C-terminal peptide were obtained (Fig. S2). The
structure of the Pol III C-terminal 9-mer (TAMN-
S1E2Q3V4E5L6E7F8D9) peptide bound to ? was solved by mo-
lecular replacement (Fig. 4A and Table S1). Superposition of the
C? atoms of the two 9-mer peptides occupying the two identical
binding sites of the ?-dimer yields a weighted rmsd value of only
0.26 Å. Hence, the Pol III 9-mer peptide binds each site in nearly
the same manner. Although all 9 aa can be modeled into the
electron density, only the C-terminal six residues fill the peptide-
binding pocket. TAMN is also clearly visible in the electron
density (Fig. S2). Residues L6, E7, and F8of the Pol III 9-mer lie
within subsite 1 and establish interactions with several side
chains of the clamp; residues L6and F8penetrate deep into this
site (Fig. 4A). Residues Q3, V4, and E5are positioned in subsite
2. The side chains of the first two residues of the 9-mer (S1and
E2) do not appear to form significant interactions with ?,
although the main-chain carbonyl of E2aligns structurally with
R10of the Pol IV peptide and forms a clear H-bond (2.7 Å) with
the amide NH of R365.
Pol II Peptide-? Structure. The Pol II peptide binds to ? with a Kd
value of 1.7 ?M (ITC; data not shown). We determined that the
structureofa Pol IIC-terminal
T1L2M3T4G5Q6L7G8L9F10) bound to ? by similar methods as for
the Pol III peptide-? complex. The Pol II peptide-? complex
crystallized in space group P1 (Fig. 4B, Fig. S2, and Table S1).
Interestingly, only one of the two peptide-binding sites in ?
contains the Pol II peptide. A similar half-site occupancy was
observed in the study of a Pol IV peptide bound to ? (10) due
to a crystal contact that occludes peptide from binding to the
second site. This may also be the case for the Pol II peptide-?
cocrystal. The Pol II ?-binding sequence consists of five residues
and thus lacks 1 aa compared with the six-residue ?-binding
N, blue; and O, red). ?-Clamp residues (ovals, side-chain interactions; rectan-
gles, main chain) are colored according to conservation as described in the
legend to Fig. 5. Hydrophilic and hydrophobic interactions are shown as
straight purple and curved black lines, respectively. (Lower) Pol III 9-mer
peptide (green) bound to ?. Peptide-free ? (light blue) is superimposed onto
peptide-bound ? (white). (B Upper) Pol II peptide atoms and ? side chains are
color coded as in A. (Lower) Pol II 10-mer peptide (blue) bound to ?. Peptide-
free ? (light blue) is superimposed onto the peptide-bound ? (wheat).
www.pnas.org?cgi?doi?10.1073?pnas.0804754105Georgescu et al.
last two C-terminal hydrophobic residues of the clamp-binding
motif. Within subsite 2, the Pol II peptide forms similar contacts
to ?, as observed for the Pol III peptide, but there are substantial
differences in the way the C-terminal sequences of the Pol II and
the five-residue Pol II ?-binding motif. The differences in subsite
1 are also due to L9, which superimposes with L7of the Pol III
9-mer. L9appears to be more efficiently buried than its Pol III
peptide homolog (L7) and establishes two additional hydropho-
bic contacts with the His175and Val361side chains of ?.
Comparison of Pol II, Pol III, Pol IV, and RU7 in the Peptide-Binding Site
of ?. Superposition of the Pol II and Pol III peptides bound to
? is shown in Fig. 5A. The Pol II and Pol III peptide residues in
subsite 2 are held in nearly the same conformation (all atoms
align within an rms of 0.125 Å). Subsite 1 is the deepest pocket
and would be expected to form the tightest interactions with
bound ligands. However, superposition of Pol II and Pol III
peptides in subsite 1 reveals a remarkable difference in the
position of the last few residues of the two peptides (see Fig. 5A).
It is interesting to note that the side chains in subsite 1 that bind
RU7 (R246, V247, S346, M362, T172, R152, and backbone amide of
P242) also bind the Pol III peptide, with the exception of R152. In
contrast, the Pol II peptide makes contact with six of these seven
residues. The Pol II peptide makes unique contacts to residues
His175and Val361in subsite 1, which do not appear to contact
RU7 or the Pol III 9-mer. Superposition of the Pol IV peptide,
which was solved in an earlier study (10), with the Pol II and Pol
III peptides show that the Pol IV peptide binds to subsite 2,
similar to Pols II and III, but the Pol IV peptide binds to subsite
1 of ? in yet a different way than either Pol II or Pol III (Fig. 5B).
Even though the Pol IV peptide is of the six-residue class of the
?-binding motif, it does not appear to contact some of the
residues that bind RU7 and the Pol III peptide. For example, E7
of the Pol III 9-mer (fifth residue of the six-residue consensus)
is a glycine in the Pol IV peptide. Hence, the two interactions
with ?, formed by the Pol III E7residue, are lacking in the Pol
IV peptide. One of the Pol III 9-mer E7interactions is with the
side chain of M362, and the second is a water-mediated H-bond
with the amide nitrogen of S346. Both of these residues contact
RU7 and thus likely contribute to the greater inhibition by RU7
of Pol III compared with Pol IV. Furthermore, Pol IV is known
to form additional contacts to ? outside of the peptide-binding
to the relative resistance of Pol IV to inhibition by the RU7
We cannot exclude the possibility that the N-terminal TAMN
may affect their conformation upon binding to ? (the structure
of Pol IV peptide-? complex did not contain an N-terminal
TAMN moiety). However, the structural differences in the three
peptides localize to the last few C-terminal residues and the
N-terminal residues are in essentially the same conformation for
underlie the structural differences in the C-terminal residues of
the polymerase peptides observed here. The TAMN moiety of
the Pol III peptide is visible in the electron density and forms a
crystal contact with an adjacent ?-clamp. However, the TAMN
moiety of the Pol II peptide is not visible in the electron density,
indicating that it may be disordered and thus may not interact
A Chemical Distinguishes Polymerase Function with ?.Thestructures
of polymerase peptides bound to ? reveal unexpected features in
the way different DNA polymerases engage the peptide-binding
pocket in the ?-clamp. Specifically, the clamp-binding peptides
of Pol II, Pol III, and Pol IV contact ? in different ways in subsite
1, the deepest part of the protein-binding pocket. The RU7
small-molecule compound binds to subsite 1 of the clamp and
distinguishes the activity of these different polymerases with ?.
The RU7 compound inhibits ?-dependent synthesis by Pol III at
least 5- and 50-fold more efficiently than it inhibits ?-dependent
synthesis by Pol II and Pol IV, respectively. We propose that the
underlying basis by which RU7 differentially inhibits Pols II, III,
and IV lies in the different ways these DNA polymerases bind to
subsite 1 of the clamp. In addition, the Pol IV-? structure shows
that Pol IV has two major points of contact with ?; Pol IV binds
the peptide-binding pocket and also interacts with the side of the
?-ring, which may contribute to the resistance of Pol IV to
Both ? and Pol III ? subunit are essential proteins, and
therefore the interaction of ? with ? cannot be eliminated by
mutagenesis without killing the cell. Thus, a chemical that
specifically disrupts this interaction may enable future chemical
genetic approaches to probe the roles of Pol II and Pol IV with
?. For example, both Pol II and Pol IV are present in normal
cells, but their role in normal growth is obscure. At low con-
centrations, RU7 will first inhibit Pol III-?, leaving only Pol II
and/or Pol IV to function with ?. Chemical genetic studies could
perhaps address whether Pol IV or Pol II play a role in
and Pol IV are induced to high levels in the SOS response to
DNA damage, and a chemical probe may be useful to address the
role of these enzymes during times of cellular stress. Chemical
genetic approaches would require further studies beyond those
reported here. For example, the chemical must enter the cell and
not be pumped back out.
The ?-Clamp As a Target for Antibacterial Compounds. Cell growth
information pathway such as transcription, translation, and
to ?. (A) Superposition of the Pol III peptide (green), Pol II peptide (blue), and
Pol IV peptide (purple; PDB ID code 1OK7) (10). (B) Superposition of Pol III
peptide (green) and the RU7 compound (orange). The surface of the ?-clamp
is colored white, and the protein-binding pocket of ? is colored according to
proceeds from red (90% conservation) to yellow (50% conservation). Circled
regions in the peptide-binding pocket indicate subsites 1 and 2. Figures were
prepared by using Pymol (27).
Georgescu et al.
August 12, 2008 ?
vol. 105 ?
no. 32 ?
replication. These pathways use numerous essential enzymes,
each of which is a potential target for an antibiotic chemical
inhibitor. Indeed, there are several well known, small-molecule
inhibitors of the transcription and translation machinery. To
mention a few, rifampicin targets RNA polymerase, and tetra-
cyclin and erythromycin target the ribosome. Precedent for
replication inhibition in antiviral and antibacterial therapies is
based in chain-terminating nucleoside analogs such as AZT
(polymerase inhibitors), topoisomerase inhibitors, and active-
site competitive inhibitors of RNA/DNA polymerases.
The ? sliding clamp would appear to be an attractive phar-
maceutical target because it is essential for cell viability yet
shares no sequence homology with the eukaryotic PCNA clamp.
Consistent with the extensive sequence difference between
PCNA and ?, RU7 does not inhibit PCNA-dependent synthesis
by yeast Pol ?. Thus, one may ask why antibacterial compounds
that target the ?-clamp have not been found long ago. A large
factor in this stems from the fact that ?1% of terrestrial
microorganisms have been isolated and cultured for study of
antimicrobial compounds (17). Another factor is that the bac-
terial replicase has historically been difficult to study because the
intracellular level is exceedingly low, and some components of
the replicase machinery separate during purification. Only re-
cently has the bacterial replication apparatus been expressed to
high levels and reconstituted for chemical screens, such as those
reported here. In contrast, RNA polymerase and ribosomes are
plentiful in cells, making these tightly associated machineries
relatively easy to purify for chemical screens and biochemical
The ?-clamp functions in a unique manner that could enhance
its suitability as an antibacterial target. For instance, Pol III and
the clamp loader bind to ? by using the same peptide-binding
pocket, and they function with ? in a very dynamic way (1, 18,
19). After the clamp loader assembles ? onto DNA, the clamp
is left on DNA and must wait for Pol III to associate with it. This
handoff from the clamp loader to Pol III provides a window of
opportunity for a chemical compound to target the unoccupied
peptide-binding pockets of the clamp. Moreover, during lagging
strand synthesis, Pol III repeatedly dissociates and reassociates
thousands of times, from one ?-clamp to another. This may
provide additional opportunity for a chemical inhibitor to bind
?. The fact that many different proteins bind ? could also make
development of resistant cells difficult. For example, if cells
mutate the peptide-binding pocket of ? to circumvent the
compound, the mutated clamp may no longer bind one or more
Comparison of ?-clamp sequences shows that residues that
bind the Pol III 9-mer peptide are rather well conserved (see Fig.
S5), as noted (10). Moreover, the structure of the S. pyogenes
?-clamp has been solved (PDB ID code 2AVT) (20), and
the Gram-negative E. coli ?-clamp and the ?-clamp of the
Gram-positive S. pyogenes bacterium, results in only minor
differences between corresponding ?-carbon atoms (i.e., up to
0.36 Å) (Fig. S5B). The structure of peptide-? complexes and
sequence alignment of bacterial ?-clamps suggests a consensus
sequence for the peptide-binding pocket of bacterial clamps
The E. coli Pol III C-terminal peptide inhibits ?-dependent
DNA synthesis by the Gram-positive Pol C replicases of S.
pyogenes and Staphylococcus aureus (21). In fact, the C-terminal
regions of the Pol C subunits of these Gram-positive replicases
also inhibit ?-dependent synthesis by E. coli Pol III core, and E.
coli ? even functions with the Pol C replicase of these Gram-
positive organisms (22). These observations support functional
conservation, in addition to sequence conservation of this
important polymerase/?-clamp connection and underscore the
possibility that a chemical compound may inhibit protein–
protein interactions from diverse organisms. Indeed, we dem-
onstrate here that some compounds that bind the Gram-negative
E. coli ?-clamp also bind to Gram-positive S. pyogenes ?.
Structure of RU7 As a Starting Point for Rational Drug Design. The
RU7 compound and its high-resolution structure with ? provide
a starting point for rational drug design. RU7 contains nine
H-bond acceptors, six of which contact side chains in ?. Most
side chains of ? that bind RU7 also form H-bonds to the Pol III
9-mer peptide. Although ? uses only 6 side chains to bind RU7,
11 side chains are used in this same vicinity to bind the Pol III
could be strategically placed on RU7 to enhance its affinity for
?. It is also interesting to note that the carboxyl moiety of RU7
is directed toward subsite 2 of the ?-binding pocket. Subsite 2
contains several conserved residues that form important con-
tacts with the Pol III 9-mer peptide. Thus additional interactive
atoms could be engineered into RU7 at the carboxyl moiety that
may fit into subsite 2 and enhance its potency.
Interestingly, some compounds are specific for the Gram-
negative clamp, indicating that the ?-clamp target offers possi-
bilities to develop either ‘‘species-limited’’ or ‘‘broad-spectrum’’
compounds (Fig. 1C). Thus, the high-resolution structure anal-
ysis of RU7 underscores the possibility that specific pharmaceu-
ticals may be developed for ?-clamps from different organisms.
Materials. HPLC-purified peptides were from Bio-Synthesis Inc.: Pol III C-
terminal 9-mer (double-underlined), 20-mer (underlined), 30-mer (italic) pep-
tides (GATWRVSPSDRLLNDLRGLIGSEQVELEFD), Pol III internal ?-binding mo-
tif (IGQADMFGV), Pol II C-terminal 10-mer (TAMN-T1L2M3T4G5Q6L7G8L9F10), S.
pyogenes PolC C-terminal 20-mer (TAMN-MGILGNMPEDNQLSLFDDFF), and S.
primed with a DNA 60-mer. Genes encoding Pol II and Pol IV were cloned into
pET11 (Novagen), followed by transformation, induction, and purification
from BL21(DE3) cells. Pol III core and Pol III* were constituted from recombi-
Structures. ?-Pol III 9-mer. ? (290 ?M) and Pol III C-terminal 9-mer (290 ?M)
and allowed to crystallize upon equilibrating 1.0 ?l of protein-peptide solu-
tion with 1.0 ?l of precipitant buffer (27.5% PEG 400, 100 mM Mes (pH 6.2),
100 mM CaCl2, ans 1% DMSO) in a hanging drop at 22°C. Trapezoidal crystals
(0.3 ? 0.5 ? 0.6 mm3) diffracted to 1.9 Å resolution at 100 K by using the X4a
beamline at the National Synchrotron Light Source, Brookhaven, NY. E. coli ?
crystallized in space group P3 (2) with two dimers in the asymmetric unit and
a solvent content of 53.4%. Data reduction was achieved by using HKL2000
(23). Subsequently, the model was rebuilt and refined at 2.0 Å by using the
program Crystallography and NMR System (CNS) (24) and ONO (25). The
structure was solved by molecular replacement by using the monoclinic struc-
protein-binding pockets of both protomers. All refinements were performed
against F0 ? 0? data, by using the CNS program suite (24).
?-Pol II 10-mer. A TAMN-labeled,10-mer peptide encompassing the C-terminal
crystallized in space group P1 with one dimer in the asymmetric unit and a
solvent content of 56.4%.
PEG 400, 100 mM Mes (pH 6.1), 100 mM CaCl2, and 3% DMSO] in a hanging
drop at 22°C. Crystals (0.3 ? 0.4 ? 0.5 mm3) formed in space group P1 and
was by molecular replacement and refined to 1.64 Å as described above.
Fluorescence Anisotropy. Increasing amounts of E. coli ? or S. pyogenes ? were
titrated into reactions containing 1 ?M TAMN-20-mer C-terminal peptide
derived from E. coli Pol III ?, S. pyogenes Pol C, or S. aureus Pol C in 20 mM
Tris?HCl (pH 7.5) and 0.5 mM EDTA at 25°C. Anisotropy was measured in a PTI
spectrofluorometer (535-nm excitation; 575-nm emission).
www.pnas.org?cgi?doi?10.1073?pnas.0804754105Georgescu et al.
High-Throughput Screening. Reactions (15 ?l) contained E. coli ? (1 ?M) and
TAMN-labeled Pol III C-terminal 20-mer peptide (5 ?M) and compound (50
The Rockefeller University chemical library contained 30,600 compounds at
the time of this work. Control wells included TAMN-labeled peptide with no
plates were gently agitated, centrifuged at 1,500 ? g, and incubated 10–15
min at 22°C. Fluorescence anisotropy was measured by using a plate reader
(excitation 535 nm; emission 575 nm). Results were stable for 16 h at 22°C.
Reactions were performed in duplicate, and compounds were analyzed for
inhibition of Pol I Klenow and for ?-dependent synthesis by Pol III* as de-
DNA Replication Assays. Compounds (0.1 ?l of 5 mM stocks) were added to
dNTPs, 30 fmol of singly primed M13mp18 ssDNA, 425 pmol of SSB, and 124
fmol of ?. DNA synthesis was initiated upon adding 80 fmol of Pol III*. Plates
were incubated at 22°C for 10 min on a shaker. The same protocol was
followed for Pol I Klenow reactions, except 2 units of Pol I Klenow was added
to initiate replication, and reactions did not contain ?. Reactions were
quenched with 25 ?l of 1% SDS/40 mM EDTA containing Quant-iT PicoGreen
(1/150 dilution) (Invitrogen) as described (26). Fluorescence intensity was
measured by using a plate reader (excitation 480 nm; emission 520 nm).
Assays comparing RU7 in yeast and E. coli replication systems were per-
formed as above except that reactions contained 20 ?M ?-32P-dTTP, and DNA
synthesis was monitored by radioactive incorporation. Yeast Pol ? replicase
was assayed by using 60 ng of PCNA, 115 ng of replication factor C (RFC), and
20 ng of Pol ?. Assays comparing RU7 with Pols II, III ,and IV were also
performed by using ?-32P-dTTP as above except that ? (100 fmol) was loaded
onto singly primed M13mp18 DNA by ?-complex (20 fmol) in a 5-min prein-
cubation before adding DNA polymerase (600 fmol per reaction). After 1 min
at 37°C, 1 ?l of RU7 was added, and replication was initiated by adding dATP
Accession Code. Coordinates are deposited in the Protein Data Bank under
ACKNOWLEDGMENTS. We thank Chuck Karan for help at the Rockefeller
University Screening facility and the staff at beamlines X4a and X25 of the
by National Institutes of Health Grants GM38839 (to M.O.D.), GM70841 (to
X.-P.K.), and GM45547 (to J.K.).
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