Fragmentation of chromosomal DNA is a critical step in apoptosis that prevents a cell from transcribing and replicating its genes and thus facilitates the cell killing process. Defects in this process can cause various pathological conditions, including autoimmune disorders and cancer. We have identified ten apoptotic nucleases and several non-nuclease factors involved in regulating and executing apoptotic chromosome fragmentation in C. elegans. They act in a sequential and CED-3 caspase-dependent manner to promote stepwise fragmentation and degradation of chromosomes. The process is initiated by a novel CED-3-mediated conversion of the dicer ribonuclease (RNase) into a deoxyribonuclease (DNase), which makes the first cuts on chromosomes. In parallel, a mitochondrial nuclease CPS-6 and its activator WAH-1 are released from mitochondria and translocated to the nucleus, where they interact and cooperate with other cell death nucleases to turn the initial cuts by dicer into double-strand DNA breaks, leading to fragmentation and degradation of chromosomes. In this proposed work, we will carry out molecular genetic, biochemical, cell biological, and structural analyses to understand these two critical events of apoptotic DNA degradation.
In Aim 1, we will investigate the molecular and structural basis underlying CED-3-mediated conversion of the dicer RNase into a DNase.
In Aim 2, we will dissect the new signaling pathway that controls cytosolic calcium increase and release of the mitochondrial apoptogeneic factors during apoptosis.
In Aim 3, we will perform molecular genetic and functional characterization of two new genes, cps-13 and cps-14, that regulate and coordinate two key cell death execution events, chromosome fragmentation and phosphatidylserine (PS) externalization. These studies should reveal the novel mechanism that controls the specificity and function switch of the dicer nuclease and new signaling mechanisms and players that control the release of the mitochondrial apoptogenic factors.

Public Health Relevance

Fragmentation of chromosomal DNA is a critical step in apoptosis, a cell suicide process that could go awry to cause multiple human diseases. Defects in apoptotic chromosome fragmentation can cause various pathological conditions, including autoimmune disorders and cancer. C. elegans is an excellent animal model for studying basic mechanisms of apoptosis and apoptotic chromosome fragmentation. This proposal seeks to understand the mechanisms that regulate activation of apoptotic nucleases and release of apoptosis factors from mitochondria through important signaling pathways. The studies may identify new genes, pathways, and mechanisms that regulate and execute apoptosis, leading to identification of new therapeutic targets or ideas to treat human disease such as Alzheimer's disease, autoimmune disorders, and cancer that are caused by abnormal apoptosis or DNA degradation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM079097-07
Application #
8626408
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Maas, Stefan
Project Start
2007-03-01
Project End
2016-02-29
Budget Start
2014-03-01
Budget End
2015-02-28
Support Year
7
Fiscal Year
2014
Total Cost
$278,394
Indirect Cost
$90,644
Name
University of Colorado at Boulder
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
007431505
City
Boulder
State
CO
Country
United States
Zip Code
80309
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