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Journal of Bacteriology, November 2000, p. 6106-6113, Vol. 182, No. 21
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Escherichia coli DNA Polymerase III tau - and gamma -Subunit Conserved Residues Required for Activity In Vivo and In Vitro

James R. Walker,* Christine Hervas,dagger Julie D. Ross,Dagger Alexandra Blinkova, Michael J. Walbridge,§ Emilynn J. Pumarega,|| Mi-Oak Park,# and Harold R. Neely

Section of Molecular Genetics and Microbiology, Institute for Cell and Molecular Biology, University of Texas at Austin, Austin, Texas 78712

Received 17 March 2000/Accepted 28 July 2000

The Escherichia coli DNA polymerase III tau  and gamma  subunits are single-strand DNA-dependent ATPases (the latter requires the delta  and delta ' subunits for significant ATPase activity) involved in loading processivity clamp beta . They are homologous to clamp-loading proteins of many organisms from phages to humans. Alignment of 27 prokaryotic tau /gamma homologs and 1 eukaryotic tau /gamma homolog has refined the sequences of nine previously defined identity and functional motifs. Mutational analysis has defined highly conserved residues required for activity in vivo and in vitro. Specifically, mutations introduced into highly conserved residues within three of those motifs, the P loop, the DExx region, and the SRC region, inactivated complementing activity in vivo and clamp loading in vitro and reduced ATPase catalytic efficiency in vitro. Mutation of a highly conserved residue within a fourth motif, VIc, inactivated clamp-loading activity and reduced ATPase activity in vitro, but the mutant gene, on a multicopy plasmid, retained complementing activity in vivo and the mutant gene also supported apparently normal replication and growth as a haploid, chromosomal allele.

* Corresponding author. Mailing address: Section of Molecular Genetics and Microbiology, Institute for Cell and Molecular Biology, University of Texas at Austin, Austin, TX 78712. Phone: (512) 471-1692. Fax: (512) 471-2088. E-mail: jrw{at}mail.utexas.edu.

dagger Present address: University of Connecticut School of Dental Medicine, Farmington, CT 06030.

Dagger Present address: University of Texas Medical Branch, Galveston, TX 77555.

§ Present address: Genome Therapeutics Corp., Waltham, MA 02453.

|| Present address: 1807 Frazier Ave., Austin, TX 78704.

# Present address: Union Square Family Dentistry, Union City, CA 94587.

Journal of Bacteriology, November 2000, p. 6106-6113, Vol. 182, No. 21
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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