dUTPases
Section 1.
Form and Function
Homotrimeric dUTPases; Structural Solutions for Specific
Recognition and Hydrolysis of dUTP Pp-287-300
Rebecca Persson, Eida S.
Cedergren-Zeppezauer and Keith S. Wilson
Glycine Rich P-loop Motif in Deoxyuridine Pyrophosphatase Pp-301-311
G. Sridhar Prasad
Section 2.
Genetics and Evolution
Evolution of the DUT Gene: Horizontal Transfer between
Host and Pathogen in all Three Domains of Life
Pp-313-324
Marcella A. McClure
Evolution of the dUTPase Gene of Mammalian and Avian
Herpesviruses Pp-325-333
Section 3.
dUTPase in Human Cells: Regulation in Healthy Tissues and Loss of
Control in
Virus-Infection and Neoplasia
The Nature of Enzymes Involved in Uracil-DNA Repair: Isoform Characteristics of
Proteins Responsible for Nuclear and Mitochondrial
Genomic Integrity Pp-335-347
Sal Caradonna and Susan Muller-Weeks
dUTPase in Human Neoplastic Cells as a Potential Target for Therapeutic
Intervention Pp-349-360
Friedrich A. Grässer, Bernd F. M. Romeike,
Gerald Niedobitek, John Nicholls and Elisabeth Kremmer
The Role of dUTPase and Uracil-DNA Repair in Cancer
Chemotherapy Pp-361-370
Robert D. Ladner
Section 4.
dUTPases in Pathogenic Organisms: Targets for Drug Design
The Herpesvirus Encoded dUTPase as a Potential Chemotherapeutic
Target Pp-371-379
A.W. Studebaker, G.K. Balendiran and
Marshall V. Williams
The Role of Retroviral dUTPases in Replication and
Virulence Pp-381-388
Susan L. Payne and John H. Elder
Trypanosomal dUTPases as Potential Targets for Drug
Design Pp-389-397
Fernando Hidalgo-Zarco and Dolores González-Pacanowska
Back to top] Homotrimeric dUTPases; Structural
Solutions for Specific Recognition and Hydrolysis of dUTP
Prevention of incorporation of dUTP into DNA is essential for maintenance of the genetic information. Prompt and specific removal of dUTP from the nucleotide pool, as expedited by the ubiquitous enzyme dUTPase, is therefore required for full viability in most biological systems. Conserved structural features perpetuate specificity in choice of substrate, which is crucial as hydrolysis of the structurally closely related nucleotides dTTP, dCTP and UTP would debilitate DNA and RNA synthesis.
The most common family of dUTPases is the homotrimeric variety where X-ray structures are available for one bacterial, one mammalian and two retroviral dUTPases. These four enzymes have similar overall structural layouts, but the interactions that stabilise the trimer vary markedly, ranging from exclusively hydrophobic to water-mediated interactions. Trimeric dUTPases contain five conserved sequence motifs, positioned at the subunit interfaces where they contribute to the formation of the active sites. Each of the three identical active sites per trimer is built of residues contributed by all three subunits. One subunit provides residues involved in base and sugar recognition, where a b-hairpin acts to maintain exquisite selectivity, while a second subunit contributes residues for phosphate interactions. The third subunit supplies a glycine-rich consensus motif located in the flexible C-terminal part of the subunit, known from crystallographic studies to cover the active site in the presence of substrate and certain substrate analogues.
All dUTPases studied require the presence of a divalent metal ion, preferably Mg2+, for optimal activity. The putative position of the essential metal ion has been identified in the structure of one retroviral dUTPase.
Structure-function studies are
essential if the properties of dUTPases are to be understood fully in relation
to their biological role. In this review the structural arrangement of the
homotrimeric dUTPases is discussed in the context of active site geometry,
achievement of specificity and subunit interactions.
Back to top] Glycine Rich P-loop Motif in
Deoxyuridine Pyrophosphatase
Deoxyuridine pyrophosphatase
(dUTPase) cleaves the a-b phosphodiester bond of dUTP to form pyrophosphate and
dUMP, preventing incorporation of uracil into DNA and providing the substrate
for dTTP synthesis. Similar to other nucleotide binding proteins, dUTPase also
consists of a sequence motif rich in glycine residues known as P-loop motif.
The P-loop motif of the nucleotide binding proteins are involved in substrate
binding, catalysis, recognition and regulation of activity. In dUTPase the
function of the P-loop motif is not well understood. One of the main reasons
for this limited information is the lack of the three-dimensional structure of
a dUTPase enzyme with an ordered Gly-rich P-loop motif with a bound substrate
and Mg2+
ion. This review presents an insight into the role of Gly-rich P-loop motif in
the function of dUTPase as revealed from the crystal structure. The analysis
reveals the Gly-rich P-loop motif of dUTPase to be the longest in terms of its
amino-acid composition as compared to other nucleotide binding proteins and
exhibit a high-degree of sequence conservation among spectrum of species. The
enzyme utilizes adaptive recognition to bind to the phosphate groups of the
nucleotide. In particular, the a-b
phosphodiester bond adopts an unfavorable eclipsed conformation in the presence
of the Gly-rich P-loop motif. This conformation may be relevant to the
mechanism of a-b phosphodiester bond
cleavage.
Back to top] Evolution of the DUT Gene: Horizontal
Transfer between Host and Pathogen in all Three Domains of Life
The ubiquity of the dut gene in
Eukarya, Eubacteria, and Archaea implies its existence in the last common
ancestor of the three domains of life. The dut gene exists as single, tandemly
duplicated, and tandemly triplicated copies. The dUTPase is encoded as an
auxiliary gene in the genomes of several DNA viruses and two distinct lineages
of retroviruses. A comprehensive analysis of dUTPase amino acid sequence
relationships explores the evolutionary dynamics of dut genes in viruses and
their hosts. The data set was comprised of representative sequences from
available Eukaryotes, Archaea, Eubacteria cells and viruses. A multiple
alignment of these protein sequences was generated using a hidden Markov model
(HMM) approach developed to align divergent data. Phylogenetic analysis revealed
that horizontal transfer from hosts to virus genomes has occurred in all three
domains of life. The evidence for horizontal transfers is particularly
interesting in Eukaryotes as these dut genes have introns, while DNA virus dut
genes do not. This implies an intermediary Retroid Agent facilitated the
horizontal transfer process, via reverse transcription, between host mRNA and
DNA viruses. The horizontal transfer of the dut gene from Eukaryotic,
Eubacterial, and Archaeal organisms to both DNA and RNA viruses is the first
documented case of host to pathogen transfer that has occurred in all three
domains of life.
Back to top] Evolution of the dUTPase Gene of
Mammalian and Avian Herpesviruses
Sequences of dUTPases encoded by
Alpha- and Gammaherpesviruses resemble other dUTPases in their possession of
five conserved motifs, but differ in having greater chain lengths (about twice
as long) and in the location of Motif 3 at an N‑terminal location
relative to the other motifs. It was proposed that the herpesvirus gene arose
by intragenic duplication of a standard dUTPase coding sequence and subsequent
loss of one copy of each motif from the double‑length chain, and that the
resulting enzyme was active as a monomer. With knowledge of the trimeric 3D
structure of standard dUTPases, it is possible to suggest transformations that
occurred in evolutionary development of the herpesvirus dUTPase. The distinct
location of Motif 3 can indeed be seen to be consistent with it contributing to
a single intramolecular active site with the other motifs. Separately, the
occurrence in herpesvirus dUTPases of around 20 to 40 additional residues
between Motifs 4 and 5 allows the C-terminal Motif 5 to reach the active site
intramolecularly. The driving force behind these evolutionary changes remains
obscure. We speculate that they may have allowed acquisition of a novel,
presently unknown function by the protein. Consistent with this idea is the
observation that in Alpha- and Gammaherpesvirus dUTPases the original locus of
Motif 3 is occupied by a distinct conserved sequence (Motif 6); perhaps this
element constitutes part of a separate functional capability. Notably, the
apparently orthologous protein in Betaherpesviruses lacks the standard motifs
while Motif 6 is still present.
Back to top] The Nature of Enzymes Involved in Uracil-DNA Repair: Isoform Characteristics of
Proteins Responsible for Nuclear and Mitochondrial
Genomic Integrity
Sal Caradonna
and Susan Muller-Weeks
The absence of uracil from DNA genomes is a consequence of enzyme functions that eliminate intracellular dUTP pools and that purposefully recognize and remove uracil moieties from DNA. These enzymatic functions are dUTP nucleotidohydrolase (dUTPase) and uracil-DNA glycosylase (UDG), respectively. There are distinct nuclear and mitochondrial isoforms of each of these enzymes in human cells. The mitochondrial isoform of dUTPase (DUT-M) begins as a 31 kilodalton precursor protein containing an arginine-rich, amino-terminal presequence required for targeting to the mitochondria. This precursor is processed into a 23 kilodalton protein that resides, in mature form, in the mitochondria. The nuclear isoform of dUTPase (DUT-N) is an 18 kilodalton protein. Both species of dUTPase are nearly identical except for their amino-termini. Analysis of protein expression reveals that DUT-M is constitutive and independent of cell cycle phase or proliferation status of the cell. In contrast, DUT-N protein and mRNA levels are tightly regulated to coincide with nuclear DNA replication. The common sequence for both nuclear and mitochondrial isoforms includes a cyclin-dependent kinase consensus site. However, only the nuclear form appears to be phosphorylated at this site in vivo. Studies on dUTPase genomic organization reveal that both isoforms are encoded by the same gene. Isoform specific transcripts arise through the use of alternate 5' exons.
Uracil-DNA glycosylase (UDG1) is but one of a growing family of enzymes that repairs potentially mutagenic events caused by uracil in DNA. Human cells contain two isoforms of UDG1 which are also nearly identical except for their amino termini. One isoform (UDG1-M), which is constitutively expressed, is targeted to the mitochondria. This form originates as a 35,000 dalton precursor and is N-terminally processed to a mature 29,000 dalton protein as it transits into the mitochondria. The other isoform is targeted to the nucleus and its expression is a function of cellular proliferation status. As with dUTPase, UDG1 isoform specific transcripts arise through the use of alternate 5’ exons. Both of these enzymatic functions are a unique illustration, in humans, of the use of alternate exons to generate differentially expressed proteins targeted to different organelles.
There are questions as to whether the nuclear isoform of UDG (UDG1-N) is also processed (at the N-terminus) to a lower molecular weight form. Polyclonal antisera generated to the unique N-terminal region of this isoform, reveals that UDG1-N exists as a 36,000 dalton protein in human cell nuclei. Since the epitope for this antibody resides in the first 24 amino acids of UDG1-N, it is apparent that the majority of this isoform is not processed and retains its amino terminus. Evidence also indicates that UDG1-N exists as a serine/threonine phosphoprotein and that phosphorylation occurs in the unique N-terminal region. This was initially deduced from the observation that nuclear UDG1-N migrates as multiple bands on SDS-PAGE and as a single band subsequent to phosphatase treatment. Cdc2 kinase is at least one of the enzymes that can phosphorylate UDG1-N.
This review will summarize the
current information on isoform characteristics of both dUTPase and uracil-DNA
glycosylase. It will also focus on evidence for phosphorylation and speculate
as to the purpose of these post-translational events.
Back to top] dUTPase in Human Neoplastic Cells as a Potential
Target for Therapeutic Intervention
Friedrich A. Grässer,
Bernd F. M. Romeike, Gerald Niedobitek, John Nicholls and Elisabeth Kremmer
With the exception of brain, most
human tissues analysed contain dUTPase protein detectable by
immunohistochemistry. Non-dividing tissues like untreated peripheral blood lymphocytes
(PBL’s) contain basal levels of cytoplasmic dUTPase and express additional,
nuclear dUTPase upon mitogenic stimulation. Normal, proliferating tissues like
intestinal mucosa or germinal centres within tonsils contain cytoplasmic as
well as nuclear dUTPase in accordance with a proposed role for dUTPase during
cell division. Notably, no dUTPase is detectable during mitosis. The failure to
stain dUTPase in normal brain tissue by immunohistochemistry while mRNA is
readily detectable by Northern blotting cannot be explained at this moment.
Epithelial tumours, such as adenocarcinoma of the lung, breast, colon, vulva or
nasopharynx contain cells which are either positive for both or only one of the
subcellular forms of the dUTPase and show variable numbers of dUTPase-positive
cells. High levels of dUTPase correlate with a poor prognosis regarding the
progression of colorectal carcinoma. Of the intracranial tumours tested,
neuroepithelial tumours show almost exclusively nuclear expression whereas
meningiomas of higher grades of malignancy (WHO grade II and III) also contain
cells with additional cytoplasmic dUTPase. The dUTPase is detectable in other
malignancies including tumours derived from lymphatic tissues like Burkitt’s
lymphoma or non-Hodgkin’s-lymphoma. The downregulation of dUTPase protein
during apoptosis or the inhibition of dUTPase during nerve cell development in
Drosophila melanogaster suggests a possible role of the enzyme during
apoptosis. In line with these observations, inhibition of dUTPase by antisense
in p53-deficient tumour cells hints at a possible route of treatment of
p53-deficient tumours which are otherwise resistant to therapies like
irradiation. The expression of the enzyme in normal tissues indicates that
sublethal levels of dUTPase inhibitors may also exert an unwanted mutagenic
effect.
Back to top] The Role of dUTPase and Uracil-DNA Repair in Cancer
Chemotherapy
Thymidylate metabolism is an important target for chemotherapeutic agents that combat a variety of neoplastic diseases including head and neck, breast and gastrointestinal cancers. Therapeutic strategies applied to this pathway target the thymidylate synthase (TS) reaction that catalyzes the reductive methylation of deoxyuridylate (dUMP) to form thymidylate (TMP). This reaction represents the sole de novo source of TMP required for DNA replication and repair. Inhibitors of this pathway include the widely utilized fluoropyrimide and antifolate classes of anti-cancer agents. Studies attempting to elucidate the molecular mechanisms of cell killing mediated by inhibitors of the TS reaction suggest that cytotoxicity results from a process known as “thymineless death”. This term describes the extreme TTP pool depletion observed following TS inhibition. Although depletion of TTP pools is clearly involved in this process, there is now considerable evidence implicating aberrant uracil-DNA metabolism as an important mechanism of toxicity. Upon TS inhibition, dUTP pools may accumulate, inducing repeated cycles of uracil misincorporation into DNA and repair-mediated DNA damage. Central to the uracil-misincorporation pathway are the enzymes deoxyuridine nucleotidohydrolase (dUTPase) (EC 3.6.1.23) and uracil-DNA glycoslyase (UDG) (EC 3.2.2.3). dUTPase catalyzes the hydrolysis of dUTP to form dUMP and pyrophosphate thereby eliminating dUTP and preventing its utilization by DNA polymerases during replication and repair. UDG initiates the base excision repair pathway effectively removing any uracil residues that may arise in DNA. Under normal conditions, uracil is precluded from DNA by the combined actions of dUTPase and UDG. However, during TS inhibition, dUTP pools may accumulate and overwhelm dUTPase, resulting in repeated cycles of uracil misincorporation and detrimental repair leading to strand breaks and cell death. Because dUTPase plays a pivotal role in regulating cellular dUTP pools, this enzyme could have profound effects on the efficacy of agents that target thymidylate biosynthesis.
This article reviews our current
understanding of the role of aberrant uracil-DNA metabolism as a contributing
mechanism of cytotoxicity initiated by chemotherapeutic agents that target de
novo thymidylate metabolism. The role of dUTPase expression in modulating
therapeutic response is presented including evidence from yeast and mammalian
cell culture models and clinical studies. The regulation of human dUTPase
isoforms in normal and neoplastic tissues will be reviewed as well as the role
of dUTPase expression as a prognostic marker for overall survival and response
to therapy in colon cancer.
Back to top] The Herpesvirus Encoded dUTPase as a Potential
Chemotherapeutic Target
A.W.
Studebaker, G.K. Balendiran and Marshall V. Williams
The human herpesviruses are a
well characterized group of viruses that are responsible for a wide spectrum of
human diseases. Included in this group of pathogens are the alphaherpesviruses
(herpes simplex types 1 and 2 and varicella-zoster virus), the
betaherpesviruses (cytomegalovirus, human herpesvirus types 6 and 7) and the
gammaherpesviruses (Epstein -Barr virus and human herpesvirus 8). An important
feature of these viruses is that they cause latent infections that can be
reactivated to cause disease. The herpesviruses encode for a large number of
structural and non-structural proteins, and several of the non-structural
proteins, such as thymidine kinase, DNA polymerase, and ribonucleotide
reductase, have been utilized as targets for the development of
anti-herpesvirus agents. Another herpesvirus encoded enzyme that has received
little attention as a potential target for the development of specific
anti-herpesvirus agents is deoxyuridine triphosphate nucleotidohydrolase
(dUTPase). Furthermore, little is known concerning the role of the
herpesviruses’ encoded dUTPases in virus replication and in modulating the
chemotherapeutic efficiency of other anti-herpes agents. Because of recent
advances in molecular virology and biochemistry, it is now possible to
rationally develop “designer” drugs based upon the structural/functional
interaction of the drug with a specific viral protein. The purpose of this
review is to describe previous studies demonstrating the potential use of the
herpesvirus encoded dUTPase as a drug target, to describe problems associated
with using the dUTPase as a target and to discuss new approaches that can be
used.
Back to top] The Role of Retroviral dUTPases in Replication and
Virulence
Susan L. Payne
and John H. Elder
Several retroviruses, including
equine infectious anemia virus (EIAV), visna virus, caprine
arthritis-encephalitis virus (CAEV) and feline immunodeficiency virus (FIV)
encode dUTPase. The role of this enzyme in the replication of these viruses has
been scrutinized, with particular emphasis on potential roles for dUTPase in
virulence and viral mutation rate. Overall, the results of these studies have
indicated a central role for dUTPase in facilitating productive viral
replication in non-dividing cells. The requirement for dUTPase in EIAV, which
replicates exclusively in macrophages, may be the most stringent. Studies of
dUTPase mutants of virulent EIAV clones suggest that the enzyme is a major
determinant of virulence. In contrast, FIV readily replicates in dividing cell
populations such as CD4+ and CD8+ T cells, and B cells as well as in
non-dividing macrophages. Thus, the virus burden and disease sequelae are
lowered in cats infected with a dUTPase-minus FIV relative to cats infected
with wild type FIV, but not totally abrogated. Growth in macrophages is
attenuated with the DU-minus FIV with evidence of a 5 to 8-fold increase in G®A
transition mutations in viral integrants present in macrophages. These findings
are consistent with an increase in uracil misincorporation in the absence of
dUTPase, resulting in transition mutations that cripple the virus. Effects on
virus replication and disease production have also been noted for
dUTPase-deleted CEAV and visna virus. While HIV and SIV do not encode dUTPase
some reports suggest that other viral and host cell factors may substitute for
its activity. Betaretroviruses also encode dUTPase and while several of these cause
significant disease, the role of dUTPase in their replication and pathogenesis
is currently unknown.
Back to top] Trypanosomal dUTPases as Potential Targets for Drug
Design
Fernando
Hidalgo-Zarco and Dolores González-Pacanowska
Parasites of the Trypanosomatidae
family are responsible for diseases that afflict several million people
worldwide. Currently there is an urgent need for new drugs against these diseases
and an approach to drug discovery is the study of biochemical and structural
properties of a potential target and the subsequent design of specific
compounds. Trypanosomatid genes coding for enzymes which distinctively
hydrolyze dUTP have been isolated by genetic complementation in Escherichia
coli mutants defective in dUTPase activity. An analysis of these sequences from
Leishmania major and Trypanosoma cruzi showed that no significant similarity
could be established with the family of known dUTPases and that the five
consensus motifs were absent. However, limited similarity was identified for
three motifs present in an enzyme related in function the dCTPase-dUTPase from
T phages and 35% identity with a putative dUTPase identified in the eubacteria
Campylobacter jejuni. T. cruzi and L. major dUTPases were highly similar and
catalyzed in a specific fashion the hydrolysis of dUTP. A detailed kinetic
study of both enzymes revealed that dUDP is also an efficient substrate of the
enzyme while other nucleotides are poorly hydrolyzed. The enzyme is essential
for viability in Leishmania and is up-regulated by inhibitors of dTMP
synthesis. Thus, a new family of dUTPases might exist in certain organisms that
bear no sequence or structure similarity with eukaryotic enzymes accomplishing
the same function and that may constitute potential drug targets for the
development of specific inhibitors.