An investigation of the biochemical basis of mutation is fundamental to human health related issues involving genetic disorders including cancer, aging, and neurodegenerative disease. An understanding of DNA polymerase fidelity is at the core of understanding how mutations are generated. Our grant, "Error Correction in DNA Synthesis: A Biochemical Study" has, for the past 35 years, focused on fundamental issues of polymerase fidelity. Initially, we developed concepts and techniques to analyze how polymerases select right from wrong bases for insertion into DNA and to eliminate errors through exonuclease proofreading. The scope of our biochemical studies expanded into the field of human immunological diversity, where we studied the properties of DNA-dependent cytidine deaminases involved in the initiation of somatic hypermutation in immunoglobulin genes and inactivation of HIV-1. While investigating the biochemical basis of SOS damaged- induced mutagenesis in E. coli, we discovered DNA polymerase V, a founding member of a new family (Y- family) of "error-prone" DNA polymerases. We showed that pol V is a heterotrimer (UmuD'2C) composed of two proteins required for UV mutagenesis. In 2009, we resolved a long-standing issue in DNA damage-induced mutagenesis in E. coli, the direct role of a RecA nucleoprotein filament (RecA*) in the replication of damaged DNA templates by pol V. We showed that the role of RecA* is to transfer a molecule of RecA7ATP from its 3'- end to convert inactive pol V into mutagenically active pol V Mut. The properties of pol V Mut (UmuD'2C- RecA7ATP) are regulated through a biochemical cycle of polymerase activation, translesion DNA synthesis, deactivation and reactivation. All forms of pol V Mut retain UmuD'2C-RecA7ATP in a bound complex. In this grant, we propose to study each conformational state of pol V Mut by examining where RecA7ATP binds in relation to UmuD'2 and to the catalytic UmuC subunit and observe the transitions between states in real-time. We will incorporate unnatural amino acids in each subunit to attach site-directed fluorescent probes. These probes will be used to investigate each stage of the pol V Mut cycle by stopped-flow FRET and rotational anisotropy techniques.
Aim 1 will determine specific interactions between the RecA7ATP, UmuD'2 and UmuC subunits in the activated and deactivated forms of pol V Mut.
Aim 2 will investigate individual kinetic steps during polymerase activation, DNA synthesis, deactivation and reactivation. To obtain a deeper understanding of the biochemical properties of pol V Mut in relation to its behavior in the cell, Aim 3 will analyze two "classical" RecA mutants, one that does not induce mutations in the presence of DNA damage and the other which causes hypermutation in the absence of DNA damage. Our proposal addresses a new model for the regulation of DNA damaged-induced mutagenesis, where the active and inactive forms of the DNA polymerase are governed by the assembly of RecA nucleoprotein filament. This new regulatory mechanism acts to ensure that error-prone pol V Mut cannot mutate the cell unnecessarily by copying undamaged DNA templates.

Public Health Relevance

In all organisms including bacteria and humans, mutations are typically deleterious, causing numerous sporadic and inherited diseases. Yet it is clearly evident that mutations are required for evolution and are essential in providing immunological diversity and general fitness. The proposed research explores the biochemical mechanisms of a completely new type of error-prone DNA polymerase, one which is activated when needed to copy damaged DNA, deactivated to keep it from mutating undamaged DNA, then reactivated again to deal with further DNA damage. This study explores biochemical mechanisms that govern the ability of error-prone DNA polymerases to copy damaged DNA that would otherwise cause a cessation of chromosome replication resulting in cell death.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM021422-36
Application #
8393486
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Reddy, Michael K
Project Start
1978-09-01
Project End
2014-11-30
Budget Start
2012-12-01
Budget End
2013-11-30
Support Year
36
Fiscal Year
2013
Total Cost
$388,481
Indirect Cost
$148,678
Name
University of Southern California
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
072933393
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
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