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Deleterious Deoxyguanosine Kinase Double Destruction
Westosha Central SMART Team: Julia Alberth, Nicholas Bielski, Dylan Clements, Jared Hollway, Evan Kirsch, Mitchell Kirsch
Becca Lawrence, Julia Mellor, Maddie Murphy, Adam Papendick, Sean Quist, AJ Reeves, Zachary Wermeling, Julia Williams
Teacher: Jonathan Kao
Mentor: Jason Kowalski, Ph.D., University of Wisconsin Parkside
I. Abstract
IV. mtDNA and Central Dogma
Mitochondrial Deficiency Syndrome (MDS) is characterized by a deficient amount of
mitochondrial DNA (mtDNA). Without sufficient copies of mtDNA, the mitochondria
cannot manufacture an adequate amount of ATP, leading to complications in energy
expensive tissues such as the brain and liver, ultimately causing death in early
infancy. Deoxyguanosine kinase (dGK), an enzymatic protein, plays a role in regulation
of mtDNA by attaching a phosphate to a sugar/nitrogen-base nucleoside. Once
phosphorylated, the assembly of mtDNA proceeds. Mutations in dGK prevent the
phosphorylation of mtDNA and lead to a decrease in mitochondrial function. Two
point mutations have been shown to have a deleterious impact on dGK: the R142K
mutation is 0.2% active when compared to the wild type, and the E227K mutation is
5.5% active when compared to the wild type. The 3D model printed by the Westosha
Central SMART (Students Modeling A Research Topic) team displays these two
specific mutations and additional mutations reported in MDS patients. Screening for
mutations in dGK may be a potential method for quickly diagnosing MDS, saving
precious time for those affected.
II. Mitochondrial
Deficiency Syndrome
Mitochondrial Deficiency Syndrome (MDS) is a disorder that arises from insufficient
copying of mitochondrial DNA (mtDNA) resulting in a lack of ATP production.
Symptoms include muscle malfunction, along with liver, muscle, kidney, and lung
failure. These are energy expensive tissues, therefore ATP deficiency has the greatest
effect on them. MDS is inherited through an autosomal recessive gene and exists in
multiple forms. Various symptoms are present in different forms, as shown in Figure
1, but a commonality between all forms of MDS is insufficient production of ATP.
Most forms of MDS have early
onset and result in death during
early infancy, however some forms
of MDS have a later onset and
symptoms may occur within five
years of birth. Cases are extremely
rare and specific to the individual;
few cases exist globally at one time,
because death occurs in the first
few years. Through studying MDS,
scientists have concluded that
organ transplants and gene therapy
are possible approaches in the
quest for a cure.
Figure 1. Symptoms of MDS.
Affected organs and tissues display
III. Biological Significance
symptoms due to inadequate ATP3
Figure 2: A) EPR spectrum of mouse
liver comprised of at least 17
overlapping metallo-protein spectra.
B) A model of individual metalloprotein spectra simulations to model
the experimental spectrum in 2A. C)
A hypothetical model of a mouse
liver affected by a mutation in dGK6.
Screening for MDS can be done with whole
tissue Electron Paramagnetic Resonance
(EPR). EPR data has demonstrated it is
possible to resolve up to 17 different
metallo-protein spectra in mouse organs,
Figure 2A. Of these 17 proteins, 11 are
found in the mitochondria. It is possible to
model individual spectra of all 17 proteins
into a simulation of the experimental data,
Figure 2B. A mouse with MDS would have
a noticeable decrease in the EPR signal of
mitochondrial proteins, Figure 2C. This is
just one of many protein specific forms of
MDS that EPR of whole tissue may be able
diagnose more quickly than traditional
methods.
The story of mitochondrial proteins is interesting because they involve two iterations of
the central dogma. The central dogma of molecular biology states that DNA codes for
proteins using RNA as an intermediate as shown in Figure 3. Nuclear proteins, such as
dGK, are responsible for the assembly of mtDNA. As expected, dGK is coded for by
chromosome 2 and transcribed to mRNA and then is produced by a ribosome in the
cytoplasm. The new protein is then transported to a mitochondrion where it constructs
mtDNA through the phosphorylation of a guanosine monomer as shown in Figure 4. This
is where the second iteration of the central dogma begins. When dGK is properly
functioning, mitochondrial DNA (mtDNA) is assembled correctly and efficiently. Improper
dGK function leads to misassembled and mutated mtDNA. Mitochondrial DNA codes for
proteins and ribosomes that are necessary for the proper formation of ATP as shown in
Figure 5.
A)
B)
C)
D)
Figure 3. The Central Dogma
A) DNA is transcribed to mRNA in the nucleus. B) Translation from a nucleic acid
sequence to an amino acid sequence occurs at the ribosome. C) Amino acid
sequences fold spontaneously based on amino acid properties. An error in the process
can cause mutations. D) The final tertiary structure of a protein determines function.
V. Point Mutations and dGK
Function
Several mutations have been reported in dGK. Almost all of these mutations
have a deleterious effect on the function of dGK. Mutations discussed in
Eriksson (2003) are highlighted in Figure 6.
The E227K mutation results when the
nucleotide 679 interchanges A  G. The
resulting codon mutates a Glutamic Acid
into a Lysine. This reduces the enzyme’s
ability to phosphorylate deoxyguanosine, a
pre-cursor to the monomer of mitochondrial
DNA. The E227K mutation is only 2.8%-5.5%
efficient when compared to wild type. This
efficiency drop creates insufficient copies of
mtDNA, leading to ATP deficiency.
Fig. 7 E227K Mutation
This mutation is the result of a Glutamic
Acid mutating to a Lysine. This mutation
occurs outside the active site but drops
the efficiency of dGK.
A)
B)
C)
D)
Figure 4. Location of Mitochondrial DNA
A) All Eukaryotic cells contain mitochondria in
the cytoplasm1. B) Mitochondria produce ATP,
the molecular currency of energy4. C) mtDNA is
a circular piece of DNA specific to the
mitochondria which codes for proteins
necessary to produce ATP. D) dGK
phosphorylates a guanosine monomer creating
guanine, a nitrogen base found in mtDNA.
Figure 5. Mitochondrial DNA
Almost the entirety of mtDNA is
devoted to coding for proteins
necessary for ATP synthesis. Incorrectly
assembled mtDNA may result in
proteins incapable of producing ATP5.
Misassembled mtDNA cannot code for proteins involved in ATP synthesis resulting in
insufficient ATP production by the mitochondria. In this case, the central dogma has failed as
mtDNA is not coding for mitochondrial proteins accurately. Lack of ATP leads to muscle
malfunction, along with liver, kidney, and lung failure which can be broadly characterized as
MDS. This sequence of events necessitates that dGK does not have deleterious mutations such
that it will not mutate or inhibit mtDNA. Proteins coded for by mtDNA act as if downstream
from the dGK gene; any earlier mutation will result in a cascade of unwanted consequences.
The R142K mutation results when nucleotide 425
interchanges A  G. The resulting codon mutates
an Arginine into a Lysine. Alteration of this specific
dGK amino acid leads to a lethal mutation in the
active site. While Arginine and Lysine are both
positively charged amino acids, Arginine is a
stronger base and will donate hydrogen more
readily than Lysine. The R142K mutant is still active
but has a decreased phosphorylation rate, slowing
it to 0.2% of the wild type, effectively terminating
the protein’s function. If phosphorylation is
terminated, then the mitochondria will be inhibited
from producing ATP and the infant with the R142K
mutation will die.
Figure 8. R142K Mutation
This mutation is the result of an Arginine
mutating to a Lysine. This mutation
affects the active site of dGK. Despite a
mutation in the active site, the similar
properties of Arginine and Lysine allow
phosphorylation to continue, albeit at a
drastically slower rate.
These are truncation mutations that are caused by a change in
nucleotide sequence such that a premature stop codon is
coded for, ending synthesis of the protein by the ribosome.
This results in an incomplete protein that has an improperly
folded active site, or no active site at all. As a result, these
mutations usually result in completely inactive proteins.
Figure 6. A Model of dGK based on file 2OCP.pdb
VI. References
1. Animal cell clipart. (n.d.). Clker.com. Retrieved February 27, 2014, from http://www.clker.com/clipart-animal-cell.html
2. Eriksson, S. (2003). Mitochondrial deoxyguanosine kinase mutations and mitochondrial DNA depletion syndrome. FEBS Letters, 554(3),
319-322. http://www.sciencedirect.com/science/article/pii/S0014579303011815
3. Hesterlee, S. (n.d.). Mitochondrial Disease in Perspective Symptoms, Diagnosis and Hope for the Future. Mitochondria Research Society.
Retrieved March 4, 2014, from http://www.mitoresearch.org/treatmentdisease.html
Fig 9. Truncation Mutations
Mutations in the amino acid sequence that result in the
early termination of the protein have been reported at
positions 105, 213, and 255.
4. Mari, M., Tooze, S. A., & Reggiori, F. (2012, February 1). The Enigmatic Membrane. The Scientist. Retrieved February 27,
2014, from http://www.the-scientist.com/?articles.view/articleNo/31626/title/The-Enigmatic-Membrane/
5. Mitochondrial DNA Map. (n.d.). Mitochondrial DNA Map. Retrieved March 10, 2014, from
http://lslab.lscore.ucla.edu/MTDNA/mtDNAMap.htm
6. Unpublished data from the lab of Dr. Brian Bennett, Medical College of Wisconsin
SMART Teams are supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Number 8UL1TR000055.
Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH