Global Patent Index - EP 1504091 A4

EP 1504091 A4 20060426 - ERROR-PRONE DNA POLYMERASE I MUTANTS AND METHODS FOR TARGETED RANDOM MUTAGENESIS IN CONTINUOUS CULTURE USING ERROR-PRONE DNA POLYMERASE I MUTANTS

Title (en)

ERROR-PRONE DNA POLYMERASE I MUTANTS AND METHODS FOR TARGETED RANDOM MUTAGENESIS IN CONTINUOUS CULTURE USING ERROR-PRONE DNA POLYMERASE I MUTANTS

Title (de)

ZU FEHLERN NEIGENDE DNA-POLYMERASE-I-MUTANTEN UND VERFAHREN ZUR GEZIELTEN ZUFALLSMUTAGENESE IN DAUERKULTUR UNTER VERWENDUNG DER ZU FEHLERN NEIGENDEN DNA-POLYMERASE-I-MUTANTEN

Title (fr)

MUTANTS DE POLYMERASE I D'ADN SUJET A L'ERREUR ET TECHNIQUES DE MUTAGENESE ALEATOIRE CIBLEE EN CULTURE CONTINUE UTILISANT CES MUTANTS DE POLYMERASE I D'ADN SUJET A L'ERREUR

Publication

EP 1504091 A4 20060426 (EN)

Application

EP 03736735 A 20030529

Priority

  • US 0316798 W 20030529
  • US 38494402 P 20020531

Abstract (en)

[origin: WO03102213A2] Mutant forms of DNA polymerase I having mutations within motif A and/or motif B in the active domain that increase error rates during replication. Expression plasmid constructs and cell lines for expressing these low-fidelity polymerase mutants are provided. Methods are also provided for utilizing these low-fidelity DNA polymerase I mutants for generating libraries of randomly-mutagenized genes, which may be prokaryotic or eukaryotic. Random mutagenesis involves the coupling of mutagenesis and selection in continuous culture for convenient iteration, which results in diverse range of base pair substitutions, widely distributed along the sequence. Some advantages include the minimization of deleterious damage to chromosomal DNA, and adaptation to strains that are amenable to complementation, which substantially facilitates the generation and identification of enzymes with altered properties.

IPC 1-7

C12N 9/12; C12N 9/00; C07K 14/00; C07H 21/04

IPC 8 full level

C12N 15/09 (2006.01); C12N 1/15 (2006.01); C12N 1/19 (2006.01); C12N 1/21 (2006.01); C12N 5/10 (2006.01); C12N 9/12 (2006.01); C12N 15/10 (2006.01); C12Q 1/02 (2006.01); C12Q 1/68 (2006.01)

CPC (source: EP)

C12N 9/1252 (2013.01); C12N 15/1024 (2013.01)

Citation (search report)

  • [X] EP 0655506 A1 19950531 - HARVARD COLLEGE [US]
  • [A] WO 9835060 A1 19980813 - LIFE TECHNOLOGIES INC [US]
  • [X] TOSAKA AKI ET AL: "O-helix mutant T664P of Thermus aquaticus DNA polymerase I: Altered catalytic properties for incorporation of incorrect nucleotides but not correct nucleotides", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 276, no. 29, 20 July 2001 (2001-07-20), pages 27562 - 27567, XP002369418, ISSN: 0021-9258
  • [X] SUZUKI MOTOSHI ET AL: "Thermus aquaticus DNA polymerase I mutants with altered fidelity. Interacting mutations in the O-helix", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 275, no. 42, 20 October 2000 (2000-10-20), pages 32728 - 32735, XP002369419, ISSN: 0021-9258
  • [X] BELL J B ET AL: "Base miscoding and strand misalignment errors by mutator Klenow polymerases with amino acid substitutions at tyrosine 766 in the O helix of the fingers subdomain", JOURNAL OF BIOLOGICAL CHEMISTRY 1997 UNITED STATES, vol. 272, no. 11, 1997, pages 7345 - 7351, XP002369420, ISSN: 0021-9258
  • [X] SHINKAI A ET AL: "In vivo mutagenesis by Escherichia coli DNA polymerase I. Ile(709) in motif A functions in base selection.", THE JOURNAL OF BIOLOGICAL CHEMISTRY. 14 DEC 2001, vol. 276, no. 50, 14 December 2001 (2001-12-14), pages 46759 - 46764, XP002369421, ISSN: 0021-9258
  • [A] PATEL P H ET AL: "Prokaryotic DNA polymerase I: evolution, structure, and ''base flipping'' mechanism for nucleotide selection", JOURNAL OF MOLECULAR BIOLOGY, LONDON, GB, vol. 308, no. 5, 18 May 2001 (2001-05-18), pages 823 - 837, XP004466165, ISSN: 0022-2836
  • [A] KAUSHIK N ET AL: "Significance of the O-helix residues of Escherichia coli DNA polymerase I in DNA synthesis: dynamics of the dNTP binding pocket.", BIOCHEMISTRY. 4 JUN 1996, vol. 35, no. 22, 4 June 1996 (1996-06-04), pages 7256 - 7266, XP002369422, ISSN: 0006-2960
  • [A] DERBYSHIRE V ET AL: "The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.", THE EMBO JOURNAL. JAN 1991, vol. 10, no. 1, January 1991 (1991-01-01), pages 17 - 24, XP002369423, ISSN: 0261-4189
  • [A] BLONDAL T ET AL: "CLONING, SEQUENCE ANALYSIS AND FUNCTIONAL CHARACTERIZATION OF DNA POLYMERASE I FROM THE THERMOPHILIC EUBACTERIUM RHODOTHERMUS MARINUS", BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY, ACADEMIC PRESS, US, vol. 34, no. 1, August 2001 (2001-08-01), pages 37 - 45, XP009061567, ISSN: 0885-4513
  • [T] CAMPS MANEL ET AL: "Targeted gene evolution in Escherichia coli using a highly error-prone DNA polymerase I.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 100, no. 17, 19 August 2003 (2003-08-19), pages 9727 - 9732, XP002369424, ISSN: 0027-8424
  • [T] CAMPS M ET AL: "When Pol I goes into high gear: Processive DNA synthesis by Pol I in the cell", CELL CYCLE 2004 UNITED STATES, vol. 3, no. 2, 2004, pages 116 - 118, XP002369425, ISSN: 1538-4101
  • See references of WO 03102213A2

Designated contracting state (EPC)

AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR

DOCDB simple family (publication)

WO 03102213 A2 20031211; WO 03102213 A3 20040617; AU 2003237269 A1 20031219; CA 2485203 A1 20031211; EP 1504091 A2 20050209; EP 1504091 A4 20060426; JP 2005528114 A 20050922

DOCDB simple family (application)

US 0316798 W 20030529; AU 2003237269 A 20030529; CA 2485203 A 20030529; EP 03736735 A 20030529; JP 2004510449 A 20030529