Biochemical, cellular and molecular identification of DNA polymerase α in yeast mitochondria

Jean-Paul Lasserre, Jacqueline Plissonneau, Christophe Velours, Marc Bonneu, Simon Litvak, Patricia Laquel, Michel Castroviejo
Biochimie. 2013-04-01; 95(4): 759-771
DOI: 10.1016/j.biochi.2012.11.003

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1. Biochimie. 2013 Apr;95(4):759-71. doi: 10.1016/j.biochi.2012.11.003. Epub 2012
Nov 14.

Biochemical, cellular and molecular identification of DNA polymerase α in yeast

Lasserre JP(1), Plissonneau J, Velours C, Bonneu M, Litvak S, Laquel P,
Castroviejo M.

Author information:
(1)MCMP, UMR 5234, CNRS, Université Bordeaux Segalen, 146 rue Léo Saignat, 33076
Bordeaux Cedex, France.

DNA replication occurs in various compartments of eukaryotic cells such as the
nuclei, mitochondria and chloroplasts, the latter of which is used in plants and
algae. Replication appears to be simpler in the mitochondria than in the nucleus
where multiple DNA polymerases, which are key enzymes for DNA synthesis, have
been characterized. In mammals, only one mitochondrial DNA polymerase (pol γ) has
been described to date. However, in the mitochondria of the yeast Saccharomyces
cerevisiae, we have found and characterized a second DNA polymerase. To identify
this enzyme, several biochemical approaches such as proteinase K treatment of
sucrose gradient purified mitochondria, analysis of mitoplasts, electron
microscopy and the use of mitochondrial and cytoplasmic markers for
immunoblotting demonstrated that this second DNA polymerase is neither a nuclear
or cytoplasmic contaminant nor a proteolytic product of pol γ. An improved
purification procedure and the use of mass spectrometry allowed us to identify
this enzyme as DNA polymerase α. Moreover, tagging DNA polymerase α with a
fluorescent probe demonstrated that this enzyme is localized both in the nucleus
and in the organelles of intact yeast cells. The presence of two replicative DNA
polymerases may shed new light on the mtDNA replication process in S. cerevisiae.

Copyright © 2012 Elsevier Masson SAS. All rights reserved.

DOI: 10.1016/j.biochi.2012.11.003
PMID: 23160073 [Indexed for MEDLINE]

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