Advisor(s)
Penny J. Beuning
Contributor(s)
David E. (David Edward) Budil, Mary Jo Ondrechen, Carolyn Lee-Parsons
Date of Award
2012
Date Accepted
3-2012
Degree Grantor
Northeastern University
Degree Level
Ph.D.
Degree Name
Doctor of Philosophy
Department or Academic Unit
College of Science. Department of Chemistry & Chemical Biology.
Keywords
chemistry, biochemistry, dimer exchange, EPR, monomer, mutagenesis, SOS response, UmuD
Subject Categories
Proteins - Conformation, DNA damage
Disciplines
Biochemistry
Abstract
The homodimeric umuD gene products play key roles in regulating the cellular response to DNA damage in Escherichia coli. UmuD is composed of 139-amino acid subunits and is upregulated as part of the SOS DNA damage response. Subsequently, damage-induced RecA:ssDNA nucleoprotein filaments mediate the slow autocleavage of the N-terminal 24-amino acid arms of UmuD yielding UmuD'. It was previously proposed that UmuD cleaves only in the trans conformation, in which the arm of one monomer utilizes that active site of the adjacent monomer for cleavage. Cleavage in trans would therefore require dimerization. However, isoenergetic models of UmuD suggested that the arms may adopt cis (intramolecular) or trans (intermolecular) conformations, and may be unbound from or bound to the globular C-terminal domain. The dynamic nature of the N-terminal arms may explain how a number of distinct protein-protein contacts that prevent and facilitate mutagenic translesion synthesis (TLS) are made. The overall goal of my research is to determine the conformation and dynamics of the UmuD proteins in order to understand its regulatory role in response to DNA damage. Chapter 1 presents the relevant background and details of structure, function and interactions of UmuD with proteins involved in DNA replication and DNA damage repair.
In an effort to learn more about the structural dynamics and functions of UmuD proteins, we designed a UmuD protein variant that is defective in dimerization. Such a variant would not only answer the question as to whether UmuD is active in the cis conformation, but also address the possibility that UmuD may be functionally active as a monomer. Although models of UmuD with the arms in the cis conformation have been proposed, evidence that this conformation is physiologically relevant has been lacking. Wild-type UmuD and UmuD' form exceptionally tight dimers in solution; however, in chapter 2 we show that the single amino-acid change N41D generates stable, active UmuD and UmuD' monomers that functionally mimic the dimeric wild-type proteins. The UmuD N41D monomer is proficient for cleavage and interacts physically with DNA polymerase IV (DinB). Furthermore, the N41D variants facilitate UV-induced mutagenesis and promote overall cell viability. Taken together, these observations show that a monomeric form of UmuD retains substantial function in vivo and in vitro.
UmuD and UmuD' can display differential interactions with their partner proteins which can lead to dramatically different cellular outcomes. These key differences may be due to the dynamics of the N-terminal arms of UmuD. Previous biochemical evidence supported a model in which the arms of UmuD are stably bound to its globular domain. However, recent experiments suggest that the N-terminal arms of UmuD are somewhat dynamic. Chapter 3 describes the use of Electron Paramagnetic Resonance (EPR) spectroscopy to probe the conformational dynamics of the N-terminal arms of the umuD gene products and variants. We determined that the arms of UmuD display a large degree of motion, are largely unbound from the globular C-terminal domain, and that the free energy of dissociation is +2.1 kJ/mol.
In chapter 4, we discuss the dimer exchange and conformation-dependent cleavage of the UmuD proteins. To further understand the dynamic regulatory roles of the umuD gene products, we monitored the kinetics of exchange and cleavage of the UmuD and UmuD' homodimers as well as of the UmuDD'; heterodimer under equilibrium conditions. We found that the heterodimer is the preferred but not exclusive dimeric protein form, and that both the heterodimer and homodimers exhibit slow exchange kinetics. In addition, the heterodimer efficiently cleaves to form UmuD'. Together, this work reveals an intricate UmuD lifecycle that involves dimer exchange and cleavage in the regulation of the DNA damage response.
Chapter 5 discusses the use of UmuD N-terminal truncations, UmuD 8 and UmuD 18, in accessing the role of the arms in regulating protein-protein interactions. Extensive characterization reveals that the loss of even the N-terminal seven amino acids results in a notable change in domain conformation, binding affinity to DinB as detected by tryptophan fluorescence, and the facilitation of UV-induced mutagenesis and UV survival. Additionally, we have also discovered a smaller version of UmuD that is also UV-inducible. Given the information above, it is plausible that this smaller UmuD may be involved in yet another level of DNA damage and repair regulation.
Document Type
Dissertation
Rights Information
Copyright 2012
Rights Holder
Jaylene N. Ollivierre
Permanent URL
Recommended Citation
Ollivierre, Jaylene N., "The umuD gene products are molecular adaptors in the regulation of DNA damage tolerance" (2012). Chemistry Dissertations. Paper 40. http://hdl.handle.net/2047/d20002405
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