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.

Description:
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&#39;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, CaCl 2  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, CaCl 2  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 &amp; 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 CaCl 2  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 Mg 2+  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 NaHCO 3  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 &gt;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 CaCl 2  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 μm 3 . 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 (Å 2 ) 
               
               
                   
                 Protein 
               
               
                   
                 β 
                 22.8 
               
               
                   
                 Peptide 
                 29.7 
               
               
                   
                 Water 
                 29.1 
               
               
                   
                 Ramachandran plot b  (%) 
               
               
                   
                 residues in core, 
                 92.4 
               
               
                   
                 allowed, 
                 6.9 
               
               
                   
                 generously allowed regions 
                 0.8 
               
               
                   
                   
               
               
                   
                   a Number in parentheses is for the last shell (1.71-1.65) 
               
               
                   
                   b Statistics 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 Å 3 /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, R 10 Q 11 L 12 V 13 L 14 G 15 L 16  (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 Q 11  residue was still easily seen while the R 10  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 (β 4′  of domain 2 and β 8″  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 (Q 1 L 2 (SD) 3 L 4 F 5 ) (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&#39;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 NaHCO 3  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 MgCl 2  (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 NaHCO 3  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 16.10 3 . 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 k on , and not from an increased k off . 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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