Abstract

The E. coli DNA polymerase III holoenzyme is a complex multisubunit enzyme that functions in natural in vitro multienzyme systems of DNA replication. Genetic and biochemical evidence have implicated the holoenzyme as being the polymerase responsible for the synthesis of the E. coli chromosome. Recent studies have led to a better understanding of the functions that give this enzyme its unique role in the replicative process. However, the lack of an understanding of the structure of this complex has hampered efforts to formulate detailed models for the holoenzyme-catalyzed reaction. Towards a solution to this problem, we propose to begin structural studies with the investigation proposed in this application. We plan to use the techniques of chemical crosslinking and fluorescence energy transfer to gain information concerning the arrangement of subunits within the replicative complex and the distance between important sites. Additionally, we propose to determine the position of the nucleic acid components within this complex. Structural tests of the dimeric replication hypothesis are also described.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM035695-04
Application #
3288751
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1985-12-01
Project End
1990-11-30
Budget Start
1988-12-01
Budget End
1989-11-30
Support Year
4
Fiscal Year
1989
Total Cost
Indirect Cost

  Institution

Name
University of Colorado Denver
Department
Type
Schools of Medicine
DUNS #
065391526
City
Aurora
State
CO
Country
United States
Zip Code
80045

  Publications

Downey, Christopher D; McHenry, Charles S (2010) Chaperoning of a replicative polymerase onto a newly assembled DNA-bound sliding clamp by the clamp loader. Mol Cell 37:481-91
Yuan, Quan; McHenry, Charles S (2009) Strand displacement by DNA polymerase III occurs through a tau-psi-chi link to single-stranded DNA-binding protein coating the lagging strand template. J Biol Chem 284:31672-9
Jarvis, Thale C; Beaudry, Amber A; Bullard, James M et al. (2005) Discovery and characterization of the cryptic psi subunit of the pseudomonad DNA replicase. J Biol Chem 280:40465-73
Bullard, James M; Pritchard, Arthur E; Song, Min-Sun et al. (2002) A three-domain structure for the delta subunit of the DNA polymerase III holoenzyme delta domain III binds delta' and assembles into the DnaX complex. J Biol Chem 277:13246-56
Glover, B P; McHenry, C S (2001) The DNA polymerase III holoenzyme: an asymmetric dimeric replicative complex with leading and lagging strand polymerases. Cell 105:925-34
Song, M S; Pham, P T; Olson, M et al. (2001) The delta and delta ' subunits of the DNA polymerase III holoenzyme are essential for initiation complex formation and processive elongation. J Biol Chem 276:35165-75
Song, M S; McHenry, C S (2001) Carboxyl-terminal domain III of the delta' subunit of DNA polymerase III holoenzyme binds DnaX and supports cooperative DnaX complex assembly. J Biol Chem 276:48709-15
Glover, B P; Pritchard, A E; McHenry, C S (2001) tau binds and organizes Escherichia coli replication proteins through distinct domains: domain III, shared by gamma and tau, oligomerizes DnaX. J Biol Chem 276:35842-6
Song, M S; Dallmann, H G; McHenry, C S (2001) Carboxyl-terminal domain III of the delta' subunit of the DNA polymerase III holoenzyme binds delta. J Biol Chem 276:40668-79
Gao, D; McHenry, C S (2001) Tau binds and organizes Escherichia coli replication proteins through distinct domains. Domain III, shared by gamma and tau, binds delta delta ' and chi psi. J Biol Chem 276:4447-53

Showing the most recent 10 out of 25 publications