Title: Spanning the In Vitro-In Vivo Gap II: Connecting intracellular DNA damage
repair to DNA polymerase molecular properties.
This project will: This project will develop an understanding of the mechanisms of DNA
repair enzymes using the significant DNA repair ability of Deinococcus radiodurans as a
model system.
Primary Faculty co-Advisors:
Vince LiCata Ph.D., Department of Biological Science and Chemistry (Biochemistry and
Biophysics)
John Battista Ph.D., Department of Biological Sciences (Microbiology)
Jacqueline M. Stephens, Ph.D., Department of Biological Sciences (Cell Biology)
Off-campus Participant: Not yet identified.
Technical Proposal:
The ability of organisms to repair damaged DNA is essential for cellular viability.
One of the greatest challenges for an organism is the ability to maintain the integrity of its
genome. Even under “normal” conditions, all living organisms are exposed to some level
of ionizing radiation on a daily basis. If efficient DNA repair were not possible, even
small amounts of radiation could have dramatic cellular effects. Indeed, deficient DNA
repair leads to many disease states, including Xeroderma Pigmentosum, Cockayne’s
disease, trichothiodystrophy. As well, many cancerous states are achieved because of a
lack of efficient DNA repair. Clearly, understanding the cellular response to DNA
lesions and the mechanism of DNA repair are important biochemical and cellular
biological questions. The aim of this team will be to learn biochemical, biophysical, and
microbiological techniques to understand the DNA damage repair response of organisms
both in vivo and in vitro. More specifically, the team will determine how the interaction
of various proteins and DNA lead to a cell’s ability to repair its genome from different
types of lesions.
Initially, the team will focus on the type I DNA polymerase (pol I) from
Deinococcus radiodurans. Deinococcus radiodurans is one of the most radiation
resistant organisms known to man. In fact, D. radiodurans routinely survives doses of radiation up to 15,000 Gy. (Battista, 1997) This is a remarkable number, as this amount
of radiation can induce approximately 200 double strand breaks, 3000 single strand
breaks, and 1000 damaged bases per cell. It is believed that D. radiodurans achieves this
remarkable ability by being able to repair its genome from dramatic damage extremely
efficiently. To date, there are 9 proteins whose absence leaves D. radiodurans
susceptible to different types of DNA damage by radiation. Deinococcus’ pol I is one of
these essential proteins, and is necessary for all types of DNA lesion repair.
Type I DNA polymerases are generally regarded as the main repair polymerase in
the cell, using intrinsic 3’-5’ exonuclease activity to proofread incorrectly mismatched
basepairs, as they transcribe DNA. In E. coli, pol I has been found to participate in both
nucleotide excision and the base excision repair pathways. The cell is able to recognize
DNA damage and excise the damaged portion by one of the two pathways mentioned.
DNA pol I fills in these gaps with very high fidelity. Thus, pol I from E. coli is deemed a
repair polymerase. Pol I is made up of three distinct structural domains. The domain
which provides replicative ability is the polymerase domain. This domain is conserved
throughout all type I DNA polymerases and is describe a half-open right hand, complete
with a finger and thumb domain that seem to “embrace” the DNA. A second structural
domain, which along with the polymerase domain make up the large fragment, is the 3’5’ exonuclease domain. This domain is believed to work as DNA is being replicated,
proofreading the newly replicated DNA. If this domain detects a mismatch in the newly
laid strand, it can excise the fragment, only to be refilled by the polymerase domain. This
3’-5’ exonuclease activity is not detectable in all type I DNA polymerases, implicating a
possible second function of these proteins in vivo. The third structural domain is the 5’3’ nuclease domain, which looks like a “tail” hanging off of the large fragment of the
protein. It’s job, presumably, is to remove fragments of DNA in the 5’-3’ direction either
on the same or opposite strand as is being synthesized. (Perler, 1996)
Determination of the important structural domains for DNA repair.
In order to determine the structural domains which are important for efficient
DNA repair, a model system will be developed using pol– mutant strains of D.
radiodurans. A pol– mutant strain is one which is sensitive to all types of ionizing
radiation, as the cell no longer has the ability to make the critical enzyme, pol I. Initial
results suggest that pol– mutant strains of D. radiodurans are viable under normal
conditions (Battista, et al., unpublished). Moreover, D. radiodurans is an excellent choice
as a model system, as they are naturally transformable. Therefore, one can introduce
foreign protein products into D. radiodurans very efficiently. (Battista, 1997) In order to
determine the structural domains of pol I which are important for DNA repair, a specific
series of experiments will be performed. These experiments will introduce a multitude of
pol I’s from other species, each of which have unique characteristics. The pol I from
Thermus aquaticus (Taq), for example, does not have any 3’-5’ exonuclease activity. The
introduction of the pol I gene from Taq into a pol - mutant strand of D. radiodurans will
be performed, and the ability of the Taq polymerase to restore the DNA repair ability of
D. radiodurans will be tested. If, for example, the Taq pol I can substitute directly, one
could deduce that the 3’-5’ activity is non-essential for DNA repair. As well, the 5’-3’
nuclease domain can be tested in exactly the same way, using deletion mutants of that
domain. (These deletion mutants are commonly referred to as Klenow for E. coli’s pol I
and Klentaq for T. aquaticus’ pol I.) As well, a long term goal of this project would be to
perform these same type of experiments in eukaryotic cells.
In addition to conducting the in vivo studies, the team will clone and purify the
Type I DNA polymerase from D. radiodurans. Having purified protein will allow this
team to determine the DNA binding characteristics of this protein. The LiCata lab has
previously characterized the DNA binding properties of four polymerases to date. (Datta,
2003) A complete biophysical characterization of this pol I will be critical in
understanding D. radiodurans DNA repair mechanisms. Purified pol I from D.
radiodurans will also allow the team to determine the nucleotide incorporation rate and
the error rate of the polymerase, as well as the thermal stability of the protein.
Number of IGERT apprentices to be recruited and probable home departments:
Allison Joubert (existing In Vitro-In Vivo Gap Team Member)
Greg Thompson (new In Vitro-In Vivo Gap Team Member)
Other students from Chemistry, Biology, and/or Engineering are desired.
Consistency with the Macromolecular Education, Research & Training theme:
Proteins are high-performance macromolecules. Their study requires many of the same
concepts (radius of gyration, polyelectrolyte effects, solution thermodynamics and
spectroscopy) needed for polymer research. Design of protein-based medical devices
requires expertise in biochemical, polymer, and cellular physiology research.
Understanding the forces that control protein function in the cellular environment
requires understanding their interaction with solvent and the impact of all intensive and
extensive system properties known to differ between the in vitro and in vivo
environments. Testing the predictions of in vitro investigations requires the ability to
work directly in the living intracellular environment. A basic understanding of the
mechanisms underlying protein structure and function is required for the exploitation of
these systems for other applications.
How does the project form a vector cross-product of existing research themes by the
participants?
Existing research directions. The research effort in the LiCata laboratory is primarily
focused on the examination of the biophysical properties of soluble proteins. The primary
focus of the Stephens’ laboratory is centered on the molecular pathogenesis of insulin
resistance and type II diabetes in adipocytes. The Battista laboratory is focused on the
genetics are microbiology of radiation resistant bacteria. Each of the participating
laboratories is supported by federal grants.
New research direction. In this research proposal, we will exploit the strengths ofeach
of the laboratories in the examination of the role of DNA polymerase and other proteins
in DNA repair. The current understanding of D. radiodurans from the Battista laboratory
will be used to clone DNA polymerase I from D. radiodurans. The protein will be
characterized at the molecular level in the LiCata laboratory. A long range goal of the
team is to test whether DNA repair mechanisms can be successfully transferred to
eukaryotic cells – such tests will require the expertise of the Stephens laboratory.
How do students benefit from the team-oriented research, beyond what would be
available to them from either advisor separately? These major approaches to
understanding a particular biological question: molecular biophysics, microbiology, and
cellular biology, form a unique span of disciplines aimed at understanding a particular
process: DNA repair. Training on this IGERT team will give students the training and
understanding to span this divide. Students trained across these disciplines will be
poised to make advances in the understanding and control of macromolecular
interactions within living systems.
Briefly describe the support level available to each individual faculty or off-campus
participant (i.e., without IGERT) Dr. LiCata is currently supported by the NSF and
NASA. Dr. Stephens is currently supported by the NIH and the ADA (American
Diabetes Association). Dr. Battista is currently supported by the NSF and NASA.
Interdisciplinary strengths of the team project: This In Vivo-In Vitro Gap Team was
already interdisciplinary with Dr. LiCata and Dr. Stephens as the faculty advisors. With
the addition of Dr. Battista and Greg Thompson to the team, the interdisciplinary span
has significantly increased. These are truly three different laboratories that would rarely
if ever interact normally. Their interactions via Allison (mostly interacting with the
LiCata and Stephens laboratories) and Greg (interacting first with the LiCata and
Battista laboratories and eventually with the Stephens laboratory) will afford one of the
widest spanning interdisciplinary training opportunities in modern biology.
Commitment of faculty & off-campus participants to work side-by-side with
apprentices:
The principal investigators on this research team are fully committed to the
overall educational paradigm of this IGERT proposal. Dr. LiCata works on a daily basis
with his research group and personally mentors his students in carrying out experimental
protocols. He has already worked side by side with Allison and Greg on small angle Xray scattering experiments related to this project. Dr. Battista and Greg Thompson have
been working closely together recently on determining the evolution of proofreading
ability in DNA polymerases. Dr. Stephens maintains an active personal research program
and interacts daily with her laboratory members, and has already interacted extensively
with Allison on the lipid binding protein part of this In Vitro-In Vivo Team (see Allison
Joubert’s Form A for a more indepth explanation of that portion of the project).
References:
Battista, J. R.; “Against all odds: the survival strategies of Deinococcus radiodurans.”
Annu Rev Microbiol, 1997, 51/203-224.
Datta, K., LiCata, V. J.; “Salt dependence of DNA binding by Thermus aquaticus and
Escherichia coli DNA Polymerases.” JBC, 2003, 278/8, 5694-5701.
Datta, K., LiCata, V. J.; “Thermodynamics of the binding of Thermus aquaticus DNA
polymerase to primed-template DNA.” Nuc Acids Res, 2003, 31/19, 1-8.
Johnson, K. A.; “Conformational coupling in DNA polymerase fidelity.” Annu Rev
Biochem,1993, 62, 685-713.
Perler, F. B., Kumar, S., Kong, H.; “Thermostable DNA polymerases.” Adv Prot Chem, 1996,
48, 377-435.