(Received for publication, October 19, 1994; and in revised form, December 16, 1994)
From the
The cell cycle-dependent transcription factor, E2F-1, regulates
the cyclin-like species of the DNA repair enzyme uracil-DNA glycosylase
(UDG) gene in human osteosarcoma (Saos-2) cells. We demonstrate,
through the deletion of the human UDG promoter sequences, that
expression of E2F-1 activates the UDG promoter through several E2F
sites. The major putative downstream site for E2F, located in the first
exon, serves as a target for E2F-1/DP1 complex binding in
vitro. We also provide evidence for the functional relationship
between the cyclin-like UDG gene product and E2F. High levels of UDG
expression in a transient transfection assay result in the
down-regulation of transcriptional activity through elements specific
for E2F-mediated transcription. Overexpression of UDG in Saos 2 cells
was observed to delay growth late in G phase and
transiently arrest these cells from progressing into the S phase. This
hypothetical model integrates one mechanism of DNA repair with the cell
cycle control of gene transcription, likely through E2F. This
implicates E2F as a multifunctional target for proteins and enzymes,
possibly, responsive to DNA damage through the negative effect of UDG
on E2F-mediated transcriptional activity.
The gene for a novel uracil-DNA glycosylase
(UDG)()has recently been cloned and characterized by DNA
sequence analysis(1) . The enzyme activity of uracil-DNA
glycosylase includes the hydrolysis of the N-glycosyl bond
from a uracil residue incorporated into the DNA strand to produce a
free base(2) . The subsequent apyrimdinic site is cleaved by
specific endonucleotidyl activity with the resulting gap filled by DNA
polymerase
(for review see (3) ). The regulation and
structural relationship of the nuclear UDG gene product to the cyclin
family of cellular factors share many common characteristics (4) . The cell cycle-dependent expression of UDG parallels that
of several cyclin and associated cdk gene products(4) . The
entire structural gene for UDG has also been characterized, with the
limits of promoter activity defined by deletion analysis of the 5` end
of the structural gene(4) . Implication that cell cycle
regulation of UDG gene transcription is mediated by E2F is supported by
the presence of several putative E2F cognate sequences dispersed
throughout the promoter region(4) .
The recent
characterization of events that occur during the cell cycle has
implicated E2F as a central mediator of transcriptional control. E2F
was initially identified as a cellular factor mediating gene
transcription of the adenovirus E2 early gene(5) . Since then,
many studies have provided evidence for the direct role of E2F as a
cellular factor required for the activation of genes necessary for the
progression into the S phase of the cell cycle(6, 7) .
The activity of E2F is mediated by the direct interaction with the
wild-type retinoblastoma susceptibility gene product (Rb) (8) and the Rb-related gene product, p107(9) . The
transcriptional activity of E2F is regulated through the interactions
with the Rb gene product and p107 in a cell cycle-restricted
manner(10, 11) . These interactions complex E2F with
other cell cycle-mediated factors, cyclins A and E as well as
cyclin-dependent kinase, cdk
2(12, 13, 14, 15, 16) . It
is considered that in the absence of normal Rb function unsequestered
E2F activity goes unchecked and, therefore, allows unrestricted
activation of many genes required for cell proliferation. This includes
many putative protooncogene products and cytokines responsible for cell
transformation (i.e. c-myc, c-myb, transforming growth factor
1(17) . Similarly, a mechanism for the deregulated
activity of E2F apparently contributes to the oncogenic potential
associated with many DNA tumor
viruses(18, 19, 20) . Gene products of many
DNA tumor viruses can actively compete for the same binding domain of
the Rb gene product as E2F. Therefore, this inhibition of E2F binding
to Rb results in free E2F and subsequent activation of many genes
responsible for cell growth(5) .
Recently, specific E2F activity has been cloned by the ability of the recombinant expression of cDNAs to interact with the Rb protein (21) and identifying one novel cDNA encoding E2F-1(22, 23, 24) . The identification of the E2F-1 gene has made available an approach to study the activation of suspect target genes by E2F-1. Despite the identification of one E2F-specific gene product, recent evidence to suggest that E2F exists as a multiple heteromeric complex has been established by the cloning and characterization of additional members of the E2F family of cellular transcription factors (25, 26) including the heterodimeric partner of E2F-1, DP1(27, 28) .
Here we take advantage of the cloned E2F-1 protein to examine the role E2F has in transactivating the transcription of a novel cell cycle-regulated DNA repair enzyme. We demonstrate that putative E2F sites, localized within the UDG promoter, contribute to the transactivation of UDG gene transcription and that the major site for E2F transactivation is localized in an E2F consensus sequence within the first exon. In addition, we have identified this sequence as a specific target for E2F-1/DP1 binding in vitro. Because of the closest relative homology of this nuclear species of UDG to the cyclin A protein, we tested the possibility that this UDG gene product modulates E2F-1 activity through an E2F-responsive target. Although the precise cellular connection of cyclin A-cdk 2 and E2F is still not clearly established, the interaction between cyclin A-cdk2 and E2F has been recently shown to have a negative role on E2F-mediated function (29) . This is likely an important component for the progression of mitotic events. We have demonstrated here the ability of this nuclear ``cyclin-like'' species of UDG to repress the activity of E2F-mediated transcription in a cotransfection assay. The likelihood of cellular factors involved in DNA repair, possibly analogous to the role cyclins may have in mediating the transcriptional activity of E2F, may represent alternative pathways regulating E2F function, thereby competing for the control of cell cycle events necessary for the restoration of damaged DNA.
A major focus in several recent genetic and biochemical studies is the intersecting relationship between DNA repair and transcriptional control. Many proposed mechanisms have suggested that common factors associated with both DNA repair and gene transcription are functionally related(30) . In addition, the role that the tumor suppressor gene product p53 has in both transcriptional control and response to damaged DNA (31) is unique in our understanding of the complex pathways that integrate gene transcription with specific factors necessary for DNA repair. We suggest one mechanism that implicates E2F-mediated transcription in modulating a cellular factor involved in DNA repair.
Plasmid expression vectors for E2F-1 and UDG were
generated by subcloning cDNA coding region fragments into the
expression vector pcDNA3 (Invitrogen). The E2F-1 and
UDG plasmid cDNAs were generously provided by William Kaelin
(Dana-Farber Cancer Center, Boston, MA) and Sal Caradonna (University
of Medicine and Dentistry of New Jersey, Stratford, NJ), respectively.
The plasmids provided appropriate DNA fragments to subclone both the
E2F-1 and UDG cDNAs into pcDNA3. Expression vector pCMV/E2F-1 was
generated by digesting the E2F cDNA with BamHI and EcoRI subcloned unidirectionally into the BamHI/EcoRI-treated pcDNA3 plasmid. The expression
vector pCMV/UDG
was similarly subcloned by insertion of the UDG
cDNA EcoRI fragment into the EcoRI site of pcDNA3.
The orientation and complete promoter sequence of the reporter minigene
constructs were confirmed by nucleotide sequence analysis using
Sequenase
II kit (United States Biochemicals Corp.,
Cleveland, OH) according to the manufacturer's instructions. The
cloning of E2F-1 and UDG cDNA inserts into expression vector pcDNA3
were also verified the complete cDNA sequence by the dideoxynucleotide
chain termination method of DNA sequencing also using
Sequenase
II kit.
The template for the PCR was
plasmid UDG-construct-D/wt. The 239-base pair PCR product was digested
with ApaI and XbaI and subcloned back into the
backbone fragment to replace the wild type ApaI/XbaI
fragment of UDG construct-D/wt with the mutation to generate minigene
UDG construct-D/mut. All the mutations were confirmed by chain
termination method of DNA sequencing using Sequenase II
according to the manufacturer's instructions.
Semi-confluent Saos 2 cells were
cotransfected with either 15 µg of expression plasmid pCMV/UDG
or pCMV/0 along with 5 µg of the B-cell surface antigen CD19 cDNA (35) expression plasmid, pcd19-hcr, which were all
directed by the CMV early gene promoter. The cotransfected cells were
cultured for 48 h in 5% CO
at 37 °C. Immunocytochemical
rosettes were formed of cells transiently expressing the B
cell-specific CD19 cell surface antigen using an anti-CD19 antibody
conjugated to a magnetic bead according to the manufacturer's
instructions (Dynal, Inc., Lake Success, NY). Cotransfected Saos2 cells
were enriched for the CD19
phenotype using the
magnetically susceptable beads and detached from the beads according to
manufacturer's directions. Cotransfected Saos 2 cells, positively
selected for CD19, were then analyzed by flow cytometric procedures.
Figure 1: Mapping of E2F-1-mediated transcription in the human UDG promoter. A, schematic illustration of a series of human UDG promoter deletion constructs fused to the CAT reporter gene. The top schematic is used as reference to indicate the position of the primary transcriptional start postion (4) and first exon relative to the promoter construct tested. Numbers below each promoter/reporter construct denote the position of the UDG promoter end points at both the 5` and 3` ends, respective of this transcriptional initiation site. The solid boxes indicate the postion of each putative E2F consensus site, and hatched boxes represent the position of putative Sp1 sites within the promoter region. Open box with an X indicates a mutation directed within an E2F consensus site. B, transcriptional effect of recombinant E2F-1 on wild type and mutant gene promoter constructs. Shown is an assay used to detect chloramphenicol acetyltransferase activity used to measure transcriptional activity mediated by a cotransfected E2F-1 expression vector. The UDG promoter constructs, fused to the CAT reporter gene, were cotransfected each with either the E2F-1 expression vector pCMV/E2F-1 or negative control vector pCMV/0. CAT assays were performed and CAT gene activity verified by an autoradiograph of the thin layer chromatograph shown.
To identify the boundaries of E2F-1 activity within the promoter, several deletions of the UDG gene promoter were constructed as reporter minigenes to account for putative E2F and Sp1 sites. 5` deletions, designated as UDG constructs-B, -C, -D/wt, -D/mut, and -F, omitted sites specific for the presumed recognition by E2F and Sp1 transcription factors (Fig. 1). These deletions extend from the 5` side of transcriptional initiation. UDG construct-E was a deletion mutant derived from UDG construct D/wt, extending from the 3` side of transcription (Fig. 1).
Minigene constructs containing deletions generated from the 5` end of the UDG promoter were cotransfected with the E2F-1 expression vector shown in Fig. 1. Reporter gene expression from the 5` deletion mutants demonstrate the relative stimulation resulting from each of the putative E2F and Sp1 sites. The CAT activities were measured with values (shown in Fig. 2) proportional to the presence of putative E2F targets extending from the 5` end. In each set of cotransfection experiments, a negative control was performed in parallel with cotransfections performed with the E2F-1 expression vector pCMV/E2F-1. Cotransfections using the parental negative control vector pCMV/0 allowed us to access the stimulation by E2F-1. The stimulation by E2F-1 was quantitated relative to cotransfections performed with pCMV/0 in each of the reporter minigene plasmids shown (Fig. 1). Results from this experiment represent the level at which E2F-1 can transactivate the individual minigene reporter constructs. Although cotransfection of these deletion mutants with the E2F-1 expression vector indicate the relative contribution of these sites in E2F-mediated transactivation of the UDG promoter (Fig. 1), the basal levels of CAT activity are also proportional to the length of the UDG promoter. This is demonstrated by the negative control expression vector pCMV/0 cotransfected with each of the deletions extending from the 5` end. Values presented as histograms (Fig. 2), indicating the relative activity of each minigene construct, show the relative contribution of each E2F site to E2F-1-mediated stimulation of UDG gene transcription. Deletion of the proximal Sp1 site reduces the activity of E2F-1-mediated transcription of the UDG promoter approximately 4-fold (Fig. 2). It is therefore consistent with other genetic models indicating the importance of Sp1 in basal stimulation and start site selection of gene transcription(38, 39) . Although the activity of this Sp1 site provides some evidence that Sp1 may synergistically costimulate E2F-1 transcriptional activity, the function of Sp1 on E2F-1 activity is likely through a basal mechanism. The role of Sp1 in UDG gene transcription remains to be further elucidated.
Figure 2: Comparative quantitation of E2F-1-mediated induction of UDG gene transcription. The histogram evaluates, comparatively, the levels of E2F-1 stimulation of transcription mediated by each of the promoter constructs tested. The graph represents the percent conversion to an acetylated form of chloramphenicol in each cotransfection performed with either pCMV/E2F-1 or the negative control pCMV/0. Numerical values, adjacent to the graph, indicate the degree of stimulation mediated by the transient expression of E2F-1 when compared to the negative control signal. The -fold stimulation of reporter gene activity was calculated by the amount of acetylation (percent) from cells transfected with pCMV/E2F-1 divided by the amount of acetylation in cells transfected with the negative control vector pCMV/0. Internal control plasmid directing the expression of the reporter human growth hormone was included to standardize transfection efficiency for each experiment performed. The ± indicates the standard error from four separate transfection experiments performed with two separate preparations of plasmid DNA.
Site-specific mutation of the UDG promoter within the core downstream of the putative E2F target was converted from the wild type 5`-TTCGCGAAAGCTTTCCCGGTT-3` to the mutant sequence 5`-TTCGCGAGAGCGCTCCCGGTT-3`. The strategy for site-directed mutagenesis was developed to destroy the affinity of E2F-specific binding to the UDG promoter at the most proximal E2F site relative to transcriptional initiation. This is consistent with previous experiments that examine the sequence-specific affinity of DNA binding by cellular E2F(40) . Cotransfections performed with pCMV/E2F-1 and the reporter minigene UDG construct-D/mut reduce vitrually all of the stimulation resulting from the transient expression of E2F-1 ( Fig. 1and Fig. 2). The site-specific mutagenesis of the UDG human promoter accounts for the majority of transactivation provided by E2F-1. Although these results do not completely account for the degenerated consensus of E2F binding, this report indicates that this triple mutation within the E2F core element is sufficient for destroying the majority of E2F-1-mediated transactivation.
Figure 3:
The E2F
complex recognizes an E2F consensus within the human UDG promoter. A
putative E2F site, located immediately downstream of transcription,
requires the heterodimer of E2F-1 and DP1 for efficient DNA binding. A, nuclear extracts prepared from Saos 2 cells, synchronized
in G, were used in an EMSA experiment to detect a specific
DNA-E2F complex. A double-stranded oligonucleotide probe, corresponding
to the region of the UDG promoter between +7 to +35, was
labeled with
P and incubated with 5 µg of nuclear
extract. All binding reactions were preincubated with nonspecific
competitor DNA (poly (dI-dC)). Gel shift analysis was also performed in
the presence of specific competitor DNA corresponding to the E2F
recognition site of the adenovirus E2 promoter (shown in the last
lane). Prior to the addition of the
P-labeled
oligonucleotide, the E2F competitor sequence from the adenovirus E2
promoter was added to the nuclear extract in 100-fold excess (relative
to the labeled oligonucleotide probe) and assayed by band mobility
shift analysis. B, SDS-PAGE, following the affinity
purification of bacterially generated malE/DP1 and GST/E2F-1
fusion proteins, was stained with Coomassie Blue stain and
photographed. Molecular mass markers adjacent to samples are indicated
in kilodaltons. C, EMSA of bacterially produced and
affinity-purified E2F-1 and DP1 fusion gene products, individually and
together, were used to analyze DNA binding to the wild type and mutant
E2F consensus elements from the UDG promoter (``Materials and
Methods''). Oligonucleotide competitor DNA, corresponding to the
E2F target of the adenovirus E2 gene promoter was used in the last
lane.
Figure 4:
Abundant overexpression of the uracil-DNA
glycosylase gene product. Immunoblot analysis of cellular extracts
prepared from enriched CD19 Saos 2 cells 48 h
following the cotransfection with a CD19 expression vector along with
either pCMV/0 or pCMV/UDG
, respectively. Approximately 4
10
Saos 2 cells, selected for the expression of
CD19
cells after cotransfections, were analyzed for
the expression of recombinant UDG. Each of three individual
cotransfections were were pooled together, then lysed, and cleared of
cellular debris. Twenty-five µg of cellular protein in each lane
were separated by SDS-PAGE and transferred to polyvinylidene difluoride
membranes. Immunoblots were performed using a primary monoclonal
antibody, mAb-u(91-243), directed against an internal cyclin-like
domain (Thr
to Ile
) of UDG. Prestained
molecular weight protein markers were used to predict the size of the
proteins detected.
Figure 5: UDG expression down-regulates human UDG gene promoter activity at an E2F site. A, examination of CAT activity mediated by a transiently expressed E2F-1 cDNA on a wild type and mutant E2F consensus element, using the UDG gene promoter target to direct expression of the CAT gene. Human UDG promoter constructs, UDG constructs-D/wt, and UDG construct-D/mut were previously shown (see also Fig. 1A). The effector plasmid expression vector pCMV/E2F-1 contains the E2F-1 cDNA. The parental minus expression vector, pCMV/0, was used as a negative control in each experimental set. Cotransfections were performed with effector expression vector and target minigene reporter (as shown). Included in each cotransfection performed was an internal control to standardize the transfection efficiency. CAT activity was evaluated as percent acetylated radiolabeled chloramphenicol as indicated by the histogram. Solid box represents the wild type E2F sequence. Open box with an X represents a mutated E2F consensus element. Open box indicates the presence of a Sp1 consensus located directly upstream of the E2F target. Standard errors were calculated from the results of five individual transfection experiments performed with two separate plasmid DNA preparations. Standard error is indicated by the error bars. B, examination of CAT activity mediated by a transiently expressed UDG cDNA on a wild type and mutant E2F consensus element, using the UDG gene promoter target to direct expression of the CAT gene. CAT activities were comparatively measured to indicate the relative amount of acetylation between each experimental set.
Figure 6:
Expression of UDG can down-regulate a
heterologous promoter in Saos 2 cells. A, schematic of the
reporter minigene construct used in the cotransfection experiment. The
adenovirus E2 promoter was used as a target for the expression of UDG,
contains E2 promoter sequences extending from -408 to + 7
relative to the site of transcription fused to the bacterial CAT gene.
Nucleotide sequence indicates the E2F protein binding consensus located
within the adenoviral E2 gene promoter. B, cotransfections
were performed using the adenovirus E2 promoter target along with
either the UDG expression plasmid, pCMV/UDG, or the control
plasmid, pCMV/0. CAT activity was measured by the phase-extraction
method and quantitated. Relative CAT activity is presented as percent
acetylation. The standard error was calculated from eight separate
transfections using two separate plasmid DNA preparations. The standard
error is represented by the error bars shown in the
histogram.
Examples of DNA repair have recently been shown to overlap
with mechanisms of transcriptional
control(46, 47, 48, 49, 50) .
The gene for a novel cyclin-like uracil-DNA glycosylase that encodes a
predicted 36-kDa cell cycle-regulated DNA repair enzyme has been
previously reported(4) . Here we have demonstrated the
relationship of the E2F-1 transcription factor to transcriptional
regulation of the UDG promoter. We have localized the sequences
responsive to E2F-1 within the UDG promoter. The regulation of UDG by
the cell cycle-dependent activity of E2F-1 provides evidence for
converging mechanisms between transcription, DNA repair and cell cycle
events leading to DNA replication. Here, we have tested the potential
for the cyclin-like species of UDG itself to be involved in
transcriptional control through an E2F-mediated regulatory element.
Recent evidence to support the role(s) of E2F-1 as an important
regulator of many cell cycle events, including apotosis, may be an
example of the ability of E2F-1 to overcome cell cycle checkpoints
mediated by both Rb and p53(51) . Thus, these results implicate
one species of UDG in the alteration of E2F-mediated transcription.
This raises the possibility that pathways that integrate
transcriptional control with cell cycle events, through E2F, may also
integrate cellular factors involved in the response to and repair of
damaged DNA. Indication of the involvement of E2F-1 in the regulation
of UDG gene transcription documents a mechanism likely involved in the
cell cycle-dependent expression of human cyclin-like UDG. Although this
does not preclude the role of other transcription factors in the cell
cycle regulation of the UDG gene, including Sp1, we suggest that E2F
may require interactions with other factors that equally contribute to
the cell cycle-dependent regulation of UDG. In surprising contrast to
our expectations, recent experiments conducted in this laboratory
suggest that transient expression of Rb significantly contributes to
the overall stimulation of UDG gene transcription through a combined
Rb/Sp1-responsive element located upstream of the UDG promoter. ()Recently, it has been shown that both Rb and Sp1 can
converge on a specific 5`-CCACCC-3` nucleotide motif in the IGF-II gene
promoter and results in the stimulation of IGF-II gene
transcription(53) . Despite the role Rb has in exerting an
inhibitory effect on E2F-1-mediated transcription by interacting with
E2F directly(10) , the complexity of the UDG promoter may
warrant contrasting and competing signals for Rb interaction. Thus, it
is possible that the mechanism for the positive regulation of UDG may
separately include both Rb and E2F-1. This model could hypothetically
account for the rapid requirements for transient UDG expression through
signals associated with detection of damaged DNA to initiating a delay
in the progression of the cell cycle by simultaneously incorporating
the activities of Rb, Sp1, and E2F-1 by competing for the affinity of
Rb.
We demonstrated that one physiological result of the
constitutive overexpression of exogenous cyclin-like species of UDG is
the accumulation of Saos2 cells delayed in late G of the
cell cycle. This following several hours after the transfection of a
UDG expression vector is shown in Table 1. It is interesting that
transient overexpression of UDG only delays the growth kinetics and
then follows in a pattern of synchronized entry into S phase of the
cell cycle (Table 1), suggesting that this process delays
progression of the cell cycle and is only temporary, possibly
superseded by more dominant mechanisms for control of the cell cycle
kinetics. Although the precise mechanism for this result has yet to be
defined, it is suspected that the cyclin-like homology of this UDG
species may be ``in effect'' a functional component of UDG,
possibly similar to that of a cyclin. Due to the vital role E2F may
have in the progression of the replicative phase of the cell
cycle(51) , one strategy to try to account for the potential of
UDG to alter the progression of the cell cycle was to examine the
effect of an abundantly transiently expressed UDG protein on
E2F-mediated transcription. The results of these experiments indicate
that E2F-mediated transcription is down-regulated by the high levels
and exogenous expression of UDG ( Fig. 5and Fig. 6). This
repression occurs through a mapped E2F target located proximal to the
transcriptional start site (Fig. 5). The apparent regulation of
UDG gene transcription by its own gene product may be analogous to that
seen by regulation of the Rb gene by the Rb gene product (54) through interactions with E2F-1. Despite the similarities
between UDG and cyclin protein kinase A to down-regulate E2F-1-mediated
transcription as recently described(29) , there are significant
differences in the timing of these activities during the cell cycle. (
)Therefore, we do not know nor do we suggest that the
individual activities demonstrated by UDG or cyclin A on E2F-1-mediated
function occur by similar biochemical mechanisms. Also, there is no
evidence in this report to suggest the cellular role of UDG to be
analogous to the function of Rb in vivo. We believe the
restricted cell growth resulting from abundant UDG expression may come
as a result of biochemical interactions between UDG and E2F-1, but have
no direct evidence for this interaction at the present time. It is also
possible that the growth inhibitory function of UDG may also be a
component of Rb or related Rb-like nuclear complexes, but this has not
been fully evaluated. We have examined the activity between UDG and
E2F-1-mediated transcription in the absence of normal Rb activity. This
defect in Rb function is a characteristic phenotype of osteosarcoma
Saos 2 cells, due to a mutation in the growth inhibitory domain of
Rb(55) . Despite the absence of a normal Rb gene product
observed in the experiments performed in this report, it may be
interesting to see if the p53 tumor suppressor gene product (51) could potentiate the down-regulation of E2F
transcriptional activity mediated by UDG.
We should note that
expression of the UDG cDNA in Saos 2 cells seems to produce a
marginally smaller UDG gene product (Fig. 4) from that
previously predicted and described(4) . We have yet to account
for this slight difference from the predicted molecular weight and
currently remains unexplained. In addition, overexpression of this cDNA
product in mammalian cells has not resulted in any increase in
enzymatic activity associated with uracil-DNA glycosylase, nor have we
been able to copurify any specific UDG enzyme activity from any
complexes associated with p107, Rb, or E2F. ()Excluding the
possibility of any cross-reaction with another antigen, it is
conceivable that the detection of this antigen may represent either a
modified enzymatically inactive form of UDG or is an alternative gene
product. We suspect that the activity of E2F-1, potentially mediated
through physical interactions with UDG, may preclude DNA enzymatic
activity of UDG and could indicate the expression of an altered or
processed protein encoded by a UDG gene. Additionally, the notion that
activities expressed by UDG may represent different enzymatic and
cellular functions through the selective expression of distinct domains
of the UDG protein could be supported through the previous examination
of the 37 kDa Ref-1 protein(52) . It was shown that Ref-1 can
mediate AP1 DNA binding and transcriptional activities through the
enzymatic reduction of a conserved cysteine residue required for DNA
binding of both the Fos and Jun transcription
factors, as well as encode specific AP endonuclease activity through
separate and non-overlapping domains of the Ref-1 protein(56) .
Through this modeled analogy, we speculate that the enzymatic
mechanisms employed by UDG may remotely resemble that of the Ref-1
protein. We intend to map the domain(s) of UDG responsible for the
growth-delayed phenotype to determine if this is linked, in any way, to
both E2F-mediated transcription and enzymatic DNA repair function.