Abstract

DNA polymerase III holoenzyme (holoenzyme), the chromosomal replicase of Escherichia coli, consists of a polymerase subunit in association with 9 accessory proteins. The beta accessory protein is a dimer and has the shape of a ring. The beta ring is placed around primed DNA by the 5- subunit gamma complex accessory protein in a reaction that requires ATP. The beta ring on DNA is tightly clamped to it by virtue of encircling the duplex and it slides freely along its surface. Beta also binds the polymerase subunit thereby tethering it down to DNA and conferring onto it the remarkable speed and processivity that distinguishes the holoenzyme as a chromosomal replicase.
The aim of this grant proposal is to fully understand the novel enzymatic mechanism by which the gamma complex assembles the beta ring around DNA. How many ATP molecules are needed and what are their roles? What structural determinants of primed DNA does the gamma complex recognize to acids of beta contribute to its sliding motion and how does it contact the gamma complex and the polymerase? The beta sliding clamp and assembly function of the gamma complex will likely generalize to the yeast and human chromosomal replicase accessory proteins (PCNA protein and 5-protein RF-C complex) and may underlie the mechanisms of other processive processes such as translation and transcription.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM038839-06
Application #
3295565
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1988-08-01
Project End
1997-07-31
Budget Start
1993-08-01
Budget End
1994-07-31
Support Year
6
Fiscal Year
1993
Total Cost
Indirect Cost

  Institution

Name
Weill Medical College of Cornell University
Department
Type
Schools of Medicine
DUNS #
201373169
City
New York
State
NY
Country
United States
Zip Code
10065

  Publications

Langston, Lance; O'Donnell, Mike (2017) Action of CMG with strand-specific DNA blocks supports an internal unwinding mode for the eukaryotic replicative helicase. Elife 6:
Georgescu, Roxana; Langston, Lance; O'Donnell, Mike (2015) A proposal: Evolution of PCNA's role as a marker of newly replicated DNA. DNA Repair (Amst) 29:4-15
Georgescu, Roxana E; Schauer, Grant D; Yao, Nina Y et al. (2015) Reconstitution of a eukaryotic replisome reveals suppression mechanisms that define leading/lagging strand operation. Elife 4:e04988
Sun, Jingchuan; Shi, Yi; Georgescu, Roxana E et al. (2015) The architecture of a eukaryotic replisome. Nat Struct Mol Biol 22:976-82
Langston, Lance D; Zhang, Dan; Yurieva, Olga et al. (2014) CMG helicase and DNA polymerase ? form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication. Proc Natl Acad Sci U S A 111:15390-5
Marzahn, Melissa R; Hayner, Jaclyn N; Finkelstein, Jeff et al. (2014) The ATP sites of AAA+ clamp loaders work together as a switch to assemble clamps on DNA. J Biol Chem 289:5537-48
Georgescu, Roxana E; Yao, Nina; Indiani, Chiara et al. (2014) Replisome mechanics: lagging strand events that influence speed and processivity. Nucleic Acids Res 42:6497-510
Georgescu, Roxana E; Langston, Lance; Yao, Nina Y et al. (2014) Mechanism of asymmetric polymerase assembly at the eukaryotic replication fork. Nat Struct Mol Biol 21:664-70
Barros, Tiago; Guenther, Joel; Kelch, Brian et al. (2013) A structural role for the PHP domain in E. coli DNA polymerase III. BMC Struct Biol 13:8
Indiani, Chiara; Patel, Meghna; Goodman, Myron F et al. (2013) RecA acts as a switch to regulate polymerase occupancy in a moving replication fork. Proc Natl Acad Sci U S A 110:5410-5

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