Book of Abstracts: Albany 2007

category image Albany 2007
Conversation 15
June 19-23 2007

The Stoichiometry and EM Structure of Human DNA Polymerase gamma

Mitochondria contain a single DNA polymerase, pol γ, responsible for replication and repair of mtDNA. Human pol γ is isolated from mitochondria as a complex containing a catalytic subunit, pol γA, of 139 kDa and an accessory subunit, pol γB, of 53 kDa. The accessory factor is itself a homodimer with remarkable structural similarity to prokaryotic tRNA synthetases (1). Our studies using gel filtration and isothermal calorimetry show that that the functional human holoenzyme contains two subunits of the processivity factor and one catalytic subunit, thereby forming a heterotrimer (3). Pol γ is mutated in individuals with genetic disorders such as chronic external opthalmoplegia and Alpers syndrome, it is an adventitious target for toxic side effects of several antiviral compounds and mutation of its proofreading exonuclease leads to accelerated aging in mouse models (2).

We used electron microscopy to examine the structure of the human DNA pol γ heterotrimer. Separate analysis of negatively stained preparations of the catalytic subunit, pol γA, and of the holoenzyme including the dimeric accessory factor, pol γB2, permitted unambiguous identification of the position of the accessory factor within the holoenzyme. One pol γB2 subunit dominates contacts with the catalytic subunit while the second B subunit is largely exposed to solvent. These EM studies of pol γ provide a clear view of residues in the accessory subunit that interact with the catalytic subunit and suggest a model for the interaction of the enzyme with DNA.

References and Footnotes
  1. Carrodeguas, J. A., Theis, K., Bogenhagen, D. F., and Kisker, C. Mol Cell 7, 43-54 (2001).
  2. Trifunovic, A., Wredenberg, A., Falkenberg, M., Spelbrink, J., Rovio, A., Bruder, C., Bohlooly-Y, M., Gidlof, S., Oldfors, A., Wibom, R., et al. Nature 429, 417-423 (2004).
  3. Yakubovskaya, E., Chen, Z., Carrodeguas, J. A., Kisker, C., and Bogenhagen, D. F. J Biol Chem 281, 374-382 (2006).

Elena Yakubovskaya
Mark Lukin
Zhixin Chen
John Berriman2, 3
Caroline Kisker1, 4
Daniel F. Bogenhagen*

Department of Pharmacological Sciences
1Center for Structural Biology
State University of New York at Stony Brook
Stony Brook, NY 11794-8651
2New York Structural Biology Center
New York, NY
3Current address:
The National Institute for Medical Research
The Ridgeway
Mill Hill, London
4Current address:
Rudolf Virchow Center for Experimental Biomedicine
University of Würzburg
97078 Würzburg, Germany

*Phone: 631-444-3068
Fax: 631-444-3218
Email: dan@pharm.sunysb.edu elena@pharm.stonybrook.edu