Download Specific inhibition of DNA polymerase (3 by its 14 kDa domain: role

Nucleic Acids Research, 1995, Vol. 23, No. 9
1597-1603
Specific inhibition of DNA polymerase (3 by its 14 kDa
domain: role of single- and double-stranded DNA
binding and 5-phosphate recognition
Intisar Husain, Bradley S. Morton, William A. Beard1, Rakesh K. Singhal1, Rajendra
Prasad1, Samuel H. Wilson1 and Jeffrey M. Besterman*
Department of Cell Biology, Glaxo Research Institute, Research Triangle Park, NC 27709, USA and 1 Sealy
Center for Molecular Science, University of Texas Medical Branch, Galveston, TX 77555-1068, USA
Recieved November 30, 1994; Revised and Accepted March 20, 1995
ABSTRACT
DNA polymerase p (P-polymerase) has been implicated
in short-patch DNA synthesis In the DNA repair
pathway known as base excision repair. The native 39
kDa enzyme is organized into four structurally and
functionally distinct domains. In an effort to examine
this enzyme as a potential therapeutic target, we
analyzed the effect of various p-polymerase domains
on the activity of the enzyme in vitro. We show that the
14 kDa N-terminal segment of p-polymerase, which
binds to both single- and double-stranded DNA, but
lacks DNA polymerase activity, inhibits p-polymerase
activity In vitro. Most importantly, the 8, 27 and 31 kDa
domains of p-polymerase do not Inhibit p-polymerase
activity, demonstrating that the inhibition by the 14 kDa
domain is specific. The inhibition of p-polymerase
activity in vitro is abolished by Increasing the concentrations of both of the substrates (template-primer
and deoxynucleoside triphosphate). In contrast, an In
vitro base excision repair assay is inhibited in a
domain specific manner by the 14 kDa domain even in
the presence of saturating substrates. The inhibition of
P-polymerase activity by the 14 kDa domain appears
specific to p-polymerase as this domain does not
inhibit either mammalian DNA polymerase a or Escherlchia coll polymerase I (Klenow fragment). These
data suggest that the 14 kDa domain could be used as
a potential inhibitor of intracellular p-polymerase and
that it may provide a means for sensitizing cells to
therapeutically relevant DNA damaging agents.
INTRODUCTION
Many cancer chemotherapeutic agents cause direct damage to
DNA and this damage is often responsible for the cytotoxicity of
these agents. The majority of the DNA lesions are repaired by
various DNA repair mechanisms inside the cell (1-3). Unrepaired lesions appear to be responsible for the cytotoxicity and
efficacy of chemotherapeutic agents. Moreover, a lack of
* To whom correspondence should be addressed
response to irradiation or chemotherapeutic agents may be a
consequence of increased DNA repair capacity (4). Specific
inhibitors of critical DNA repair enzymes could, therefore, be
used to potentiate the cytotoxicity of existing chemotherapeutic
agents. Indeed, cells from patients with repair deficiency
syndromes are hypersensitive to radiation and various DNA
damaging agents (5-7).
In base excisionrepair,mismatchrepair,and nucleotide excision
repair of DNA damage, the repair process is a sequential
multienzyme event (2,3). Following damage and excision, the
re-synthesis of the nucleotide sequence is catalyzed by a DNA
polymerase before the nick is sealed by DNA ligases. Therefore,
DNA polymerases play a critical role in each DNArepairpathway.
Mammalian cells contain five known DNA polymerases: a, P,
y, 8, e (8). Two mammalian DNArepairsystems have been shown
to require P-polymerase for filling short gaps in vitro (9,10) and
it has been suggested that p-polymerase is involved in repair of
the short gaps (i.e., base excision repair) induced by bleomycin
and y-radiation (11). In addition, over-expression of P-polymerase has also been implicated in resistance to cisplatin (12-14).
However, P-polymerase may also function in DNA replication
because it can substitute for Pol I in Escherichia coli and can
catalyze the joining of Okazaki fragments (15). As well,
p-polymerase is essential for the conversion of single-stranded to
double-stranded DNA in Xenopus extracts (16).
Since human mutant cell lines deficient in p-polymerase are not
yet available, we wanted to define a strategy for determining the
role of this enzyme in repair of therapeutically relevant DNA
damage. In the past, a number of strategies have been utilized to
define the role of P-polymerase with limited success. For
example, the role of P-polymerase during in vivo gap filling
synthesis has been defined in intact and permeabilized cells using
inhibitors against other cellular polymerases and an inhibitor of
P-polymerase, dideoxynucleoside 5'-triphosphate (11,17-20).
Because the specificity of these inhibitors is not absolute, the
issue of which DNA polymerase(s) is involved in gap filling in
the different DNA repair pathways is not settled. However, DNA
polymerase P has been clearly identified as the polymerase
involved in base excision repair pf G:U mismatches (21).
1598
Nucleic Acids Research, 1995, Vol. 23, No. 9
Attempts to reduce the intracellular P-polymerase levels using
an antisense expression approach have not been fully successful
since enzyme levels were only partially depleted (22). In an
alternative approach, mutated protein and DNA binding domains
have been utilized to inhibit intracellular resident activity of other
DNA repair proteins. For example, over-expression of mutated
ERCC-1 protein in repair proficient cells competes with the wild
type protein in the repair process resulting in a mutated cell
phenotype (23). These cells demonstrate higher sensitivity to
mitomycin C as compared to wild type cells. In another recent
study, introduction of the human poly (ADPR) polymerase DNA
binding domain, either as a purified polypeptide or over-expression from an expression vector in transfected cells, selectively
interferes and inhibits resident poly (ADPR) polymerase activity
(24). This blocking property of the DNA binding domain depends
absolutely on binding to DNA breaks through zinc fingers.
Therefore, we determined whether a similar approach utilizing
domains of p-polymerase, which lack DNA polymerase activity,
could be used to inhibit the activity of this enzyme in vitro.
Mammalian DNA polymerase p, a 39 kDa monomeric protein,
exhibits distributive DNA synthesis on a DNA substrate in which
a primer is annealed to a single-stranded template and processive
gap-filling on short-gapped (up to 6 nt) duplex DNA substrates
which have a 5'-phosphate on the downstream oligonucleotide
(25). The intact enzyme is capable of binding both single- and
double-stranded nucleic acids. The chemical and proteolytic
cleavage of P-polymerase generates domains that are devoid of
catalytic activity, but retain DNA binding capacity. Of these
domains, only the N-terminal 14 kDa domain binds both singleand double-stranded DNA. Based on these observations, we
wanted to know whether the 14 kDa domain could inhibit
p-polymerase activity in vitro.
MATERIALS AND METHODS
Materials
Deoxynucleoside triphosphates, poly(dA), p(dT)i6, and p(dT)2o
were purchased from Pharmacia. [a-32P]dTTP (3000 Ci/mmol)
was obtained from DuPont/New England Nuclear Corporation.
Klenow fragment and bovine serum albumin were purchased
from Gibco-BRL. Immobilon-S membrane was obtained from
Millipore (Bedford, MA). The catalytic subunit of a-polymerase,
which was expressed in baculovirus and purified, was a generous
gift from Dr William Copeland (NIEHS). HPLC purified
heteropolymeric oligomers of defined sequence were obtained
from Operon Technologies, Inc. T4 polynucleotide kinase was
from US Biochemicals and Nensorb-20 columns were obtained
from DuPont
DNA polymerase P domains
The 8 kDa fragment of P-polymerase and intact P-polymerase
were overexpressed in E.coli and purified as described earlier
(26-28). To facilitate analysis of the 14 and 31 kDa domains,
expression plasmids were constructed with the coding sequences
of each domain, residues 1-140 and 87-334, respectively. These
were overexpressed in E.coli and purified to homogeneity. The 16
kDa domain (residues 18-154) was obtained by CNBr treatment
of the holoenzyme (29) and the 27 kDa domain of P polymerase
was prepared by chymotrypsin digestion of purified enzyme.
Both fragments were purified as described previously (27-29).
DNA polymerase reactions
DNA synthesis by P-polymerase was measured with
poly(dA)539/p(dT)i6 template-primer (T-P). The T-P was constructed by annealing p(dT) | g to poly(dA)s39 at a nucleotide ratio
of 4.2 (template to primer) by heating the mixture to 95 °C and
slowly cooling to room temperature over several hours to prevent
stacking of the oligo(dT) on the template (30). The final reaction
mixture contained 50 mM Tris-HCl pH 7.5, 5 mM MnCl2, 25
mM KG (unless otherwise indicated), 2% glycerol, 5 nM
p-polymerase, 125 nM dTTP and 5 nM poly(dA)-p(dT)i6
(expressed as primer 3'-OH termini) unless otherwise indicated.
The concentration of the P-polymerase domains in each reaction
is indicated in the figures. Reaction mixtures were incubated at
25 °C for 10 min and stopped by adding EDTA to a final
concentration of 50 mM. The reaction mixtures were filtered
through a manifold with an Immobilon-S membrane. Unincorporated [a-32P]dTTP was removed by five washes of 0.3 M
ammonium formate pH 8. The dried filters were cut into
individual wells and counted in 3 ml Ecolume scintillation fluid.
Alternatively, radioactive products were collected on Whatman
DE81 filter disks as described previously (31).
The polymerase activity of Klenow fragment was determined
as above but with 10 mM MgCl2 or MnCl2. DNA polymerase a
activity was also determined as described above. Further details
are provided in the figure legends.
The apparent equilibrium dissociation constants (i.e., K^^) for
the binding of heteropolymeric DNA template-primers were
determined by inhibition of DNA polymerase activity on a
homopolymeric template-primer as described previously (31).
Lyophilized heteropolymeric oligonucleotides were resuspended in
10 mM Tris-HCl pH 7.4 and 1 mM EDTA, and the concentrations
determined from their UV absorbance at 260 run. Template-primers
were annealed by heating a solution of 5 |iM template (expressed as
3'-OH) with an equivalent concentration of primer to 90°C for 3
min, incubating the solution for an additional 30 min at 50-60 °C,
followed by slow cooling to room temperature. The sequences of the
oligonucleotides used were: P], 5'-CGAGCCATGGCCGCTAG-3';
P2, 5'-TTTTTTGCGGTGCCAGG-3'; T, 5'-CCTGGCACCGCAAAAAATCTGCCTAGCGGCCATGGCTCG-3'. Dephosphorylated primers were labeled by T4 polynucleotide kinase as described
(31).
Enzyme activities were determined using a standard reaction
mixture (50 ^1) containing 50 mM Tris-HCl pH 7.4, 100 mM
KC1, 5 mM MnCl2 or MgCl2 (Klenow fragment), 25 \M
[a-32P]dTTP, the indicated concentration of poly(dA)-p(dT)2o
(expressed as 3'-OH primer termini), and 1 ^M of competitor
heteropolymeric DNA. Nucleotide incorporation does not occur
with the competitor substrate since the correct nucleotide to be
incorporated (i.e., dGTP when P] is annealed to T, see above) is
not included in the reaction mixture. Reactions were initiated by
addition of polymerase, incubated at room temperature for 12 min
and stopped by the addition of 20 \x\ of 0.5 M EDTA. Quenched
reaction mixtures were spotted on DE81 filter disks and dTMP
incorporation was determined as described above.
The apparent dissociation constant, Kd^p, for the heteropolymeric duplex was calculated from:
Nucleic Acids Research, 1995, Vol. 23, No. 9 1599
Apparent
M.W.
singfe
-334
39*
1.0
<0.01
-86
<0.01
87-
-334
31*
0.05
<aoi
16
<ooi
27
<001
-154
140-
334
Figure 1. Schematic representation of DNA polymerase f3 and its functional domains obtained after limited proteolysis of purified enzyme. Relative polymcrase activity
was taken from Kumar et al. (27). (*) represents domains which are cloned and over-expressed in E.coli. The 16 kDa domain was prepared from CNBr treatment of
P-polymerase (29).
s+
where Km and V,™, for the homopolymeric template-primer (S)
were determined in the absence of heteropolymeric competitor
DNA (C). The A:m for poly(dA)-p(dT)2o was determined to be 40,
230 and » 2 0 0 0 nM for P-polymerase, Klenow fragment, and the
catalytic subunit of DNA polymerase a, respectively, for our
assay conditions. Therefore, the poly(dA)-p(dT)2o concentration
for the competition studies was 30, 200 and 1000 nM primer
3'-OH for P-polymerase, Klenow fragment and DNA polymerase
a, respectively.
Base excision repair assay
An in vitro uracil base excision repair assay utilizing bovine
nuclear extract has recently been developed (21). It utilizes a
synthetic 51 bp oligonucleotide substrate containing a U residue,
at position 22, opposite G. Repair of the G:U mismatch by the
bovine nuclear extract results in incorporation of [a-32P]dCMP
into the uracil containing strand. Subsequent ligation results in the
radioactive labeling of the 51mer repaired oligonucleotide (Fig.
6). Preparation of the bovine nuclear extract and specific reaction
conditions for the base excision assay were as described (21).
RESULTS AND DISCUSSION
Mammalian DNA polymerase P exhibits distributive DNA
synthesis on a DNA substrate in which a primer is annealed to a
single-stranded template, and processive gap-filling on a shortgapped duplex DNA substrate which has a 5'-phosphate on the
downstream oligonucleotide (25). The intact enzyme is capable
of binding both single- and double-stranded nucleic acids. The
chemical and proteolytic cleavage of P-polymerase indicated that
the enzyme is organized into two highly-folded functionally
distinct domains of 8 and 31 kDa. The N-terminal 8 kDa domain
has no catalytic activity. It binds strongly to single-stranded DNA,
but only weakly to double-stranded DNA. The C-terminal 31 kDa
domain, on the other hand, binds only to double-stranded DNA
and has - 5 % of the catalytic activity of the holoenzyme (27,28).
A recombinant 14 kDa fragment (residues 1-140) and a
CNBr-derived 16 kDa fragment (residues 18-154) both span the
protease-sensitive site and can bind both single- and doublestranded nucleic acids (29; Prasad and Wilson, unpublished data).
These domains, however, do not show any detectable polymerase
activity. To prepare large amounts of protein, a plasmid carrying
the coding sequence of the N-terminal 14 kDa domain (residues
1-140) of rat pVpolymerase was constructed and overexpressed
in E.coli. Schematic representation of P-polymerase and its
different domains is depicted in Figure 1.
In an attempt to establish a new approach for assessing the role
of P-polymerase in DNA repair, we evaluated the effect of
isolated purified P-polymerase domains on polymerase activity in
vitro. We first determined whether the 14 kDa domain inhibited
P-polymerase activity. We anticipated that the 14 kDa domain
would be the most likely domain to specifically inhibit DNA
polymerase P as it not only possesses both single- and doublestranded DNA binding activity, but is formed, in part, by the 8
kDa domain which directs P-polymerase to the downstream
5'-phosphate in a gap (31). To establish the in vitro assay,
P-polymerase activity was determined over a range of enzyme
concentrations. The reaction was linear with up to 20 nM enzyme
for 10 min (data not shown). With 5 nM enzyme, the polymerase
activity was linear for 20 min (data not shown). Thus, in all
subsequent experiments, P-polymerase activity was determined
with 5 nM enzyme for 10 min.
The catalytic activity of P-polymerase was measured in the
presence of varying concentrations of the 14 kDa domain. As seen
in Figure 2, -50% of the P-polymerase activity was inhibited with
90 nM of the 14 kDa domain at a total ionic strength of 90 mM.
At higher ionic strength (165 mM), however, 50% inhibition
required greater than 700 nM of this domain. Thus, the 14 kDa
domain could inhibit P-polymerase holoenzyme activity effectively at a moderate ionic strength. In order to determine whether
the inhibition of holoenzyme activity by the 14 kDa domain was
specific to this domain or whether other domains could also
inhibit the activity of the holoenzyme, the catalytic activity of
p-polymerase in the presence of the 8, 27 and 31 kDa domains
was examined. As illustrated in Figure 3, the other domains did
not inhibit p-polymerase activity significantly up to a concentration of 600 nM. hi addition, BSA did not inhibit P-polymerase
activity (data not shown). The 16 kDa domain (residues 18-154)
also inhibited P-polymerase activity to the same extent as the 14
kDa recombinant fragment (residues 1—140; data not shown).
Thus, the inhibition of holoenzyme appears torequireboth single-
1600
Nucleic Acids Research, 1995, Vol. 23, No. 9
100
200
300
400
500
600
700
50
[14-kDa Domain] (nM)
100
150
200
(T/P](nM)
Figure 2. Effect of ionic strength on 14 kDa dependent inhibition of
f}-polymerase activity. DNA polymerase (3 activity was determined as outlined
in Materials and Methods in the presence of 90 (A) or 165 (A) mM ionic
strength. Normalized activity (V/Vo) represents enzyme activity determined in
the presence of the 14 kDa domain (Vj) compared to in its absence (Vo). The
data in the presence of 165 mM ionic strength represents the weighted average
and standard error of two independent experiments. The concentration of 14
kDa domain which inhibited activity 50% (Kb-S) w a s 90 and 1050 nM in the
presence of 90 or 165 mM ionic strength, respectively.
100
200
300
400
500
600
[T/P] (nM)
O D
100
200
300
400
500
600
700
Figure 4. Effect of substrate concentration on the 14 kDa domain inhibition of
pVpolymerase activity. DNA polymerase P activity was determined as outlined
in Materials and Methods in the absence (A) and presence (O) of 300 nM 14
kDa domain. The apparent Km for T-P was determined from a non-linear least
squares fit of the data to the Michaelis-Menton equation. (A) With 0.1 |iM
tfl'lV, ^nvapparcni = 16 ± 4 and 61 ± 23 nM in the absence and presence of 14
kDa domain,respectively.(B) With 15 nM dTTP, Km_appmat = 45 ± 15 and 64
± 16 nM in the absence and presence of 14 kDa domain, respectively.
[Domain] (nM)
Figure 3. Influence of DNA polymerase P domains on activity of intact P
polymerase. DNA polymerase P activity was determined as outlined in
Materials and Methods in the presence of increasing concentrations of 8 ( • ) ,
14 (A), 27 (O) and (A) 31 kDa domains. Normalized activity (V/V o ) represents
enzyme activity determined in the presence of the domain (V;) compared to in
its absence (Vo). Addition of an equivalent amount of BSA had no influence on
enzyme activity (data not shown).
and double-stranded DNA binding capacity since the 8 and 31
kDa domains, which possess either single- or double-stranded
DNA binding activity alone, do not inhibit p-polymerase activity.
The 27 kDa domain, which does not bind to DNA, also had no
inhibitory effect on the holoenzyme.
In order to define the mechanism of inhibition by the 14 kDa
domain, the polymerase activity of the holoenzyme was determined in the presence of a fixed concentration of the 14 kDa
domain while varying the substrate (primer) concentration.
Figure 4A shows that by increasing the primer concentration, the
inhibition by the 14 kDa domain is diminished, suggesting that
the 14 kDa domain competes with pVpolymerase for primer
termini.
The effect of the 14 kDa domain on the (J-polymerase activity
in all experiments described above was determined in the
presence of 0.1 fiM dTTP. The Km for dTTP under the assay
conditions used is ~2 (iM. To determine the effect of dTTP
concentration on inhibition of |}-polymerase activity by the 14
kDa domain, we compared the kinetics of inhibition at concentrations of dTTP below (0.1 ^M) and above (15 uM) its Km (Fig. 4).
At 15 (iM dTTP, no inhibition was observed at low
poly(dA)-p(dT)i6 concentrations, whereas significant inhibition
was observed at 0.1 (iM dTTP (compare Fig. 4A and B). Thus,
these experiments suggest that inhibition by the 14 kDa domain
is not simple, in that both substrates [poly(dA)-p(dT)i6 and
dTTP] can abolish inhibition at high concentrations. One
explanation for these observations is that in the presence of a high
concentration of dTTP, the holoenzyme binds to the DNA
substrate with a much higher apparent affinity than when the
dTTP concentration is limiting. Therefore, under conditions of
high dTTP, the 14 kDa domain competes less effectively with the
holoenzyme for the DNA substrate. Such an increase in apparent
DNA binding affinity upon nucleotide binding has been shown to
occur with HIV-1 reverse transcriptase (32,33) and is expected
for a mechanism which follows an ordered binding of substrates
as with DNA polymerase P (34).
To characterize the specificity of inhibition by the 14 kDa
domain further, we determined the ability of the 14 kDa domain
to inhibit the polymerase activity of the Klenow fragment of
E.coli polymerase I and mammalian DNA polymerase a . As seen
Nucleic Acids Research, 1995, Vol. 23, No. 9 1601
o
BSA
8 kDa
14 kDa
51Klenow fragment
OJ
o0
1 0 O 2 O O 3 O O 4 O O 3 O O 6 O O 7 O O
[14-kDa Domain] (nM)
22-
0
50
100
150
200
250
300
350
[Domain] (nM)
Figure 5. Effect of DNA polymerase P domains on activity of mammalian
DNA polymerase a and Klenow fragment The polymerase acDvity was
determined using 5 nM enzyme as outlined in Materials and Methods (A) The
effect of the 14 kDa domain on DNA polymerase a and Klenow fragment
polymerase activity Normalized activity (V/VQ) represents enzyme activity
determined ui the presence of the 14 kDa domain (VJ compared to in its
absence (Vn) Klenow fragment was assayed with MgCl2 Klenow fragment
activity represents the weighted average and standard error of two independent
experiments and the data for polymerase a was compiled from 2-9 independent
experiments The activity of Klenow fragment was not influenced by 14 kDa
domain, however, normalized polymerase a activity was increased 2 9-fold
with 50 nM 14 kDa domain giving half maximal stimulation (B) DNA
polymerase a activity in the presence of increasing concentrations of 8 (A), 27
(O) and (D) 31 kDa domains
in Figure 5A, the 14 kDa domain did not inhibit either Klenow
fragment or cc-polymerase activity. DNA polymerase a activity
was rather stimulated modestly (2- to 3-fold) by the 14 kDa
domain. The concentration of the 14 kDa domain giving
half-maximal stimulation was 50 nM. The stimulatory effect on
a-polymerase activity was specific to the 14 kDa domain, as the
8, 27 and 31 kDa domains neither inhibited nor stimulated
a-polymerase activity (Fig. 5B). This observation suggests the
possibility of a physical interaction between these DNA polymerases in vitro and may be important in a sequential polymerase
mechanism postulated for filling of large gaps in vivo
(19,20,31,35-37). Alternatively, the 14 kDa domain may inhibit
non-productive binding of a polymerase so as to increase the
active fraction of enzyme.
The specificity of the 14 kDa domain toward p*-polymerase is
probably explained as follows: unlike other DNA polymerases,
(J-polymerase utilizes both the 3'-OH end of the template-primer
and the 5'-PO4 terminus of the downstream oligonucleotide.
P-polymerase binds to the 5'-PO4 of the downstream oligonucleotides through the 8 kDa domain on short-gapped (6-8 nt)
Figure 6. Domain specific inhibition of uracil base excision repair Reactions
were assembled and repair of a G U mismatch was determined as described
(21) Repair of the 51mer synthetic heteropolymenc duplex with a GU
mismatch at position 22 is followed by appearance of a radioactive band
(51mer) in the autoradiogram due to the excision of the uracil and the
incorporation of [a-32P]dCMP by DNA polymerase P Lane I, no additions,
lanes 2-3, 0 2 ng and 3 ng BSA, respectively, lanes 4-6, 0 2, 2 and 20 nM 8
kDa domain, respectively; lanes 7-9, 0 2, 2 and 20 nM 14 kDa domain,
respectively
substrates and carries out processive synthesis (25,31). p"-polymerase activity on substrates in which the downstream oligonucleotide is not phosphorylated is dramatically reduced (31 and
data not shown). In vitro, on substrates with larger gaps, such as
used in this study (average gap size of 50 nt), the majority of
p"-polymerase molecules would bind to the 5'-PC>4 and only a
small proportion would bind to the 3'-OH ends of the upstream
primer (31). Those molecules of fJ-polymerase bound to the
3'-OH would carry out distributive synthesis. Once the large gap
is reduced to a size of 6-8 nt, P-polymerase carries out processive
synthesis using the 3'-OH ends to fill the remaining gap. This
unique mode of P-polymerase binding probably explains the
specificity of inhibition of (i-polymerase activity by the 14 kDa
domain. In other words, the unique ability of the 14 kDa domain
to bind to both single- and double-stranded DNA is required to
identify the 5'-PC>4 in a gap as well as to bind to the 3'-OH of the
nearby (short gap) upstream pnmer.
To determine if the polymerase specificity can be attributed to
the ability of P-polymerase to bind tightly and specifically to a
5'-PC>4 in a gap, the apparent binding affinity of Klenow fragment
and DNA polymerase a binding to the 3'-OH or 5'-PO4 of
synthetic heteropolymenc template-primers was determined
(Table 1). As illustrated in Table 1, a 17mer primer was either
annealed to the 3'- or 5'-end of a 39mer template (P) and P2,
respectively). This results in two template-primer substrates
where the primer 3'-OH or 5'-PC>4 is at a blunt end or adjacent to
single-stranded template. As observed previously (31), P-polymerase binds specifically and tightly to the template-primer
(T-P2) which has a 5'-PO4 adjacent to the single-stranded
template (Table 1). P-polymerase does not have significant
affinity for this substrate if ?j is not 5'-phosphorylated (31). In
1602
Nucleic Acids Research, 1995, Vol. 23, No. 9
TaWe 1. Effect of 3' and 5' heteropolymeric primer position on DNA
polymerase template-primer binding affinity
Duptexb
DNA Polymerui
B
»1000
a
»1000
K hitow Pnfmcot
3'10
S
23
a
85*
Kknow Fragment
100
"Apparent dissociation constants for the heteropolymeric template-primers
were determined from the inhibition of DNA polymerase activity on
poly(dA)-p(dT)2o as described in Materials and Methods.
b
A 39mer heteropolymeric template was annealed with a 17mer primer as
outlined in Materials and Methods.
°Due to the high Km for poly(dA)-p(dT)2o under our assay conditions, this
value represents a lower estimate of the apparent binding affinity.
contrast, Klenow fragment selectively binds to the templateprimer where the 3'-OH is next to the single-stranded template
(i.e., T-P]), as compared to the substrate where the 3'-OH is at the
blunt end of the template-primer (Table 1). DNA polymerase a
has very low affinity for a DNA primer annealed in such a way
to create a potential 3'-OH for nucleotide incorporation. However, it does show modest affinity, although 3- to 4-fold lower
than for p-polymerase to the 5'-PO4 on a downstream oligonucleotide (T-P2, Table 1) and is similar to that observed for
Klenow fragment. Thus, of the three polymerases, P-polymerase
has the highest affinity for the 5'-PO4 in a 'gapped' DNA
substrate and therefore would be most sensitive to competition by
the 14 kDa domain. The 8 kDa domain, because of its weak
binding to only single-stranded DNA, does not compete with the
holoenzyme since it lacks the capacity to bind double-stranded
DNA.
An in vitro base excision repair assay has recently been
developed which relies exclusively on DNA polymerase P for
DNA synthesis activity in the repair of a G:U mismatch (21). This
assay relies on endogenous P-polymerase activity, as well as other
enzymatic activities of nuclear extracts from bovine testis such as
uracil-DNA glycosylase, endonuclease and DNA ligase believed
to be required for base excision repair. Repair of a 51 bp synthetic
DNA substrate containing a single G:U mismatch at position 22
can be followed by the radioactive-labelling of the duplex after
excision of uracil, P-polymerase dependent incorporation of
[32P]dCMP, and sealing of the gap. An autoradiogram illustrating
the effect of the 8 and 14 kDa domains on the formation of the
labelled 51 mer is shown in Figure 6. B S A and 8 kDa domain (^20
(iM) did not influence repair of the duplex with the G:U mismatch
(Fig. 6, lanes 1-6). In contrast, 20 |iM 14 kDa domain inhibited
base excision repair over 75% (Fig. 6, lane 9). The 14 kDa domain
at 200 nM nearly abolished repair activity (data not shown).
The structure of the 39 kDa holoenzyme with bound substrates
has recently been determined (38,39). As predicted by proteolysis
(27,28), these crystal structures show that P-polymerase is
composed of two domains; an N-terminal 8 kDa domain and a 31
kDa polymerase domain. The structure of the polymerase domain
of P-polymerase is similar to the structure of other polymerases
in that it has finger, palm and thumb subdomains which form a
DNA binding site. However, the 8 kDa domain was not observed
to be interacting with DNA, possibly due to the nature of the DNA
substrate (i.e., an ungapped DNA substrate lacking a downstream
5'-phosphate) used in the crystallization (39). It has recently been
demonstrated that the 8 kDa domain recognizes the 5'-phosphate
in gapped DNA (31) and postulated that lysine 72 of the 8 kDa
domain binds specifically to the downstream 5'-phosphate (38).
The 14 kDa domain corresponds to the 8 kDa domain connected
to the 'fingers' subdomain of P-polymerase (38).
In summary, our data show that the 14 kDa domain of
P-polymerase inhibits p-polymerase activity in vitro, as well as in
an in vitro base excision repair assay, probably by competing with
intact enzyme for DNA binding. Other domains of p-polymerase
(8, 27 and 31 kDa) do not have any significant inhibitory effect
on the activity of the holoenzyme. The inhibitory effect of the 14
kDa domain on P-polymerase activity is specific since this
domain does not inhibit the activity of either Klenow fragment or
mammalian a-polymerase. Indeed, the 14 kDa domain stimulates
a-polymerase activity 2- to 3-fold. The specific inhibition of
P-polymerase by the 14 kDa domain in vitro and in base excision
repair suggests that this domain could be used as an inhibitor of
resident P-polymerase activity in intact cells if high intracellular
concentrations could be achieved. This could be done by
microinjection of the domain or its over-expression by expression
vectors. Thus, the 14 kDa domain of p-polymerase may provide
a useful tool for assessing the role of this enzyme in repair of
various forms of DNA damage. Furthermore, as the crystal
structure of the P-polymerase has been solved (38-40), it may be
possible to design structure-based inhibitors of P-polymerase as
potential anti-cancer agents. Experiments to address these
questions are underway.
ACKNOWLEDGEMENTS
We would like to thank Dr William Copeland for the generous gift
of mammalian a-polymerase catalytic subunit, Dr Jingwen Chen
and Peter Leitner for helpful discussions, and Mary Ellen
Buchheit for preparing the manuscript.
REFERENCES
1
2
3
4
5
6
7
8
9
Sancar, A. and Sancar, G.B. (1988) Ann. Rev. Biochem. 57, 29-67.
Hoeijmakers, J.HJ. (1993) Trends Genet. 9, 211-217.
Hoeijmakers, J.HJ. (1993) Trends Genet. 9, 173-177.
Carmichael, J. and Hickson, I. D. (1991) Int. J. RadiaL Oncology Biology
Physics 20, 197-202.
Webster, D., Arlett, C.F., Harcourt, S.A., Teo, LA. and Henderson, L.
(1982) In Bridges, B.A. and Hamden, D.G. (eds), Ataxia-telangiectasia - A
cellular and molecular link between cancer. Neurvpaihology and Immune
Deficiency. John Wiley and Sons Ltd.
Friedberg, EC. (1985) DNA repair. W.H. Freeman and Company, New
York. Chapter 9, pp. 505-574.
Barnes, D.E., Tomkinson, A.E, Lehmann, A.R., Webster, D.B. and
Lindahl, T. (1992) Cell 69, 495-503.
Wang, T.S-F. (1991) Ann. Rev. Biochem. 60, 513-552.
DiaDOV, G., Price, A. and Lindahl, T. (1992) MoL Cell. BioLU,
1605-1612.
Nucleic Acids Research, 1995, Vol. 23, No. 9 1603
10 Wicbaucr, K. and Jiricny. J. (1990) Pmc. Nail. Acad. Sci. USA 87,
5842-5845.
11 DiGiuseppe, J.A. and Dresler, S.L. (1989) Biochemistry 28, 9515-9520.
12 Scanlon, KJ., Jiao, L., Funato, T., Wang, W., Tone, T., Rossi, JJ., and
Kashani-Sabet, M. (1991) Proc. Natl. Acad Sci. USA 88, 10591-10595.
13 Scanlon, KJ., Kashani-Sabet, M. and Sowers, L.C. (1989) Cancer
Commun. 1, 269-275.
14 Vazquez-Padna, M.A., Stames, M.C. and Chen, Y.C. (1990) Cancer
Commun. 2, 55—62.
15 Sweasy, J.B. and Loeb, LA. (1992) J. Biol. Chem. 267, 1407-1410.
16 Jenkins, T.M., Saxena, J.K., Kumar, A., Wilson, S.H. and Ackerman, EJ.
(1992) Science 258, 475-^»78.
17 Dresler, S.L. and Lieberman, M.W. (1983) J. Biol. Chem. 258, 9990-9994.
18 Miller, M.R. and Chinault, D.N. (1982) J. Biol. Chem. 257, 10204-10209.
19 Smith, C A. and Okumoto, D.S. (1984) Biochemistry 23, 1383-1390.
20 Hammond, R.A., McClung, J.K. and Miller, M.R. (J990) Biochemistry 29,
286-291.
21 Singhal, R.K., Prasad, R. and Wilson, S.H. (1995) J. Biol Chem. 270,
949-957.
22 Zmudzka, EZ. and Wilson, S.H. (1990) Som. Cell Mol. Gen. 16, 311-320.
23 Belt, P.B.G.M., Van Oosterwijk, M.F.V., Odijk, H., Hoeijmakers, J.HJ. and
Backendorf, C. (1991) Nucleic Acids Res. 19, 5633-5637.
24 Mohncte, M., Vermeulen, W., Burkle, A., Menissier-de Murca, J., Kupper,
J.H., Hoeijmakers, J.HJ. and Murcia ,G.D. (1993) EMBO J. 12,
2109-2117.
25 Singhal, R.K. and Wilson, S.H. (1993)7. Biol. Chem. 268, 15906-15911.
26 Prasad, R., Kumar, A., Widen, S.G., Casas-Finet, J.R. and Wilson, S.H.
(1993)/ Biol. Chem. 268, 22746-22755.
27 Kumar, A., Abbotts, J., Karawya, EM. and Wilson, S.H. (1990)
Biochemistry 29, 7156-7159.
28 Kumar, A., Widen, S.G., Williams, K.R., Kedar, P., Karpel, R.L. and
Wilson, S.H. (1990)7. Biol. Chem. 265, 2124-2131.
29 Casas-Finet, J.R., Kumar, A., Karpel, R.L. and Wilson, S.H. (1992)
Biochemistry 31, 10272-10280.
30 Mesner, L.D. and Hockensmith, J.W. (1992) Proc. Natl. Acad. Sci. USA
89,2521-2525.
31 Prasad, R., Beard, W.A. and Wilson, S.H. (1994) J. BioL Chem. 269,
18096-18101.
32 Kati, W.M., Johnson, K.A., Jerva, L.F. and Anderson, K.S. (1992) J. Biol.
Chem. 267, 25988-25997.
33 Miiller, B., Restle, T., .Reinstein, J. and Goody, R.S. (1991) Biochemistry
30,3709-3715.
34 Tanabe, K., Bohn, W. and Wilson, S.H. (1979) Biochemistry 18,
3401-3406.
35 Keyse, SM. and Tyrrell, R.M. (1985) MM. Res. 146, 109-119.
36 Mosbaugh, D.W. and Linn, S. (1984) J. Biol. Chem. 259, 10247-10251.
37 Yamada, K., Hanaoka, F. and Yamada, M. (1985) /. BioL Chem. 260,
10412-10417.
38 Sawaya, M.R., Pelleuer, H., Kumar, A., Wilson, S.H. and Kraut, J. (1994)
Science 264, 1930-1935.
39 Pelletier, H., Sawaya, M.R., Kumar, A., Wilson, S.H. and Kraut, J. (1994)
Science 264, 1891-1903.
40 Davies n, J.F., Almassy, RJ., Hostomska, Z., Feme, R.A. and Hostomsky,
Z.(1994)CW/76, 1123-1133.