Abstract

The RB69 DNA polymerase binding-pocket residue Tyr567 was shown previously to be a key determinant of fidelity: the replacement Y567A causes a powerful mutator activity. Ser565 is a nearby component of the binding pocket. Unlike Y567A, the replacement S565G has only a small impact on fidelity. However, combining Y567A and S565G reverses most of the mutator activity of Y567A. This is a unique and instructive observation in structural studies of polymerase fidelity, and we are working through a detailed examination of the mutational propensities of these constructs both in vivo and in vitro.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Intramural Research (Z01)
Project #
1Z01ES061054-11
Application #
7734473
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
11
Fiscal Year
2008
Total Cost
$26,268
Indirect Cost

  Institution

Name
National Institute of Environmental Health Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code

  Publications

Burch, Lauranell H; Zhang, Leilei; Chao, Frank G et al. (2011) The bacteriophage T4 rapid-lysis genes and their mutational proclivities. J Bacteriol 193:3537-45
Trzemecka, Anna; Jacewicz, Agata; Carver, Geraldine T et al. (2010) Reversal of a mutator activity by a nearby fidelity-neutral substitution in the RB69 DNA polymerase binding pocket. J Mol Biol 404:778-93
Jacewicz, Agata; Makiela, Karolina; Kierzek, Andrzej et al. (2007) The roles of Tyr391 and Tyr619 in RB69 DNA polymerase replication fidelity. J Mol Biol 368:18-29
Zhang, Hong; Rhee, Chanu; Bebenek, Anna et al. (2006) The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Biochemistry 45:2211-20
Bebenek, Anna; Carver, Geraldine T; Kadyrov, Farid A et al. (2005) Processivity clamp gp45 and ssDNA-binding-protein gp32 modulate the fidelity of bacteriophage RB69 DNA polymerase in a sequence-specific manner, sometimes enhancing and sometimes compromising accuracy. Genetics 169:1815-24
Bebenek, Anna; Carver, Geraldine T; Dressman, Holly Kloos et al. (2002) Dissecting the fidelity of bacteriophage RB69 DNA polymerase: site-specific modulation of fidelity by polymerase accessory proteins. Genetics 162:1003-18
Bebenek, A; Dressman, H K; Carver, G T et al. (2001) Interacting fidelity defects in the replicative DNA polymerase of bacteriophage RB69. J Biol Chem 276:10387-97