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Sequence-specific stalling of DNA polymerase gamma and the effects of mutations causing progressive ophthalmoplegia

Journal article
Authors N Atanassova
Javier Miralles Fusté
Sjoerd Wanrooij
Bertil Macao
S Goffart
Stefan Bäckström
G Farge
I Khvorostov
NG Larsson
JN Spelbrink
Maria Falkenberg
Published in Human Molecular Genetics
Volume 20
Issue 6
Pages 1212-1223
ISSN 0964-6906
Publication year 2011
Published at Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology
Pages 1212-1223
Language en
Links dx.doi.org/10.1093/hmg/ddq565
Subject categories Cell and Molecular Biology

Abstract

A large number of mutations in the gene encoding the catalytic subunit of mitochondrial DNA polymerase γ (POLγA) cause human disease. The Y955C mutation is common and leads to a dominant disease with progressive external ophthalmoplegia and other symptoms. The biochemical effect of the Y955C mutation has been extensively studied and it has been reported to lower enzyme processivity due to decreased capacity to utilize dNTPs. However, it is unclear why this biochemical defect leads to a dominant disease. Consistent with previous reports, we show here that the POLγA:Y955C enzyme only synthesizes short DNA products at dNTP concentrations that are sufficient for proper function of wild-type POLγA. In addition, we find that this phenotype is overcome by increasing the dNTP concentration, e.g. dATP. At low dATP concentrations, the POLγA:Y955C enzyme stalls at dATP insertion sites and instead enters a polymerase/exonuclease idling mode. The POLγA:Y955C enzyme will compete with wild-type POLγA for primer utilization, and this will result in a heterogeneous population of short and long DNA replication products. In addition, there is a possibility that POLγA:Y955C is recruited to nicks of mtDNA and there enters an idling mode preventing ligation. Our results provide a novel explanation for the dominant mtDNA replication phenotypes seen in patients harboring the Y955C mutation, including the existence of site-specific stalling. Our data may also explain why mutations that disturb dATP pools can be especially deleterious for mtDNA synthesis.

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