# Patent

## Title
Protein crystal comprising the processivity clamp factor of DNA polymerase and a ligand, and its uses

## Abstract

A protein crystal having the processivity clamp factor of DNA polymerase that is the β subunit of DNA polymerase III of Escherichia coli and a peptide of about 3 to about 30 amino acids, in particular of about 16 amino acids. The peptide includes all or part of the processivity clamp factor binding sequence of a processivity clamp factor interacting protein, such as prokaryotic Pol I, Pol II, Pol III, Pol IV, Pol V, MutS, ligase I, α subunit of DNA polymerase, UmuD or UmuD′, or eukaryotic pol ε, pol δ, pol η, pol ι, pol κ.

## Background
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a protein crystal comprising the processivity clamp factor of DNA polymerase and a peptide comprising all or part of the processivity clamp factor binding sequence of a processivity clamp factor interacting protein, and its uses, in particular for the screening, the design or the modification of ligands of the processivity clamp factor of DNA polymerase.
2. Description of the Related Art
The presence of lesions on DNA may severely impair its replication and have dramatic consequences on cells survival. Beside the activity of efficient repair processes, which remove most of the lesions from DNA before replication occurs, the replisome is able to cope with replication blocking DNA lesions, thanks to specialized biochemical processes referred to as damaged DNA tolerance pathways. Translesion synthesis (TLS) is one of these mechanisms which requires the incorporation of a nucleotide opposite and past the lesion. Depending on the nature of the incorporated nucleotide relative to the parental sequence, the TLS process is error-free or mutagenic. TLS has recently gained much understanding, with the discovery of specialized DNA polymerases, which are able to replicate through lesions which otherwise impede the progression of DNA polymerases involved in replication. These new polymerases have been found in both prokaryotes and eukaryotes and most of them have been classified in the Y superfamily (Ohmori et al., 2001). In Escherichia coli, two such polymerases have been identified, Pol IV (DinB) (Wagner et al., 1999) and Pol V (Tang et al., 1999; Reuven et al., 1999), whereas Pol II polymerase has also been shown to perform TLS, although it belongs to the B family (Napolitano et al., 2000; Becherel et al., 2001; Fuchs et al, 2001). Interestingly, all these three polymerase genes are part of the SOS network and are induced upon the arrest of replication due to the presence of replicase blocking lesions onto DNA.
The discovery of translesional polymerases (Ohmori et al., 2001) resulted in a major modification of the molecular model of TLS and resulting lesion induced mutagenesis. The previous model, essentially built on genetic experiments in E. coli (Bridges and Woodgates, 1985) suggested that the replicative polymerase stalled at blocking lesions was assisted by SOS induced proteins, whose functions were expected to facilitate the polymerase progression through the lesion by increasing its anchoring onto modified DNA or by reducing its fidelity either by alteration of the correct nucleotide selection process and/or by inhibition of its proofreading activity. The current new model (Cordonnier et al., 1999) proposes that the blocked replicative polymerase is replaced by one or several TLS polymerases that cooperate at different steps of the translesional process, namely incorporation opposite the lesion and elongation of the lesion terminus, to ensure an efficient bypass of the lesion. These polymerases further dissociate from the DNA substrate and the replicative enzyme resumes its synthesis function.
It was demonstrated that prokaryotic and eukaryotic replicative polymerases (Pol III holoenzyme of E. coli, pol C, eukaryotic pol δ and pol ε) physically interact with their respective processivity clamp factor, also called sliding clamp. Moreover, all prokaryotic and most eukaryotic TLS polymerases also interact with their processivity clamp factor (Lenne-Samuel et al., 2002; Wagner et al., 2000; Becherel et al., 2002; Haracska et al., 2002; Haracska et al., 2001a; Haracska et al., 2001b). These clamps, which act by increasing the replicative polymerase processivity (Bruck and O'Donnel, 2001), are homodimeric (β of E. coli) or homotrimeric (gp45 of T4/RB69 or PCNA in eukaryotes) toroid-shape molecules that are loaded onto DNA near primer-template junctions, by specific clamp loader complexes (e.g. the so-called γ complex in E. coli and RFC in eukaryotes). The β and PCNA monomers fold into structurally similar subdomains (3 and 2, respectively), despite a lack of internal homology in their amino acids sequences, so that the ring presents a pseudo-six-fold symmetry. A consensus pentapeptidic sequence, QL(SD)LF, conserved among eubacteria, was identified in most of the β-binding proteins as the motif mediating their connection with the clamp, through hydrophobic interactions (Dalrymple et al., 2001). Similarly, a eukaryotic PCNA (or alternative sliding clamps) consensus binding sequence has been identified. A recent study in E. coli demonstrated that the integrity of this motif is absolutely required for the inducible polymerases to perform TLS: Pol IV and Pol II mutant proteins deleted for their β-clamp binding motif retain their polymerase activity, but loose their functions in the TLS process in vivo, highlightening the fact that their functional interaction with β is crucial for translesion DNA synthesis and mutagenesis (Becherel et al., 2002; Lenne-Samuel et al., 2002).
The presence of several TLS polymerases within a single organism has remained a puzzling question. Analysis of the TLS process in E. coli indicated that, depending on both the nature of the lesion and the local DNA sequence, one or several TLS polymerases may participate to a single TLS event (Napolitano et al., 2000; Wagner et al., 2002). TLS appears as a complex process where a pool of low fidelity polymerases replace the highly stringent replisome and eventually exchange mutually to accommodate the large variety of DNA lesions and to ensure ultimately the completion of DNA replication. Whether this polymerase switching process is somehow coordinated or simply occurs on the basis of competition between the different TLS polymerases is not yet known.
BRIEF SUMMARY OF THE INVENTION
An object of the invention is to provide a method to obtain ligands of the processivity clamp factor which would impair the interaction between the sliding clamp and its interacting proteins.
Such ligands might be useful for the preparation of drugs for the treatment of bacterial diseases or of proliferative disorders.
The invention follows on from the solving by the Inventors of the structure of a co-crystal obtained between the β clamp of E. coli and the 16 residues C-terminal peptide of Pol IV DNA polymerase (P16) of E. coli containing its β-binding sequence, from the identification of the peptide binding site on β and from the description of the interactions between P16 and β residues.
The Invention also follows on from the results of experiments carried out by the Inventors showing that P16 competes with Pol IV, but also with the α subunit of the E. coli replicative Pol III holoenzyme, for binding to β, thus inhibiting their β dependent polymerase activity.
The present invention relates to a protein crystal comprising the processivity clamp factor of DNA polymerase and a peptide of about 3 to about 30 amino acids, in particular of about 16 amino acids, said peptide comprising all or part of the processivity clamp factor binding sequence of a processivity clamp factor interacting protein, such as prokaryotic Pol I, Pol II, Pol III, Pol IV, Pol V, MutS, ligase I, α subunit of DNA polymerase, UmuD or UmuD′, or eukaryotic pol ε, pol δ, pol η, pol ι, pol κ.
Other processivity clamp factor interacting proteins are notably described in Tsurimoto et al. (1999).
The expression “processivity clamp factor of DNA polymerase” refers to dnaN genes products and their functional analogs in prokaryotes, and PCNA genes products and their functional analogs and orthologs in eukaryotes. It can also be referred to as a sliding clamp. It is notably described in Kong et al. (1992) and Gulbis et al. (1996).
“Pol I”, “Pol II”, “Pol III”, “Pol IV”, “Pol V” respectively refer to DNA polymerase I, II, III, IV and V, in bacteria, such as E. coli, as reviewed in Friedberg et al. (2000a), and Friedberg et al. (2000b).
“MutS” refers to the product of the mutS gene in E. coli, and functional analogs and orthologs thereof, involved in mismatch repair.
“Ligase I” refers to the product of the lig gene in E. coli, and functional analogs and orthologs thereof.
“α subunit of DNA polymerase” refers to the product of the dnaE gene in E. coli, and functional analogs and orthologs thereof.
“UmuD” refers to the product of the umuD gene in E. coli, and functional analogs and orthologs thereof.
“Pol ε”, “pol δ”, “pol η”, “pol ι”, “pol κ” refer to eukaryotic polymerases as reviewed in Friedberg et al. (2000a), and Friedberg et al. (2000b).
The invention more particularly relates to a protein crystal as defined above, wherein the processivity clamp factor of DNA polymerase is the β subunit of DNA polymerase, in particular the β subunit of DNA polymerase III of Escherichia coli, and the peptide has the following sequence:

VTLLDPQMERQLVLGL  (SEQ ID NO: 1)

The β subunit of DNA polymerase III of Escherichia coli is in particular described in Kong et al. (1992).
The invention more particularly relates to a protein crystal as defined above, comprising the β subunit of DNA polymerase III of Escherichia coli and the peptide of SEQ ID NO: 1, said crystal belonging to the triclinic space group P1 and its cell dimensions being approximately a=41.23 Å, b=65.22 Å, c=73.38 Å, α=73.11°, β=85.58°, γ=85.80°.
The expression “triclinic space group P1” refers to a nomenclature well known to the man skilled in the art, it is in particular described in “International tables for X-ray crystallography”, Vol. 1 (The Kynoch press, Birmingham, England, 1968)
The expression “cell dimensions” refers to the geometrical description of the smallest volume being repeated in the three dimensions to build the crystal.
The invention more particularly relates to a protein crystal as defined above, characterized by the atomic coordinates such as obtained by the X-ray diffraction of said crystal, said atomic coordinates being represented in FIG. 1.
The expression “atomic coordinates” refers to the three coordinates X, Y, Z (given in Å, 1 Å=10−10 m) necessary to describe the exact position of each atom in the molecule.
The expression “X-ray diffraction” refers to the phenomenon following which X-rays are scattered in a specific way by a crystal.
Two major X-ray sources can be used: a rotating anode, which is a usual laboratory equipment and/or a synchrotron which is a large-scale equipment, such as the European Synchrotron Radiation Facility (ESRF) in Grenoble, France.
The general methodology to obtain atomic coordinates from X-ray diffraction of a crystal is well known to man skilled in the art, briefly it consists in measuring the intensities of the numerous secondary X-rays beams resulting from the diffraction by the crystal of an incident X-ray beam.
The invention more particularly relates to a protein crystal as defined above, characterized by the atomic coordinates representing the peptide and the peptide binding site of the β subunit of DNA polymerase III of Escherichia coli, and being as follows:























ATOM
4045
N
LEU
B
155
5.874
17.816
22.109
1.00
1.00
B


ATOM
4046
CA
LEU
B
155
6.029
16.359
22.087
1.00
1.00
B


ATOM
4047
CB
LEU
B
155
5.055
15.686
23.064
1.00
1.00
B


ATOM
4048
CG
LEU
B
155
5.260
16.046
24.536
1.00
1.00
B


ATOM
4049
CD1
LEU
B
155
4.256
15.237
25.360
1.00
1.00
B


ATOM
4050
CD2
LEU
B
155
6.686
15.757
24.980
1.00
1.00
B


ATOM
4051
C
LEU
B
155
5.808
15.776
20.682
1.00
1.00
B


ATOM
4052
O
LEU
B
155
6.177
14.613
20.431
1.00
1.00
B


ATOM
4177
N
THR
B
172
9.112
11.246
22.902
1.00
1.00
B


ATOM
4178
CA
THR
B
172
8.212
10.730
23.917
1.00
1.00
B


ATOM
4179
CB
THR
B
172
8.776
11.014
25.344
1.00
1.00
B


ATOM
4180
OG1
THR
B
172
7.931
10.400
26.328
1.00
1.00
B


ATOM
4181
CG2
THR
B
172
8.870
12.532
25.619
1.00
1.00
B


ATOM
4182
C
THR
B
172
6.805
11.269
23.709
1.00
1.00
B


ATOM
4183
O
THR
B
172
6.588
12.352
23.145
1.00
1.00
B


ATOM
4192
N
GLY
B
174
4.562
10.770
26.397
1.00
1.00
B


ATOM
4193
CA
GLY
B
174
3.992
10.745
27.737
1.00
1.00
B


ATOM
4194
C
GLY
B
174
3.762
9.337
28.266
1.00
1.00
B


ATOM
4195
O
GLY
B
174
3.667
9.141
29.489
1.00
1.00
B


ATOM
4196
N
HIS
B
175
3.650
8.349
27.375
1.00
1.00
B


ATOM
4197
CA
HIS
B
175
3.440
6.953
27.796
1.00
1.00
B


ATOM
4198
CB
HIS
B
175
2.313
6.309
26.977
1.00
1.00
B


ATOM
4199
CG
HIS
B
175
0.992
6.997
27.119
1.00
1.00
B


ATOM
4200
CD2
HIS
B
175
0.106
7.435
26.193
1.00
1.00
B


ATOM
4201
ND1
HIS
B
175
0.420
7.255
28.345
1.00
1.00
B


ATOM
4202
CE1
HIS
B
175
−0.763
7.817
28.170
1.00
1.00
B


ATOM
4203
NE2
HIS
B
175
−0.977
7.938
26.875
1.00
1.00
B


ATOM
4204
C
HIS
B
175
4.706
6.135
27.641
1.00
1.00
B


ATOM
4205
O
HIS
B
175
4.990
5.212
28.403
1.00
1.00
B


ATOM
4207
CA
ARG
B
176
6.711
5.768
26.422
1.00
18.30
B


ATOM
4208
CB
ARG
B
176
6.575
4.633
25.398
1.00
19.53
B


ATOM
4209
CG
ARG
B
176
6.329
5.094
23.954
1.00
22.88
B


ATOM
4210
CD
ARG
B
176
4.876
4.888
23.657
1.00
22.11
B


ATOM
4211
NE
ARG
B
176
4.435
5.312
22.314
1.00
22.09
B


ATOM
4212
CZ
ARG
B
176
4.555
4.591
21.202
1.00
20.17
B


ATOM
4213
NH1
ARG
B
176
5.159
3.403
21.213
1.00
17.04
B


ATOM
4214
NH2
ARG
B
176
3.914
4.977
20.120
1.00
20.02
B


ATOM
4215
C
ARG
B
176
7.684
6.807
25.902
1.00
17.30
B


ATOM
4216
O
ARG
B
176
7.255
7.860
25.374
1.00
18.10
B


ATOM
4217
N
LEU
B
177
8.957
6.504
26.080
1.00
17.97
B


ATOM
4218
CA
LEU
B
177
10.049
7.360
25.633
1.00
17.85
B


ATOM
4219
CB
LEU
B
177
10.664
8.095
26.827
1.00
18.29
B


ATOM
4220
CG
LEU
B
177
11.921
8.955
26.611
1.00
16.28
B


ATOM
4221
CD1
LEU
B
177
11.819
10.163
27.559
1.00
19.52
B


ATOM
4222
CD2
LEU
B
177
13.191
8.172
26.839
1.00
19.12
B


ATOM
4223
C
LEU
B
177
11.110
6.517
24.964
1.00
18.45
B


ATOM
4224
O
LEU
B
177
11.291
5.329
25.281
1.00
18.33
B


ATOM
4710
N
PRO
B
242
11.254
17.279
27.890
1.00
1.00
B


ATOM
4711
CD
PRO
B
242
9.987
16.826
27.286
1.00
1.00
B


ATOM
4712
CA
PRO
B
242
11.660
16.404
28.997
1.00
1.00
B


ATOM
4713
CB
PRO
B
242
10.688
15.230
28.874
1.00
1.00
B


ATOM
4714
CG
PRO
B
242
9.448
15.869
28.336
1.00
1.00
B


ATOM
4715
C
PRO
B
242
13.124
15.947
28.987
1.00
1.00
B


ATOM
4716
O
PRO
B
242
13.728
15.748
27.925
1.00
1.00
B


ATOM
4748
N
ARG
B
246
16.133
11.840
33.560
1.00
1.00
B


ATOM
4749
CA
ARG
B
246
15.239
11.808
34.707
1.00
1.00
B


ATOM
4750
CB
ARG
B
246
14.755
13.227
34.984
1.00
1.00
B


ATOM
4751
CG
ARG
B
246
15.880
14.252
35.113
1.00
1.00
B


ATOM
4752
CD
ARG
B
246
16.443
14.295
36.529
1.00
1.00
B


ATOM
4753
NE
ARG
B
246
15.374
14.318
37.524
1.00
1.00
B


ATOM
4754
CZ
ARG
B
246
14.316
15.126
37.477
1.00
1.00
B


ATOM
4755
NH1
ARG
B
246
14.169
15.992
36.481
1.00
1.00
B


ATOM
4756
NH2
ARG
B
246
13.396
15.067
38.430
1.00
1.00
B


ATOM
4757
C
ARG
B
246
14.022
10.889
34.566
1.00
1.00
B


ATOM
4758
O
ARG
B
246
13.384
10.536
35.560
1.00
1.00
B


ATOM
4759
N
VAL
B
247
13.695
10.532
33.327
1.00
1.00
B


ATOM
4760
CA
VAL
B
247
12.553
9.675
33.018
1.00
1.00
B


ATOM
4761
CB
VAL
B
247
12.061
9.942
31.585
1.00
1.00
B


ATOM
4762
CG1
VAL
B
247
10.930
8.991
31.216
1.00
1.00
B


ATOM
4763
CG2
VAL
B
247
11.624
11.391
31.462
1.00
1.00
B


ATOM
4764
C
VAL
B
247
12.962
8.218
33.133
1.00
1.00
B


ATOM
4765
O
VAL
B
247
12.125
7.334
33.308
1.00
1.00
B


ATOM
4996
M
PHE
B
278
−7.702
−1.352
24.244
1.00
1.00
B


ATOM
4997
CA
PHE
B
278
−6.698
−1.155
25.300
1.00
1.00
B


ATOM
4998
CB
PHE
B
278
−7.318
−1.432
26.663
1.00
1.00
B


ATOM
4999
CG
PHE
B
278
−8.431
−0.459
27.021
1.00
1.00
B


ATOM
5000
CD1
PHE
B
278
−8.142
0.882
27.268
1.00
1.00
B


ATOM
5001
CD2
PHE
B
276
−9.760
−0.869
27.021
1.00
1.00
B


ATOM
5002
CE1
PHE
B
278
−9.177
1.816
27.508
1.00
1.00
B


ATOM
5003
CE2
PHE
B
278
−10.795
0.052
27.258
1.00
1.00
B


ATOM
5004
CZ
PHE
B
278
−10.496
1.391
27.500
1.00
1.00
B


ATOM
5005
C
PHE
B
278
−5.403
−1.957
25.131
1.00
1.00
B


ATOM
5006
O
PHE
B
278
−4.356
−1.582
25.677
1.00
1.00
B


ATOM
5332
N
ASN
B
320
0.635
−2.143
27.431
1.00
1.00
B


ATOM
5333
CA
ASN
B
320
−0.051
−1.983
26.158
1.00
1.00
B


ATOM
5334
CB
ASN
B
320
−0.055
−0.504
25.796
1.00
1.00
B


ATOM
5335
CG
ASN
B
320
−0.561
−0.259
24.407
1.00
1.00
B


ATOM
5336
OD1
ASN
B
320
−0.226
−0.997
23.481
1.00
1.00
B


ATOM
5337
ND2
ASN
B
320
−1.362
0.791
24.242
1.00
1.00
B


ATOM
5338
C
ASN
B
320
0.927
−2.745
25.249
1.00
1.00
B


ATOM
5339
O
ASN
B
320
2.093
−2.350
25.102
1.00
1.00
B


ATOM
5353
N
TYR
B
323
2.932
−0.853
22.482
1.00
1.00
B


ATOM
5354
CA
TYR
B
323
4.110
−0.088
22.908
1.00
1.00
B


ATOM
5355
CB
TYR
B
323
3.878
0.590
24.259
1.00
1.00
B


ATOM
5356
CG
TYR
B
323
2.813
1.668
24.294
1.00
1.00
B


ATOM
5357
CD1
TYR
B
323
2.397
2.314
23.127
1.00
1.00
B


ATOM
5358
CE1
TYR
B
323
1.458
3.374
23.170
1.00
1.00
B


ATOM
5359
CD2
TYR
B
323
2.284
2.093
25.509
1.00
1.00
B


ATOM
5360
CE2
TYR
B
323
1.354
3.166
25.567
1.00
1.00
B


ATOM
5361
CZ
TYR
B
323
0.957
3.790
24.399
1.00
1.00
B


ATOM
5362
OH
TYR
B
323
0.112
4.886
24.453
1.00
1.00
B


ATOM
5363
C
TYR
B
323
5.327
−1.018
23.041
1.00
1.00
B


ATOM
5364
O
TYR
B
323
6.468
−0.646
22.726
1.00
1.00
B


ATOM
5519
N
VAL
B
344
3.837
−1.100
39.291
1.00
1.00
B


ATOM
5520
CA
VAL
B
344
3.324
0.227
39.030
1.00
1.00
B


ATOM
5521
CB
VAL
B
344
2.676
0.818
40.318
1.00
1.00
B


ATOM
5522
CG1
VAL
B
344
1.474
−0.026
40.725
1.00
1.00
B


ATOM
5523
CG2
VAL
B
344
3.687
0.847
41.456
1.00
1.00
B


ATOM
5524
C
VAL
B
344
4.405
1.163
38.512
1.00
1.00
B


ATOM
5525
O
VAL
B
344
4.199
2.365
38.405
1.00
1.00
B


ATOM
5532
N
SER
B
346
7.618
2.151
35.615
1.00
21.53
B


ATOM
5533
CA
SER
B
346
8.060
2.002
34.239
1.00
21.50
B


ATOM
5534
CB
SER
B
346
8.655
3.320
33.722
1.00
21.47
B


ATOM
5535
OG
SER
B
346
9.793
3.703
34.474
1.00
26.08
B


ATOM
5536
C
SER
B
346
9.107
0.914
34.106
1.00
20.70
B


ATOM
5537
O
SER
B
346
9.755
0.521
35.078
1.00
21.55
B


ATOM
5632
N
VAL
B
360
11.730
3.546
27.545
1.00
1.00
B


ATOM
5633
CA
VAL
B
360
11.023
3.501
28.812
1.00
1.00
B


ATOM
5634
CB
VAL
B
360
11.276
4.794
29.641
1.00
1.00
B


ATOM
5635
CG1
VAL
B
360
10.448
4.742
30.934
1.00
1.00
B


ATOM
5636
CG2
VAL
B
360
12.753
4.923
29.937
1.00
1.00
B


ATOM
5637
C
VAL
B
360
9.562
3.381
28.501
1.00
1.00
B


ATOM
5638
O
VAL
B
360
9.008
4.188
27.753
1.00
1.00
B


ATOM
5639
N
VAL
B
361
8.905
2.372
29.069
1.00
19.72
B


ATOM
5640
CA
VAL
B
361
7.488
2.188
28.831
1.00
18.92
B


ATOM
5641
CB
VAL
B
361
7.216
0.872
28.069
1.00
18.99
B


ATOM
5642
CG1
VAL
B
361
5.743
0.769
27.716
1.00
18.31
B


ATOM
5643
CG2
VAL
B
361
8.065
0.839
26.786
1.00
17.76
B


ATOM
5644
C
VAL
B
361
6.793
2.100
30.167
1.00
19.47
B


ATOM
5645
O
VAL
B
361
7.232
1.362
31.038
1.00
16.90
B


ATOM
5646
N
MET
B
362
5.737
2.885
30.316
1.00
1.00
B


ATOM
5647
CA
MET
B
362
4.962
2.882
31.540
1.00
1.00
B


ATOM
5648
CB
MET
B
362
4.226
4.206
31.682
1.00
1.00
B


ATOM
5649
CG
MET
B
362
3.918
4.589
33.122
1.00
1.00
B


ATOM
5650
SD
MET
B
362
5.405
4.806
34.163
1.00
1.00
B


ATOM
5651
CE
MET
B
362
4.575
4.880
35.731
1.00
1.00
B


ATOM
5652
C
MET
B
362
3.949
1.731
31.471
1.00
1.00
B


ATOM
5653
O
MET
B
362
3.385
1.438
30.410
1.00
1.00
B


ATOM
5654
N
PRO
B
363
3.698
1.069
32.599
1.00
1.00
B


ATOM
5655
CD
PRO
B
363
4.521
1.025
33.818
1.00
1.00
B


ATOM
5656
CA
PRO
B
363
2.729
−0.038
32.579
1.00
1.00
B


ATOM
5657
CB
PRO
B
363
3.155
−0.883
33.776
1.00
1.00
B


ATOM
5658
CG
PRO
B
363
3.665
0.160
34.754
1.00
1.00
B


ATOM
5659
C
PRO
B
363
1.272
0.395
32.672
1.00
1.00
B


ATOM
5660
O
PRO
B
363
0.959
1.574
32.311
1.00
1.00
B


ATOM
5661
N
MET
B
364
0.368
−0.568
32.537
1.00
1.00
B


ATOM
5662
CA
MET
B
364
−1.037
−0.272
32.674
1.00
1.00
B


ATOM
5663
CB
MET
B
364
−1.780
−0.391
31.332
1.00
1.00
B


ATOM
5664
CG
MET
B
364
−1.636
−1.670
30.568
1.00
1.00
B


ATOM
5665
SD
MET
B
364
−2.386
−1.510
28.872
1.00
1.00
B


ATOM
5666
CE
MET
B
364
−4.155
−1.253
29.308
1.00
1.00
B


ATOM
5667
C
MET
B
364
−1.602
−1.218
33.725
1.00
1.00
B


ATOM
5668
O
MET
B
364
−0.999
−2.251
34.035
1.00
1.00
B


ATOM
5669
N
ARG
B
365
−2.732
−0.836
34.307
1.00
1.00
B


ATOM
5670
CA
ARG
B
365
−3.383
−1.655
35.324
1.00
1.00
B


ATOM
5671
CB
ARG
B
365
−4.029
−0.756
36.394
1.00
1.00
B


ATOM
5672
CG
ARG
B
365
−4.785
−1.490
37.505
1.00
1.00
B


ATOM
5673
CD
ARG
B
365
−3.859
−2.316
38.398
1.00
1.00
B


ATOM
5674
NE
ARG
B
365
−4.571
−2.956
39.505
1.00
1.00
B


ATOM
5675
CZ
ARG
B
365
−3.984
−3.707
40.434
1.00
1.00
B


ATOM
5676
NH1
ARG
B
365
−2.678
−3.913
40.385
1.00
1.00
B


ATOM
5677
NH2
ARG
B
365
−4.698
−4.247
41.418
1.00
1.00
B


ATOM
5678
C
ARG
B
365
−4.459
−2.492
34.648
1.00
1.00
B


ATOM
5679
O
ARG
B
365
−5.449
−1.961
34.150
1.00
1.00
B


ATOM
5680
N
LEU
B
366
−4.267
−3.801
34.609
1.00
41.59
B


ATOM
5681
CA
LEU
B
366
−5.272
−4.665
33.996
1.00
44.25
B


ATOM
5682
CB
LEU
B
366
−4.615
−5.908
33.366
1.00
45.24
B


ATOM
5683
CG
LEU
B
366
−3.640
−5.701
32.202
1.00
45.46
B


ATOM
5684
CD1
LEU
B
366
−4.331
−5.029
31.031
1.00
47.09
B


ATOM
5685
CD2
LEU
B
366
−2.489
−4.856
32.678
1.00
46.71
B


ATOM
5686
C
LEU
B
366
−6.263
−5.080
35.092
1.00
45.55
B


ATOM
5687
O
LEU
B
366
−6.424
−6.296
35.333
1.00
46.32
B


ATOM
5688
OXT
LEU
B
366
−6.868
−4.169
35.704
1.00
46.33
B


ATOM
5689
CB
ARG
C
10
−5.663
0.205
32.737
0.76
1.00
C


ATOM
5690
CG
ARG
C
10
−7.073
−0.397
32.771
0.76
1.00
C


ATOM
5691
CD
ARG
C
10
−7.748
−0.383
31.408
0.76
1.00
C


ATOM
5692
NE
ARG
C
10
−8.728
−1.462
31.268
0.76
1.00
C


ATOM
5693
CZ
ARG
C
10
−9.992
−1.301
30.875
0.76
1.00
C


ATOM
5694
NH1
ARG
C
10
−10.464
−0.093
30.582
0.76
1.00
C


ATOM
5695
NH2
ARG
C
10
−10.779
−2.365
30.749
0.76
1.00
C


ATOM
5696
C
ARG
C
10
−4.106
2.152
32.497
0.76
1.00
C


ATOM
5697
O
ARG
C
10
−3.278
1.863
33.369
0.76
1.00
C


ATOM
5698
N
ARG
C
10
−6.417
2.186
31.464
0.76
1.00
C


ATOM
5699
CA
ARG
C
10
−5.587
1.727
32.625
0.76
1.00
C


ATOM
5700
N
GLN
C
11
−3.805
2.853
31.408
0.76
1.00
C


ATOM
5701
CA
GLN
C
11
−2.458
3.321
31.094
0.76
1.00
C


ATOM
5702
CB
GLN
C
11
−2.423
3.866
29.662
0.76
1.00
C


ATOM
5703
CG
GLN
C
11
−1.047
4.361
29.231
0.76
1.00
C


ATOM
5704
CD
GLN
C
11
−0.039
3.245
29.174
0.76
1.00
C


ATOM
5705
OE1
GLN
C
11
−0.263
2.232
28.494
0.76
1.00
C


ATOM
5706
NE2
GLN
C
11
1.082
3.415
29.876
0.76
1.00
C


ATOM
5707
C
GLN
C
11
−1.895
4.396
32.038
0.76
1.00
C


ATOM
5708
O
GLN
C
11
−2.494
5.467
32.217
0.76
1.00
C


ATOM
5709
N
LEU
C
12
−0.732
4.111
32.618
0.76
1.00
C


ATOM
5710
CA
LEU
C
12
−0.065
5.046
33.519
0.76
1.00
C


ATOM
5711
CB
LEU
C
12
0.754
4.277
34.561
0.76
1.00
C


ATOM
5712
CG
LEU
C
12
−0.036
3.305
35.450
0.76
1.00
C


ATOM
5713
CD1
LEU
C
12
0.907
2.681
36.468
0.76
1.00
C


ATOM
5714
CD2
LEU
C
12
−1.184
4.040
36.153
0.76
1.00
C


ATOM
5715
C
LEU
C
12
0.845
5.948
32.680
0.76
1.00
C


ATOM
5716
O
LEU
C
12
1.111
5.653
31.510
0.76
1.00
C


ATOM
5717
N
VAL
C
13
1.317
7.044
33.273
0.76
1.00
C


ATOM
5718
CA
VAL
C
13
2.166
7.987
32.543
0.76
1.00
C


ATOM
5719
CB
VAL
C
13
1.473
9.371
32.386
0.76
1.00
C


ATOM
5720
CG1
VAL
C
13
0.217
9.239
31.523
0.76
1.00
C


ATOM
5721
CG2
VAL
C
13
1.113
9.929
33.750
0.76
1.00
C


ATOM
5722
C
VAL
C
13
3.542
8.211
33.174
0.76
1.00
C


ATOM
5723
O
VAL
C
13
3.740
8.050
34.381
0.76
1.00
C


ATOM
5724
N
LEU
C
14
4.498
8.595
32.339
0.76
1.00
C


ATOM
5725
CA
LEU
C
14
5.860
8.846
32.803
0.76
1.00
C


ATOM
5726
CB
LEU
C
14
6.836
8.819
31.619
0.76
1.00
C


ATOM
5727
CG
LEU
C
14
6.972
7.481
30.889
0.76
1.00
C


ATOM
5728
CD1
LEU
C
14
7.666
7.705
29.557
0.76
1.00
C


ATOM
5729
CD2
LEU
C
14
7.744
6.495
31.769
0.76
1.00
C


ATOM
5730
C
LEU
C
14
6.010
10.186
33.517
0.76
1.00
C


ATOM
5731
O
LEU
C
14
5.238
11.126
33.284
0.76
1.00
C


ATOM
5732
N
GLY
C
15
7.000
10.263
34.396
0.76
1.00
C


ATOM
5733
CA
GLY
C
15
7.264
11.510
35.090
0.76
1.00
C


ATOM
5734
C
GLY
C
15
8.263
12.275
34.234
0.76
1.00
C


ATOM
5735
O
GLY
C
15
9.472
12.210
34.462
0.76
1.00
C


ATOM
5736
N
LEU
C
16
7.750
12.995
33.241
0.76
1.00
C


ATOM
5737
CA
LEU
C
16
8.576
13.756
32.306
0.76
1.00
C


ATOM
5738
CB
LEU
C
16
7.732
14.157
31.094
0.76
1.00
C


ATOM
5739
CG
LEU
C
16
7.258
12.955
30.269
0.76
1.00
C


ATOM
5740
CD1
LEU
C
16
6.303
13.411
29.171
0.76
1.00
C


ATOM
5741
CD2
LEU
C
16
8.467
12.233
29.690
0.76
1.00
C


ATOM
5742
C
LEU
C
16
9.263
14.982
32.898
0.76
1.00
C


ATOM
5743
O
LEU
C
16
10.182
15.515
32.231
0.76
1.00
C


ATOM
5744
OXT
LEU
C
16
8.870
15.398
34.009
0.76
1.00
C


END









wherein atoms 4045 to 5688 represent the peptide binding site and atoms 5689 to 5748 represent the peptide.

The atomic coordinates are represented in protein data bank (pdb) format. Such a format is well known to the man skilled in the art.
According to another embodiment, the invention relates to a method to purify the processivity clamp factor of DNA polymerase, in particular the β subunit of DNA polymerase III of Escherichia coli, comprising the following steps:

    
    
        elution of a solution containing the processivity clamp factor of DNA polymerase, in particular the β subunit of DNA polymerase III of Escherichia coli, through a cation exchange column, in particular a SP sepharose column;
        elution of a solution containing the processivity clamp factor of DNA polymerase, in particular the β subunit of DNA polymerase III of Escherichia coli, in particular as obtained by the preceding step, through an anion exchange column, in particular a Mono Q column;
        elution of a solution containing the processivity clamp factor of DNA polymerase, in particular the β subunit of DNA polymerase III of Escherichia coli, in particular as obtained by the preceding step, through a cation exchange column, in particular a Mono S column.
    
    


The expression “purify” relates to the process of separating a protein of interest from substantially all the other components of a solution containing said protein of interest, such as a bacterial extract.
Assessment of the purity of the protein of interest can be carried out by methods well known to the man skilled in the art, such as polyacrylamide gel electrophoresis analysis and Coomassie Blue staining or other type of protein staining (e.g. silver staining), mass spectrometry, protein sequencing, HPLC (high performance liquid chromatography). Quantification can be measured by absorbance spectroscopy, Bradford colorimetric assay, or protein sequencing.
The SP sepharose column, Mono Q column and Mono S column are obtained from Pharmacia (Uppsala, Sweden).
Alternatively, columns carrying ion exchange groups with properties similar to those of the SP sepharose column, Mono Q column and Mono S column can also be used.
The above mentioned column can be used with a FPLC system (Pharmacia), and possesses a high protein binding capacity. Advantageously, the SP sepharose column is used during the initial steps of the purification process because it is usually not clogged by dirty samples. The Mono Q and Mono S column are used during the last steps of the purification process, they are highly resolutive columns, but they are easily clogged by dirty samples.
The invention also relates to a method to obtain a protein crystal as defined above, comprising the following steps:

    
    
        mixing a solution of processivity clamp factor of DNA polymerase, with a solution of a peptide of about 3 to about 30 amino acids, in particular of about 16 amino acids, said peptide comprising all or part of the processivity clamp factor binding sequence of a processivity clamp factor interacting protein, such as prokaryotic Pol I, Pol II, Pol III, Pol IV, Pol V, MutS, ligase I, α subunit of DNA polymerase, UmuD or UmuD′, or eukaryotic pol ε, pol δ, pol η, pol τ, pot κ, and with a solution of MES pH 6.0 0.2 M, CaCl2 0.2 M, PEG 400 60%, to obtain a crystallisation drop,
        letting the crystallisation drop concentrate against a solution of MES pH 6.0 0.1 M, CaCl2 0.1 M, PEG 400 30%, by vapour diffusion, to obtain a protein crystal.
    
    


The expression “vapour diffusion” refers to a crystallization method for macromolecules well known to the man skilled in the art, it is in particular described in “Crystallization of nucleic acids and proteins”, pp. 130-145. A. Ducruix & R. Giegé eds., 1999, Oxford University Press.
MES refers to 2-(N-morpholino)-ethane sulfonic acid.
PEG 400 refers to polyethylene glycol 400.
Advantageously MES, PEG and CaCl2 can be obtained from Hampton Research, (Laguna Niguel, USA).
The invention more particularly relates to a method to obtain a protein crystal as defined above, wherein the processivity clamp factor of DNA polymerase is the β subunit of DNA polymerase, in particular the β subunit of DNA polymerase III of Escherichia coli, in particular as purified according the abovementioned methods of purification, and the peptide has the following sequence:

VTLLDPQMERQLVLGL  (SEQ ID NO: 1)

According to a preferred embodiment the β subunit of DNA polymerase III of Escherichia coli and the peptide of SEQ ID NO: 1 are mixed in a molar ratio of about 1:1 to about 1:3 in particular about 1:1.5
According to another preferred embodiment the concentration of the β subunit of DNA polymerase III of Escherichia coli is from about 8 mg/ml to about 50 mg/ml, in particular about 34 mg/ml.
According to another preferred embodiment the concentration of the peptide of SEQ ID NO: 1 is from about 0.5 mg/ml to about 1.2 mg/ml, in particular about 1.1 mg/ml.
According to another embodiment, the invention relates to the use of the atomic coordinates as defined above, for the screening, the design or the modification of ligands of the processivity clamp factor of DNA polymerase, in particular of the β subunit of DNA polymerase III of Escherichia coli. 
The expression “ligand” refers to a compound which is liable to bind to the processivity clamp factor of DNA polymerase.
The invention also relates to the use as defined above, for the screening, the design or the modification of ligands liable to be used for the preparation of pharmaceutical compositions useful for the treatment of bacterial diseases or diseases originating from DNA synthesis processes, such as fragile X syndrome, or proliferative disorders, such as cancers.
The expression “bacterial diseases” refers to diseases which are caused by bacterial influences, such as infections.
The expression “proliferative disorders” refers to disorders which are linked to abnormal cell multiplication, such as cancers.
The invention also relates to a method to screen ligands of the processivity clamp factor of DNA polymerase, said method comprising the step of assessing the interaction of tridimensional models of the ligands to screen with the structure of the β subunit of DNA polymerase as defined by the atomic coordinates as defined above, and in particular with the structure of the peptide binding site as defined by the atomic coordinates defined above, and more particularly with at least nine of the following amino acids: Leu 155, Thr 172, Gly 174, His 175, Arg 176, Leu 177, Pro 242, Arg 246, Val 247, Phe 278, Asn 320, Tyr 323, Val 344, Ser 346, Val 360, Val 361, Met 362, Pro 363, Met 364, Arg 365, Leu 366.
Assessing the interaction can be done by methods such as molecular dynamics, energy calculation, continuum electrostatics, semi-empirical free energy functions and other related methods well known to the man skilled in the art. Several packages and softwares are available for these purposes such as CHARM, UHBD, or SYBILL.
The invention more particularly relates to a method as defined above, to screen ligands liable to be used for the preparation of pharmaceutical compositions useful for the treatment of bacterial diseases or diseases originating from DNA synthesis processes, such as fragile X syndrome, or proliferative disorders, such as cancers.
The invention also relates to a method to design or to modify compounds liable to bind to the processivity clamp factor of DNA polymerase, said method comprising the step of designing or modifying a compound, so that the tridimensional model of said compound is liable to interact with the structure of the β subunit of DNA polymerase as defined by the atomic coordinates as defined above, and in particular with the structure of the peptide binding site as defined by the atomic coordinates as defined above, and more particularly with at least nine of the following amino acids: Leu 155, Thr 172, Gly 174, His 175, Arg 176, Leu 177, Pro 242, Arg 246, Val 247, Phe 278, Asn 320, Tyr 323, Val 344, Ser 346, Val 360, Val 361, Met 362, Pro 363, Met 364, Arg 365, Leu 366.
The invention more particularly relates to a method as defined above, to design or to modify ligands liable to be used for the preparation of pharmaceutical compositions useful for the treatment of bacterial diseases or diseases originating from DNA synthesis processes, such as fragile X syndrome, or proliferative disorders, such as cancers.
According to another embodiment, the invention relates to a peptide of the following sequence:











VTLLDPQMERQLVLGL.
(SEQ ID NO: 1)







According to a preferred embodiment, said peptide comprises non-hydrolysable bonds between amino-acids and/or non-amide bonds between amino-acids.
The invention also relates to a pharmaceutical composition comprising as active substance the peptide of SEQ ID NO: 1, in association with a pharmaceutically acceptable carrier.
Examples of pharmaceutically acceptable carrier are well known to the man skilled in the art.
According to a preferred embodiment, said peptide comprises non-hydrolysable bonds between amino-acids and/or non-amide bonds between amino-acids.
According to another embodiment the invention relates to the use of the peptide of SEQ ID NO: 1, as an anti-bacterial compound.
The expression “anti-bacterial compound” refers to a compound which has bactericidal or bacteriostatic properties, such as an antibiotic.
According to a preferred embodiment, said peptide comprises non-hydrolysable bonds between amino-acids and/or non-amide bonds between amino-acids.
The invention more particularly relates to the use of the peptide of SEQ ID NO: 1 for the manufacture of a medicament for the treatment of bacterial diseases or diseases originating from DNA synthesis processes, such as fragile X syndrome, or proliferative disorders, such as cancers.
According to another embodiment the invention relates to a method to test in vitro the inhibitory effect of compounds on the processivity clamp factor-dependant activity of DNA polymerase, in particular of Pol IV DNA polymerase of Escherichia coli, or of the α subunit of Pol III DNA polymerase of Escherichia coli, comprising the following steps:

    
    
        adding to assay solutions comprising a labelled nucleotidic primer, a template DNA, and DNA polymerase, in particular Pol IV DNA polymerase of Escherichia coli, or the α subunit of Pol III DNA polymerase of Escherichia coli, a compound to test at a given concentration for each assay solution, in the presence or the absence of the processivity clamp factor of DNA polymerase, in particular the β subunit of DNA polymerase in particular the β subunit of DNA polymerase III of Escherichia coli, 
        electrophoretically migrating the abovementioned assay solutions,
        comparing the migration pattern of each assay solutions in the presence or the absence of the processivity clamp factor of DNA polymerase, in particular the β subunit of DNA polymerase, in particular the β subunit of DNA polymerase III of Escherichia coli. 
    
    


According to a preferred embodiment of the above defined in vitro test method, the assay solutions also comprise a clamp loader, in particular the γ complex of E. coli, adenosine triphosphate (ATP), the divalent cation Mg2+ and single strand binding protein (SSB) of E. coli. 
According to another preferred embodiment of the above mentioned in vitro test method, the compounds to be tested are such that their tridimensional models have been screened, modified or designed with respect to, the structure of the β subunit of DNA polymerase, according to the corresponding above defined screening, modifying or designing methods.
The invention also relates to the use of the in vitro test method defined above, for the screening of compounds liable to be used for the preparation of pharmaceutical compositions useful for the treatment of bacterial diseases or diseases originating from DNA synthesis processes, such as fragile X syndrome, or proliferative disorders, such as cancers.



BRIEF DESCRIPTION OF THE DRAWING FIGURES
FIG. 1
FIG. 1 represents the atomic coordinates in protein databank (pdb) format of the crystallographic structure of the complex between Escherichia coli β subunit of DNA polymerase III and the 16 C-terminal residues of the β binding peptide of E. coli Pol IV DNA polymerase (P16)
FIG. 2
FIG. 2 represents a ribbon representation of the β subunit of DNA polymerase III of E. coli complexed with the P16 peptide (boxed) as obtained from the crystallographic structure of the complex.
FIG. 3A, FIG. 3B, FIG. 3C and FIG. 3D
FIG. 3A and FIG. 3B represent the inhibition of β dependant activity of Pol IV by the Pol IV β binding peptide, P16
FIG. 3C and FIG. 3D represent the inhibition of β dependant activity of Pol III α subunit by the Pol IV β binding peptide, P16.
FIG. 3A represents the migration pattern of an electrophoresis gel. β free (lanes 1-4 and 9-12) or β loaded (lanes 5-8 and 13-16) labelled primer/template hybrids are incubated with increasing amounts of control peptide (CLIP) (lanes 1-8) or P16 peptide (lanes 9-16). Concentrations of peptides are as follows: 0 μM, lanes 1, 5, 9 and 13; 1 μM, lanes 2, 6, 10 and 14; 10 μM, lanes 3, 7, 11 and 15; 25 μM, lanes 4, 8, 12 and 16. This mixture is then submitted to the enzymatic activity of Pol IV (1.5 nM) in the presence of each four dNTPs for 1 minute at room temperature. Beside the overall increase in DNA synthesis activity, the β-dependent activity of the polymerase is characterised by the apparition of synthesis products longer than 12 nucleotides (β dependent synthesis), β independent synthesis is characterised by products shorter than 12 nucleotides. The broader band at the bottom of the gel corresponds to the primer. FIG. 3B represents the quantitative analysis of the relative amounts of each β-independent (incorporation of 1 up to 12 nucleotides) and β-dependent (12 and more nucleotides incorporation) activities observed in lanes 5-8 and 13-16. Black and white rectangles represent the ratio of β-dependent to β-independent polymerase activities (vertical axis) in the presence of specified amounts of CLIP and P16 peptides (horizontal axis), respectively. Decrease in this ratio value actually indicates a specific inhibition of the β-dependent polymerase activity.
FIGS. 3C and 3D respectively correspond to the same experiments than those represented in FIGS. 3A and 3B, except that the polymerase used is the purified α subunit of Pol III (6 nM).
FIG. 4
FIG. 4 represents the growth rate of E. coli transformed by IPTG inducible plasmids expressing either the wild type Pol IV (pWp4) (triangles) or the Pol IVD5 mutant of Pol IV lacking the 5 C-terminal amino-acids (pD5p4) (squares, dotted line) in the presence of IPTG. The vertical axis represents the OD at 600 nm and the horizontal axis the time in minutes.
FIG. 5A and FIG. 5B
FIG. 5A represents the growth rate of independent E. coli clones harbouring the P403FL vector in the absence (diamonds, triangles, crosses) or the presence (squares, dashes, circles) of 0.1 mM IPTG.
FIG. 5B represents the growth rate of independent E. coli clones harbouring the P403D5 vector in the absence (diamonds, triangles, crosses) or the presence (squares, dashes, circles) of 0.1 mM IPTG.
The vertical axis represents the O.D. at 600 nm and the horizontal axis represents the time (in minutes).
FIG. 6
FIG. 6 represents Petri dishes containing an agarose-based nutritive medium supplemented with 0.05 mM IPTG and plated with E. coli cells harbouring P403FL (top) or with E. coli cells harbouring P403D5 (bottom).



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Examples
Example 1
Crystallographic Study of the Escherichia coli β Sliding Clamp Complexed with the β Binding Peptide of Pol IV DNA Polymerase of E. coli. 
1. β Binding Peptide Synthesis and Purification
The 16-mer peptide sequence VTLLDPQMERQLVLGL (P16) (SEQ ID NO: 1), representing the 16 last residues of Pol IV DNA polymerase of E. coli, was obtained purified from Neosystem (Illkirch, France) and the 22-mer control peptide. RPVKVTPNGAEDESAEAFPLEF (CLIP) (SEQ ID NO: 2) was a gift from Dr J. P. Briand (Strasbourg, France). P16 was resuspended at 1.1 mg/ml in a buffer containing Tris HCl 20 mM, pH 7.5, 5 mM EDTA, 20% glycerol, and kept at −80° C. CLIP was resuspended in 20 mM NaHCO3 buffer, pH 9, at concentrations of 250, 100 and 10 pmoles/μl
2. β Protein Purification
The dnaN gene encoding E. coli β sliding clamp (hereafter referred to as β protein) was cloned into the pET15b plasmid (Invitrogen). The β protein was expressed in a transformed E. coli BL21(DE3)pLysS/(pET15b-dnaN) and was purified as described (Johanson et al., 1986) with the following modifications. A SP Sepharose column (Pharmacia, Upsalla, Sweden) was used instead of the SP Sephadex column. A Mono Q column (Pharmacia, Upsalla, Sweden) followed by a Mono S column (Pharmacia, Upsalla, Sweden) were performed after the SP Sepharose column step. The β protein was purified to >99% purity, as judged by Coomassie gel analysis, and concentrated using Centriplus YM-30 concentrators (Amicon) to 34.2 mg/ml in a buffer containing 20 mM Tris-HCl pH 7.5, 0.5 mM EDTA and 20% (v/v) glycerol, as determined by Bradford assay, using BSA as a standard.
3. Crystalization Conditions
Drops were obtained by mixing 0.92 μL of β protein at 34.2 mg/ml (775 pmoles) with 1.89 μl of P16 at 1.1 mg/ml (1136 pmoles) and 1 μl of 2× reservoir solution. Reservoir solution contains 0.1 M MES pH 6.0, 0.1M CaCl2 and 30% PEG 400 (Hampton Research, Laguna Niguel, Calif., USA). The peptide/β monomer molar ratio was 1.46. Co-crystals were grown by vapour diffusion in hanging drops at 20° C. They typically grew within three days and reached 200×100×40 μm3. Crystals were mounted in loops (Hampton Research, Laguna Niguel, Calif., USA), frozen in liquid ethane and kept in liquid nitrogen before collection of crystallographic data.
4. Data Collection and Structure Determination
Diffraction data were collected at beam line ID 14-EH4 (ESRF, Grenoble, France). The data were integrated with DENZO and normalized with SCALEPACK (Z. Otwinowski and W. Minor “Processing of X-ray Diffraction Data Collected in Oscillation Mode”, Methods in Enzymology, Volume 276; Macromolecular Crystallography, part A, p. 307-326, 1997, C. W. Carter, Jr. and R. M. Sweet, Eds., Academic Press (New York)). The structure was solved by molecular replacement with MOLREP (CCP4, COLLABORATIVE COMPUTATIONAL PROJECT, NUMBER 4. (1994) “The CCP4 Suite: Programs for Protein Crystallography”. Acta Cryst. D50, 760-763.), using the known β protein structure as a search model (Kong et al., 1992). The peptide was built with the graphics program O (Copyright 1990 by Alwyn Jones, DatOno AB, Blueberry Hill, S-75591 Uppsala, Sweden) and the model was refined with O and CNS (Brunger et al., 1998) (Copyright© 1997-2001 Yale University).
The results are summarized in following Table 1:







TABLE 1





Crystal structure data and refinement statistics







Data collection










Space group
P1



Cell parameters
a = 41.23 Å; b = 65.22 Å;




c = 73.38 Å; α = 73.11°;




β = 85.58°; γ = 85.80°



X-ray source
ID14eh4



Wavelength (Å)
0.93922



Asymetric unit
1 dimer



Resolution (Å)
1.65



Number of observations



Unique
85999



Total
231008



Completeness (%)
96.7 (95.4)a



Rsym
0.051 (0.254)a



Mean I/σ
15.5 (4.3)a







Refinement










Resolution range (Å)
500-1.65



R-factor, reflections
20.87, 80566



Rfree, reflexions
23.71, 4226



Number of atoms



Protein
5744



Water
443



R.m.s deviation



Bond angles (°)
1.59



Bond lenghts (Å)
0.013



Average atomic B-value (Å2)



Protein



β
22.8



Peptide
29.7



Water
29.1



Ramachandran plotb (%)



residues in core,
92.4



allowed,
6.9



generously allowed regions
0.8







aNumber in parentheses is for the last shell (1.71-1.65)



bStatistics from PROCHECK (Laskowski et al., 1993)






The results obtained indicate that the crystal is triclinic, with cell dimensions a=41.23 Å, b=65.22 Å, c=73.38 Å, α=73.11°, β=85.58°, γ85.79°. These cell parameters lead to a quite usual value of 2.36 Å3/Dalton for two molecules (i.e. one ring) per asymmetric unit. The present structure was solved by molecular replacement with the program MOLREP and was refined up to 1.65 Å resolution, which represents an important improvement in comparison to the 2.5 Å resolution obtained for the structure published previously (Kong et al., 1992). The atomic coordinates of the structure solved by the Inventors are given in FIG. 1 in pdb format. The superposition of the present structure onto the previous one yields an overall rmsd of 1.22 Å for the Cα chain, which indicates that both structures are very similar, although numerous side chains and several mobile loops were rebuilt and a better description of the solvent was achieved. A more sensible superposition, systematically downweighting too distant residues (as those in the rebuilt loops), yields a weighted rmsd of 0.78 Å, which is more significant than the former value.
A density related to the presence of the peptide could be located after several rounds of refinement in a “simulated annealing composite omit map” (Brunger et al., 1998). The seven C-terminal residues of the P16 peptide, R10Q11L12V13L14G15L16 (SEQ ID NO: 6) encompassing the β binding sequence were built into the density map (FIG. 2). This map extended slightly toward the N-terminus of the peptide but rapidly faded, so that the Q11 residue was still easily seen while the R10 was built in a poor density region. The rest of the peptide, probably disordered, was not visible. The seven C-terminal amino acids of the P16 peptide bind onto the β surface within two distinct but adjacent domains: one deep crevice, located between sub-domains 2 and 3 (area 1), and a second area which extends over the third β subdomain, close to the C-terminal extremity of the β chain (area 2) (FIG. 2).
In the first area (area 1) of the peptide P16 binding site, two β strands of the clamp (β4′ of domain 2 and β8′′ of domain 3) align. Some of their residues (L177 and V360, respectively), along with residues of the subdomain connecting loop (P242 and V247), form a hydrophobic pocket at the surface of the β monomer. The P16 residues L16 and L14 bind in this crevice. The hydrophobic nature of the interactions is revealed by the removal, upon peptide binding, of water molecules nested inside the free pocket. However, L14 and L16 are also involved in interactions with other adjacent residues like L155, T172, H175, R176, S346 and M362 (Table 2). The residue G15 has no interaction with any residues of the pocket and serves as a connector between L14 and L16. Consequently, the L16 residue which, according to the pentapeptidic consensus motif (Q1L2(SD)3L4F5) (Dalrymple et al., 2001), was not considered to belong to the β-binding sequence, actually fully participates to the interaction.
In the second binding area (area 2), the four other P16 residues, V13, L12, Q11 and R10 establish mostly hydrophobic interactions with residues H175, N320, Y323, V344, M362, P363 and M364 of the β monomer (Table 2). Among the four P16 residues located within this region, the Q residue is highly conserved within the binding motifs of the various β ligands, to the same extent as residues that bind into the hydrophobic crevice (L14 and L16) (Dalrymple et al., 2001). Particularly, it forms interactions, directly or mediated by two water molecules with β residues M362 and E320. These contacts might prime the binding of the peptide with the β surface and facilitate the formation of interactions of the C-terminal residues within the hydrophobic pocket of area 1. Thus the peptide would be anchored on the β surface by two points located on each extremity of the binding sequence.










TABLE 2







β residues
Interacting P16 residues









M364
R10, Q11, L12



P363
Q11, L12



M362
Q11, L12, V13, L14



V361
L14



V344
L12



Y323
Q11



N320
Q11



V360
L14



S346
L14



V247
L14, L16



P242
L16



L177
L14, L16



R176
L14



H175
Q11, L12, V13, L14



T172
L14, L16



L155
L16







Interactions between the β residues and the peptide P16 residues. All considered distances between β and peptide P16 residues are between 3 and 3.8 Å, except those (P16 residues in bold) between L155: L16, T172: L14, L177: L16 and V361: L14 which are larger than 4 Å.






5. N-Terminal Sequencing of the Protein

The crystal was recovered after data collection, washed several times in the well solution, and dissolved in 10 μl water. The proteins contained within the crystal were derivatized and sequenced by automated Edman's degradation using a PE Applied Biosystems 492 cLC Protein Sequencer allowing the identification and precise quantitative analysis of the amino acids released at each step of degradation.
6. Improvement of the P16-β Clamp Interaction
Preliminary in silico docking experiments carried out with modified versions of the P16 peptide suggest that its interaction with the β clamp could be strengthened by replacing Leu 12 and Leu 14 by aromatic amino acids, or by extending the lateral chain of Gln 11. Thus, these modifications show the way to designing new high affinity β clamp interaction inhibitors.
Example 2
In vitro Study of the β Clamp-β Binding Peptide of Pol IV Interaction by Competition Assays
In order to ascertain the biological relevance of the P16 peptide-β clamp interaction observed in the crystallographic structure, an in vitro assay based on the activity of Pol IV DNA polymerase was designed. This assay relies on the observation that the in vitro activity of Pol IV is greatly enhanced by the presence of the β subunit loaded onto a primer/template DNA substrate (Wagner et al., 2000) (FIG. 3A, compare lanes 1 and 5 or 9 and 13), while the enzyme alone incorporates nucleotides in a distributive mode (Wagner et al, 1999).
Briefly, P16 peptide and a control peptide (CLIP) were diluted in 20 mM NaHCO3 at concentrations of 250, 100 and 10 pmol/μl. 5′ end radiolabelling, purification and annealing of synthetic primers were performed as previously described (Wagner et al., 1999). The 30/90 nucleotide synthetic construct (Wagner et al., 2000) was obtained by annealing the 30 nucleotide primer (5′GTAAAACGACGGCCAGTGCCAAGCTTAGTC) (SEQ ID NO: 3) with the 90 nucleotide template (5′CCATGATTACGAATTCAGTCATCACCGGCGC CACAGACTAAGCTTGGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACC CTGG) (SEQ ID NO: 4) to form a double stranded structure with 5′ and 3′ single stranded DNA overhangs of 25 and 35 nucleotides, respectively.
All replication experiments (10 μl final volume) were carried out in buffer E (40 mM HEPES pH 7.5, 80 mM potassium glutamate, 160 μg/ml BSA, 16% glycerol, 0.016% NP40, 8 mM DTT). The 30/90 nucleotide hybrid was first incubated with single strand binding proteins (SSB; Sigma; 90 nM final concentration) in the presence of ATP (200 μM) and MgCl2 (7.5 mM) at 37° C. for 10 min. When specified, the γ complex (1 nM final concentration) (gift from Dr. C. S. McHenry, Denver, USA), and the β clamp (5 nM as dimer final concentration) were added at that stage, and incubation was carried out at 37° C. for 10 min. Then, 7 μl of the mixture was added to 1 μl of either 20 mM NaHCO3 or 1 μl of peptide solution (1, 10 or 25 μM final concentration), incubated 20 min. at room temperature and farther 2 hours at 4° C. 1 μl of polymerase was then added (1.5 nM of Pol IV or 6 nM of α subunit (gift from Dr. H. Maki, Nara, Japan) final concentrations), incubated 5 min. at room temperature and finally, the whole reaction was mixed with 1 μl of a dNTPs solution (200 μM each dNTP final concentration) and let to react for 1 min. at room temperature. Reactions were quenched by the addition of 20 μl of 95% formamide/dyes solution containing 7.5 mM EDTA, heat-denatured and analysed by chromatography on 12% denaturing polyacrylamide gels. Radiolabelled products were visualised and quantified using a PhosphorImager 445 SI (Molecular Dynamics) and the ImageQuant software.
As shown in FIG. 3A and FIG. 3B, increasing amounts of P16 inhibits the β-dependent activity of Pol IV (lane 13 to 16). At the highest P16 concentration tested (25 μM), the β-dependent Pol IV activity is decreased by a factor around 30, as indicated on the graphic. On the other hand, the control peptide (CLIP) has no effect on this activity even at the highest concentration tested (FIG. 3A, lane 8). Also, neither P16 nor CLIP peptides do affect the intrinsic activity of Pol IV characterised by the distributive incorporation of one to up to 12 nucleotides (FIG. 3A, lanes 1-4, 9-12, FIG. 3B). Thus P16 specifically inhibits the β-Pol IV DNA polymerase interaction in solution, which demonstrate that the site we identified actually corresponds to the Pol IV DNA polymerase binding site on β.
The polymerase activity of the α subunit of the replicative DNA Polymerase III of E. coli is greatly enhanced by its interaction with the β clamp (Marians et al., 1998) (FIG. 3C, compare lanes 1 and 5 or 9 and 13), and the putative β binding peptide of the α subunit has been identified through bioinformatics analysis (Dalrymple et al., 2001) and is a variant of the pentapeptide consensus motif. In order to determine if the replicative DNA polymerase interact with the β monomer within the same site than Pol IV, the ability of P16 peptide to inhibit the β-dependent activity of the α subunit was tested. The dose dependent inhibition of the α subunit β-dependent activity (FIG. 3C, lane 13 to 16, FIG. 3D) strongly suggest that this is the case. To achieve a high level of inhibition, the concentration of P16 peptide should exceed the polymerase concentration by a factor of 4 to 16.103. The need for such a high excess of peptide may reflect a higher affinity of the whole protein for the DNA-β substrate, mediated by other polymerase-β and/or polymerase-DNA interactions, but also a high entropic factor of the free peptide as opposed to the same fragment folded in the whole protein. Therefore, the lower peptide affinity would result from a lower kinetic constant kon, and not from an increased koff. Overall, this biochemical analysis indicates that (i) the P16-β structure we solved is of biological significance as indicated by the competitive inhibition of the β dependent activity of Pol IV DNA polymerase by peptide P16 and (ii) that peptide P16 also competes with and inhibits the β dependent activity of the α subunit of the DNA Polymerase III of E. coli which suggests that (iii) if not identical, the Pol IV and α subunit interaction sites on β subunit overlap.
Example 3
In vivo Study of the Inhibition of Bacterial Growth by the β Binding Peptide of Pol IV
Plasmids bearing either the wild type Pol IV (pWp4) or the Pol IV mutant deleted for the 5 last C-terminal residues (pD5p4) coding sequences under the IPTG inducible lac promoter were transformed into recipient E. coli cells (BL21(DE3, pLys)). These transformed cells were then allowed to grow in LB medium at 37° C. with aeration and without or with (FIG. 4) addition of the protein expression inducer IPTG (0.1 mM). Growth rates were monitored by measuring the optical density of the cultures (OD 600 nm) at different time points.
The growth rates of both cultures without artificial protein expression were identical whether the cells contain the wild type Pol IV expression plasmid (pWp4) or the Pol IVD5 mutant (pD5p4). On the other hand, when protein expression was induced by the adjunction of low IPTG concentration in the culture medium (FIG. 4), a clear growth inhibition was observed for the culture expressing the wild type Pol IV protein compared to the one expressing the mutant protein. As the mutant protein (expressed from pD5p4) lacks essential amino acids for the interaction with the β-clamp, the observed cytotoxicity may be rationalised by the fact that the wild type Pol IV protein interacts with the β clamp and, because of its relative high concentration, interfere and/or compete with the binding of the replicative DNA polymerase, thereby inhibiting chromosome replication and culture growth.
In other words, these preliminary results indicate that site-specific β binding molecules (such as the Pol IV β binding motif) may serve as antimicrobial agents.
Example 4
In vivo Study of the Inhibition of Bacterial Growth by the β Binding Peptide of Pol IV
A DNA sequence encoding a catalytically inactive version of DNA polymerase IV of E. coli has been cloned into a vector to form P403FL which enable the IPTG inducible expression of the corresponding inactive enzyme. Similarly, a DNA sequence encoding the catalytically inactive version of DNA polymerase IV of E. coli depleted of the 5 last C-terminal residues (which are essential residues for the interaction with the β clamp) has been cloned into the same IPTG inducible vector to form P403D5.
Three independently isolated clones of E. coli containing either P403FL or P403D5 were cultured in a selective medium until an optical density (O.D.) of 0.2 at 600 nm was reached, 15 ml of a selective medium containing 0 or 0.1 mM IPTG were then inoculated with a quantity corresponding to 0.02 O.D. unit of the culture and bacterial growth was followed by the measure of the optical density at 600 nm during 5 hours.
The results indicate that in the absence of IPTG the three cultures of the independent clones carrying P403FL grow normally, however, in the presence of 0.1 mM IPTG the growth of these clones is completely halted (FIG. 5A). Conversely, the three independent clones carrying P403D5 grow normally, irrespective of the presence or not of IPTG (FIG. 5B).
Furthermore, about 1000 E. coli cells harbouring either P403FL or P403D5 were plated on nutritive agarose dishes containing 0.05 mM IPTG. The results shown in FIG. 6 indicate that, whereas essentially no P403FL carrying cells are growing, essentially all P403D5 carrying cells are growing.
As in Example 3, those results confirm that site-specific β binding molecules (such as the Pol IV β binding motif) may serve as antimicrobial agents.
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## Claims

1. A protein crystal comprising a processivity clamp factor of DNA polymerase that is the subunit of DNA polymerase III of Escherichia coli which has the amino acid sequence of SEQ ID NO: 5; and a peptide of 16 amino acids having the amino acid sequence of VTLLDPQMERQLVLGL (SEQ ID NO: 1), wherein said protein crystal is in triclinic space group P1 and has cell dimensions of: a=41.23 Å, b=65.22 Å, c=73.38 Å, α=73.11°, β=85.58°, and γ=85.80°.

2. The protein crystal according to claim 1 having a three-dimensional structure represented by atomic coordinates obtained by X-ray diffraction of said protein crystal, wherein said atomic coordinates are shown in FIG. 1.

3. The protein crystal according to claim 1, wherein the three-dimensional structure of the peptide of SEQ ID NO: 1 is defined by atoms 5689-5744 as shown in FIG. 1, and the peptide binding site of the β subunit of DNA polymerase III is defined by amino acid residues Leu 155, Thr 172, Gly 174, His 175, Arg 176, Leu 177, Pro 242, Arg 246, Val 247, Phe 278, Asn 320, Tyr 323, Val 344, Ser 346, Val 360, Val 361, Met 362, Pro 363, Met 364, Arg 365 and Leu 366 having the atomic coordinates as shown in FIG. 1.

4. A method to obtain the protein crystal of claim 1, said method comprising:
(a) mixing a solution comprising the β subunit of DNA polymerase III of Escherichia coli having the amino acid sequence of SEQ ID NO: 5, with a solution of the peptide of 16 amino acids having the amino acid sequence of VTLLDPQMERQLVLGL (SEQ ID NO: 1), and with a solution of 0.2 M 2-(N-morpholino)ethane sulfonic acid (MES) at pH 6.0, 0.2 M CaCl2, 60% PEG 400, to obtain a crystallization drop; and
(b) allowing the crystallization drop to concentrate against a solution of 0.1 M MES pH 6.0, 0.1M CaCl2, 30% PEG 400, by vapor diffusion, to obtain the protein crystal.