Patent EP-1639509-B1 (EP)
Inventor: BURNOUF DOMINIQUE YVES JOEL
IPC: C12N9/12
Filed: 2004-06-25
Granted: 2011-01-12
Abstract: A protein crystal (C1) comprises a processivity clamp factor of DNA polymerase and a peptide of 3-30 amino acids, preferably 16 amino acids, where the peptide has 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, alpha subunit of DNA polymerase, UmuD or UmuD', or eukaryotic pol epsilon, delta , eta or tau or pol kappa. Independent claims are also included for: (1) purifying the processivity clamp factor of DNA polymerase, preferably beta subunit of DNA polymerase III of Escherichia coli, involves eluting a solution containing the processivity clamp factor of DNA polymerase III of E.coli, through a cation exchange column, preferably SP sepharose column, eluting a solution containing the processivity clamp factor of DNA polymerase III of E.coli, in particular as obtained by the above step, through an amino exchange column, preferably Mono Q column, and eluting a solution containing the processivity clamp factor of DNA polymerase III of E.coli, in particular as obtained by the above step, through a cation exchange column, preferably Mono S column; (2) obtaining (C1), involves mixing a solution of processivity clamp factor of DNA polymerase, with a solution of a peptide of 3-30 amino acids, preferably 16 amino acids, where the peptide has 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, alpha subunit of DNA polymerase, UmuD or UmuD', or eukaryotic pol epsilon, delta , eta or tau or pol kappa, and with a solution of MES (pH 6.0) (0.2 M), CaCl 2 (0.2 M), PEG 400 (60%), to obtain a crystallization drop, and letting the crystallization drop concentrate against, a solution of MES (pH 6.0) (0.1 M), CaCl 2 (0.1 M), PEG 400 (30%) by vapor diffusion, to obtain (C1); (3) use of atomic coordinates (AC) of (C1) for screening, designing or modifying of ligands of the processivity clamp factor of DNA polymerase, preferably beta subunit of DNA polymerase III of E.coli; (4) screening (M1) ligands of the processivity clamp factor of DNA polymerase, involves assessing the interaction of tridimensional models of the ligands to screen with the structure of the beta subunit of DNA polymerase as defined by the atomic coordinates of (C1), especially with the structure of peptide binding site as defined by the atomic coordinates of (C1), preferably with nine or more of the amino acids such as Leu155, Thr172, Gly174, His175, Arg176, Leu177, Pro242, Arg246, Val247, Phe278, Asn320, Tyr323, Val344, Ser346, Val360, Val361, Met360, Pro363, Met364, Arg365, and Leu366; (5) designing or modifying (M2) compounds liable to bind to the processivity clamp factor of DNA polymerase, involves designing or modifying a compound, such that the tridimensional model of the compound is liable to interact with the structure of (C1), especially with the structure of peptide binding site as defined by the atomic coordinates of (C1), preferably with nine or more of the amino acids such as Leu155, Thr172, Gly174, His175, Arg176, Leu177, Pro242, Arg246, Val247, Phe278, Asn320, Tyr323, Val344, Ser346, Val360, Val361, Met360, Pro363, Met364, Arg365, and Leu366; (6) a peptide (P1) comprising a sequence (S1) of Val-Thr-Leu-Leu-Asp-Pro-Gln-Met-Glu-Arg-Gln-Leu-Val-Leu-Gly-Leu; (7) a composition (C2) comprising (P1) in association with a carrier; and (8) in vitro testing of the inhibitory effect of compounds on the processivity clamp factor-dependent activity of DNA polymerase, preferably Pol IV DNA polymerase of E.coli, or of the alpha subunit of Pol III DNA polymerase of E.coli, involves adding a compound to test at a given concentration, in the presence or the absence of the processivity clamp factor of DNA polymerase, preferably the beta subunit of DNA polymerase III of E.coli, to assay solutions comprising a labeled nucleotidic primer, a template DNA, and DNA polymerase, preferably Pol IV DNA polymerase of E.coli, or the alpha subunit of Pol III DNA polymerase of E.coli, electrophoretically migrating the above mentioned assay solutions, comparing the migration pattern of each assay solutions in the presence or the absence of the processivity clamp factor of DNA polymerase, preferably the beta subunit of DNA polymerase III of E.coli. ACTIVITY : Antibacterial; Analgesic; Antianginal; Cytostatic. No supporting data is given. MECHANISM OF ACTION : None given.