The long-term objective of the proposed work is the elucidation of the mechanism of DNA replication in animal mitochondria, and its relationship to mitochondrial mutagenesis and human disease. A combined approach of current methods in biochemistry, structural biology and molecular genetics will be pursued to study the mechanism, structure and physiology of the mitochondrial replisome, with a focus on the replicative DNA helicase and the mitochondrial single-stranded DNA-binding protein. In combination with the extensive studies by us and others on the key replicative enzyme in mitochondria, DNA polymerase 3, we expand the scope of prior research to study the assembly and function of proteins at the mitochondrial DNA replication fork, and to probe the modes of mitochondrial DNA replication in vivo and in vitro. Mitochondria are the energy-producing organelle in animals and mitochondrial function impacts nearly every aspect of cellular function. Thus, mitochondria play a major role in human health and likewise, mitochondrial dysfunction is intricately associated with human disease. Mitochondrial dysfunction is implicated in a wide range of neurological diseases, in metabolic diseases, in muscular dystrophies and nephropathies, and in a broad spectrum of named disorders. Defects in mitochondrial biogenesis lead to mitochondrial DNA mutation, depletion and deletion syndromes that result in loss of mitochondrial and subsequent cellular function. The prevalence of mitochondrial genetic disease and recent recognition of the mitochondrial toxicity of antiviral and antimicrobial drugs the critical need for an in-depth understanding of the structure and functions of the mitochondrial DNA replication apparatus.

Public Health Relevance

Narrative: A common feature in diverse chronic, age-related diseases is a breakdown or disturbance in the cell's ability to produce energy. Central to the cellular energy production is the mitochondrion, an organelle that plays a crucial role in multiple metabolic processes, and whose dysfunction is linked directly to many pathologies, including those most represented in an aging population. Therefore, a clear understanding of mitochondrial biogenesis and energy production and how these processes are deregulated in common disease states, is essential to treating them effectively (and economically).

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM045295-20
Application #
8468712
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Barski, Oleg
Project Start
1991-07-01
Project End
2014-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
20
Fiscal Year
2013
Total Cost
$323,163
Indirect Cost
$108,281
Name
Michigan State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
193247145
City
East Lansing
State
MI
Country
United States
Zip Code
48824
Stiban, Johnny; Farnum, Gregory A; Hovde, Stacy L et al. (2014) The N-terminal domain of the Drosophila mitochondrial replicative DNA helicase contains an iron-sulfur cluster and binds DNA. J Biol Chem 289:24032-42
Farnum, Gregory A; Nurminen, Anssi; Kaguni, Laurie S (2014) Mapping 136 pathogenic mutations into functional modules in human DNA polymerase ? establishes predictive genotype-phenotype correlations for the complete spectrum of POLG syndromes. Biochim Biophys Acta 1837:1113-21
Martínez-Azorín, Francisco; Calleja, Manuel; Hernández-Sierra, Rosana et al. (2013) Muscle-specific overexpression of the catalytic subunit of DNA polymerase ? induces pupal lethality in Drosophila melanogaster. Arch Insect Biochem Physiol 83:127-37
Fernandez-Moreno, Miguel A; Hernandez, Rosana; Adan, Cristina et al. (2013) Drosophila nuclear factor DREF regulates the expression of the mitochondrial DNA helicase and mitochondrial transcription factor B2 but not the mitochondrial translation factor B1. Biochim Biophys Acta 1829:1136-46
Oliveira, Marcos T; Kaguni, Laurie S (2011) Reduced stimulation of recombinant DNA polymerase ? and mitochondrial DNA (mtDNA) helicase by variants of mitochondrial single-stranded DNA-binding protein (mtSSB) correlates with defects in mtDNA replication in animal cells. J Biol Chem 286:40649-58
Euro, Liliya; Farnum, Gregory A; Palin, Eino et al. (2011) Clustering of Alpers disease mutations and catalytic defects in biochemical variants reveal new features of molecular mechanism of the human mitochondrial replicase, Pol ýý. Nucleic Acids Res 39:9072-84
Makowska-Grzyska, Magdalena M; Ziebarth, Tawn D; Kaguni, Laurie S (2010) Physical analysis of recombinant forms of the human mitochondrial DNA helicase. Methods 51:411-5
Oliveira, Marcos T; Garesse, Rafael; Kaguni, Laurie S (2010) Animal models of mitochondrial DNA transactions in disease and ageing. Exp Gerontol 45:489-502
Oliveira, Marcos T; Kaguni, Laurie S (2010) Functional roles of the N- and C-terminal regions of the human mitochondrial single-stranded DNA-binding protein. PLoS One 5:e15379
Palin, Eino J H; Lesonen, Annamari; Farr, Carol L et al. (2010) Functional analysis of H. sapiens DNA polymerase gamma spacer mutation W748S with and without common variant E1143G. Biochim Biophys Acta 1802:545-51

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