MITOCHONDRIAL DNA POLYMERASE--MECHANISM AND STRUCTURE
线粒体 DNA 聚合酶——机制和结构
基本信息
- 批准号:2838575
- 负责人:
- 金额:$ 18.78万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:1991
- 资助国家:美国
- 起止时间:1991-07-01 至 2000-11-30
- 项目状态:已结题
- 来源:
- 关键词:DNA binding protein DNA directed DNA polymerase DNA footprinting DNA replication Drosophilidae enzyme mechanism enzyme reconstitution enzyme structure enzyme substrate gel mobility shift assay genetically modified animals immunoprecipitation laboratory rabbit mitochondria mitochondrial DNA mitochondrial membrane nucleic acid sequence polymerase chain reaction polymerization protein sequence protein structure function site directed mutagenesis western blottings
项目摘要
DESCRIPTION: The long term objective is the elucidation of mechanisms of
DNA replication in animal mitochondria. This may be accomplished, among
other ways, when purified enzymes are reconstituted in vitro to yield
replication complexes capable of initiation and elongation of DNA synthesis.
The studies will provide the framework for research to determine the
regulation of these processes, as they occur in vivo. The proposal focuses
on the major replicative enzyme in Drosophila mitochondria, DNA Polymerase
gamma (Pol gamma). A combined approach of biochemistry and molecular
biology is being employed, to pursue mechanistic and structure-function
analyses of this p125:p35 heterodimeric DNA polymerase, to overexpress and
purify the two subunits both together and individually, and to elucidate the
role of the small subunit in enzyme function. Additional goals are to
generate altered enzyme forms for comparative studies, and to examine the
role of mitochondrial single-stranded DNA-binding protein (SSB) in Pol gamma
function. The applicant will also use a transgenic fly model to examine
possible relationships between Pol gamma function, mitochondrial DNA
replication fidelity, aging, and disease.
The control of animal cell reproduction during normal development, and the
loss of control during cancerous development, is of central importance in
the processes of human growth, aging and disease. Mitochondrial biogenesis
proceeds in parallel with cell proliferation, but it is neither tightly
coupled to mitochondrial DNA replication nor to the cell cycle.
Nevertheless, because both the DNA content of the mitochondrion and the
number of mitochondria in cell remain relatively constant, specific
regulatory mechanisms are likely required to couple mitochondrial DNA
replication and biogenesis, to nuclear DNA replication and cell division. A
detailed analysis of the key enzyme involved in mitochondrial DNA
replication will represent a major contribution toward an eventual
understanding of mitochondrial biogenesis and function in normal and
diseased tissues.
A variety of mitochondrial DNA diseases have recently been documented. This
and an increased recognition that antiviral and antitumor drugs frequently
affect mitochondrial DNA function, and in particular, the activity of Pol
gamma demonstrate a critical need for an in-depth understanding of this DNA
polymerase. The Specific Aims as follows: 1. To continue mechanistic
analysis of Pol gamma to examine the mechanisms and fidelity of nucleotide
polymerization, and the role of mitochondrial (mtSSB) in Pol gamma function;
2. To overexpress and purify recombinant forms of the two subunits of Pol
gamma in E. coli and in bacculovirus-infected insect cells; 3. To continue
structure-function studies of Pol gamma involving native, recombinant and
altered forms, to elucidate the role of the small subunit in enzyme
function, to dissect functional domains in the catalytic subunit, and begin
to elucidate the molecular architecture of the enzyme; and 4. To initiate
the development of a transgenic fly model to examine the relationship
between Pol gamma function, mitochondrial DNA replication fidelity, aging
and disease.
DESCRIPTION: The long term objective is the elucidation of mechanisms of
DNA replication in animal mitochondria. This may be accomplished, among
other ways, when purified enzymes are reconstituted in vitro to yield
replication complexes capable of initiation and elongation of DNA synthesis.
The studies will provide the framework for research to determine the
regulation of these processes, as they occur in vivo. The proposal focuses
on the major replicative enzyme in Drosophila mitochondria, DNA Polymerase
gamma (Pol gamma). A combined approach of biochemistry and molecular
biology is being employed, to pursue mechanistic and structure-function
analyses of this p125:p35 heterodimeric DNA polymerase, to overexpress and
purify the two subunits both together and individually, and to elucidate the
role of the small subunit in enzyme function. Additional goals are to
generate altered enzyme forms for comparative studies, and to examine the
role of mitochondrial single-stranded DNA-binding protein (SSB) in Pol gamma
function. The applicant will also use a transgenic fly model to examine
possible relationships between Pol gamma function, mitochondrial DNA
replication fidelity, aging, and disease.
The control of animal cell reproduction during normal development, and the
loss of control during cancerous development, is of central importance in
the processes of human growth, aging and disease. Mitochondrial biogenesis
proceeds in parallel with cell proliferation, but it is neither tightly
coupled to mitochondrial DNA replication nor to the cell cycle.
Nevertheless, because both the DNA content of the mitochondrion and the
number of mitochondria in cell remain relatively constant, specific
regulatory mechanisms are likely required to couple mitochondrial DNA
replication and biogenesis, to nuclear DNA replication and cell division. A
detailed analysis of the key enzyme involved in mitochondrial DNA
replication will represent a major contribution toward an eventual
understanding of mitochondrial biogenesis and function in normal and
diseased tissues.
A variety of mitochondrial DNA diseases have recently been documented. This
and an increased recognition that antiviral and antitumor drugs frequently
affect mitochondrial DNA function, and in particular, the activity of Pol
gamma demonstrate a critical need for an in-depth understanding of this DNA
polymerase. The Specific Aims as follows: 1. To continue mechanistic
analysis of Pol gamma to examine the mechanisms and fidelity of nucleotide
polymerization, and the role of mitochondrial (mtSSB) in Pol gamma function;
2. To overexpress and purify recombinant forms of the two subunits of Pol
gamma in E. coli and in bacculovirus-infected insect cells; 3. To continue
structure-function studies of Pol gamma involving native, recombinant and
altered forms, to elucidate the role of the small subunit in enzyme
function, to dissect functional domains in the catalytic subunit, and begin
to elucidate the molecular architecture of the enzyme; and 4. To initiate
the development of a transgenic fly model to examine the relationship
between Pol gamma function, mitochondrial DNA replication fidelity, aging
and disease.
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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LAURIE SIMON KAGUNI其他文献
LAURIE SIMON KAGUNI的其他文献
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{{ truncateString('LAURIE SIMON KAGUNI', 18)}}的其他基金
MITOCHONDRIAL DNA POLYMERASE: MECHANISM AND STRUCTURE
线粒体 DNA 聚合酶:机制和结构
- 批准号:
7933151 - 财政年份:2009
- 资助金额:
$ 18.78万 - 项目类别:
MITOCHONDRIAL DNA REPLICATION FIDELITY & CARDIAC DISEASE
线粒体 DNA 复制保真度
- 批准号:
2771634 - 财政年份:1997
- 资助金额:
$ 18.78万 - 项目类别:
MITOCHONDRIAL DNA REPLICATION FIDELITY & CARDIAC DISEASE
线粒体 DNA 复制保真度
- 批准号:
2469853 - 财政年份:1997
- 资助金额:
$ 18.78万 - 项目类别:
MITOCHONDRIAL DNA REPLICATION FIDELITY & CARDIAC DISEASE
线粒体 DNA 复制保真度
- 批准号:
6056484 - 财政年份:1997
- 资助金额:
$ 18.78万 - 项目类别:
MITOCHONDRIAL DNA POLYMERASE: MECHANISM AND STRUCTURE
线粒体 DNA 聚合酶:机制和结构
- 批准号:
6625082 - 财政年份:1991
- 资助金额:
$ 18.78万 - 项目类别:
Mitochondrial DNA Replisome: Mechanism, Structure and Physiology
线粒体 DNA 复制体:机制、结构和生理学
- 批准号:
8061977 - 财政年份:1991
- 资助金额:
$ 18.78万 - 项目类别:
MITOCHONDRIAL DNA POLYMERASE--MECHANISM AND STRUCTURE
线粒体 DNA 聚合酶——机制和结构
- 批准号:
2183051 - 财政年份:1991
- 资助金额:
$ 18.78万 - 项目类别:
MITOCHONDRIAL DNA POLYMERASE--MECHANISM AND STRUCTURE
线粒体 DNA 聚合酶——机制和结构
- 批准号:
3304709 - 财政年份:1991
- 资助金额:
$ 18.78万 - 项目类别:
MITOCHONDRIAL DNA POLYMERASE: MECHANISM AND STRUCTURE
线粒体 DNA 聚合酶:机制和结构
- 批准号:
6476519 - 财政年份:1991
- 资助金额:
$ 18.78万 - 项目类别:
MITOCHONDRIAL DNA POLYMERASE--MECHANISM AND STRUCTURE
线粒体 DNA 聚合酶——机制和结构
- 批准号:
3304710 - 财政年份:1991
- 资助金额:
$ 18.78万 - 项目类别: