From the Laboratory of Molecular Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9072
Received for publication, May 23, 2000, and in revised form, October 4, 2000
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ABSTRACT |
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The Escherichia coli dinB gene
encodes DNA polymerase (pol) IV, a protein involved in increasing
spontaneous mutations in vivo. The protein-coding region of
DINB1, the human ortholog of DNA pol IV, was fused to
glutathione S-transferase and expressed in insect cells.
The purified fusion protein was shown to be a template-directed DNA
polymerase that we propose to designate pol We previously reported the cloning and characterization of the
human DINB1 and mouse Dinb1 genes, mammalian
orthologs of the Escherichia coli dinB gene (1) and members
of the UmuC/DinB superfamily of DNA polymerases (2). Expression of the
E. coli dinB gene is tightly regulated by the SOS system
(3). Following exposure of E. coli cells to DNA-damaging
agents such as ultraviolet (UV) radiation, induction of dinB
results in enhanced spontaneous (untargeted) mutagenesis of phage Human DINB1 cDNA is predicted to encode a polypeptide
with a molecular mass of 99 kDa, which shares extensive amino acid
sequence homology with E. coli DNA pol IV, including
proposed catalytic domains (1, 8). As is the case for the E. coli
dinB gene (5), overexpression of the mouse Dinb1
cDNA in murine cells is associated with an approximately 10-fold
increase in spontaneous mutations (8). These observations suggested
that the protein encoded by the human DINB1 gene might also
function as a DNA polymerase. In the present study we have used a
baculovirus expression system to express and purify a
GST1-human DinB1 fusion
protein from insect cells and show that the purified protein is a
template-directed DNA polymerase in vitro. The enzyme has no
detectable 3' Media and Biochemical Reagents--
Insect cell TMN-FH media was
purchased from Pharmingen. The Klenow fragments of E. coli
DNA polymerase I (exo+ and exo Expression of Wild-type and Mutant GST/pol
The C-terminal deletion mutant was made by high-fidelity polymerase
chain reaction with primers HDinB5' and HDinB
These plasmids were co-transfected into SF9 cells with BaculoGold DNA
using a BaculoGold transfection kit (Pharmingen). Expression of both
wild-type and mutant GST/pol Purification of GST/pol DNA Substrates--
The oligonucleotide derived primer-templates
used as substrates in the DNA polymerase assays (24/44, 25/44, 27/44,
30/44, and 31/44) were the same as those described by Wagner et
al. (6). The 20/54 primer-template consisted of oligonucleotides
P4-OX-RS (5'-GAATTCCTGCAGCCCAGGAT-3') and T1-OX-WT
(5'-ATTCCAGACTGTCAATAACACGGTGGACCAGTCGATCCTGGGCTGCAGGAATTC-3'). Primers were purified by denaturing polyacrylamide gel electrophoresis (PAGE). Five pmol of each primer was 5' end-labeled with T4
polynucleotide kinase (New England Biolabs) in the presence of
[ Preparation of AAF-DNA Template--
A synthetic DNA oligo of
the sequence 5'-TCCTTCTGTCTCTT-3' (site of modification
underlined) was purified by denaturing PAGE and desalted using a
Sep-Pak C18 cartridge (Waters, manufacturer's instructions). 0.38 µmol of purified DNA was dissolved in TE buffer (45 mM Tris-HCl, 1 mM Na2EDTA, pH
8.0) to a concentration of 0.2 mM and incubated with AAAF
(1 mM, 10% ethanol, 37 °C, 12 h). Adducted DNA was
admixed with an equal volume 8 M urea and purified by denaturing PAGE. The AAF-modified oligo has retarded gel mobility relative to the unmodified oligo, allowing efficient removal of unmodified DNA. DNA was eluted and concentrated using a Sep-Pak C18 cartridge.
The presence of the AAF-modified deoxyguanosine residue was confirmed
by electrospray mass spectrometry (ESI-MS) and by HPLC analysis of
enzymatically digested DNA. HPLC separation of the enzymatic
hydrolysate returned peaks corresponding to deoxycytidine, thymidine,
and a strongly retained substance that co-eluted with the product of
the reaction between deoxyguanosine and AAAF. HPLC analysis was
conducted similarly to a previous report (9). The ESI-MS of each of
these co-eluted substances was shown to correspond to
N-(deoxyguanosin-8-yl)-N-(2-acetylamino)fluorene ([M
7.6 pmol of the AAF-modified oligo was admixed with 1.1 molar
equivalents of each of the two flanking DNA sequences
5'-TTTCCCAGTCACGACG-3' and 5'-TCAGTGAATTCGAGCTCGGAGCC-3' and
approximately 0.9 equivalents of the splinting DNA oligo,
5'-CACTGAAAGAGACAGAAGGACGTCGT-3'. DNAs were heated briefly (90 °C, 1 min) and annealed by cooling to room temperature on the benchtop (5 min). Annealed DNAs were ligated with T4 DNA ligase (1 × in
manufacturer's buffer, 16 °C, 12 h), ethanol precipitated, and
purified by denaturing PAGE. The 53-mer product band
(5'-TTTCCCAGTCACGACGTCCTTCTGTCTCTTTCAGTGAATTCGAGCTCGGAGCC-3', site of AAF modification underlined) was identified by mobility relative to marker oligos and to mock-treated reactions in which one or
more of the splicing components had been left out of the mixture.
An unmodified 53-mer oligonucleotide of the above sequence was used as
a wild-type control template. The unmodified and AAF-modified templates
were annealed to either a gel-purified running start oligo, P1-PF-RS,
5'-GGCTCCGAGCTCGAATTCAC-3' or a gel-purified standing start oligo,
P2-alk-SS, 5'-GGCTCCGAGCTCGAATTCACTGAAAGAGA-3' as described previously.
Preparation of Platinated DNA Template--
A synthetic DNA
oligo of the sequence 5'-TCCTTCGGTCTCTT-3' (site of
modification underlined) was purified by denaturing PAGE and desalted
using a Sep-Pak C18 cartridge. An aqueous solution of
cis-diamminedichloroplatinum(II) (1 mg/ml) was freshly
prepared and preincubated in the dark (25 °C, 12 h). 0.043 µmol of purified DNA was dissolved in water to a concentration of 0.5 mM, admixed with 1 molar equivalent of cisplatin from the
preincubated solution and incubated (5 h, 37 °C, wrapped in foil).
Crude cisplatin-modified DNA was gel-purified. The presence of the
cisplatin adduct was confirmed by matrix-assisted laser desorption
ionization-mass spectrometry and was presumed to be a 1,2-intrastrand
d(GpG) cross-link based on previous reports that cisplatin modifies N-7
of purines (reviewed in Ref. 13). A 5' end-labeled aliquot of the
sample confirmed that the modified oligonucleotide migrated as a single band on a denaturing gel and that its mobility was distinct from unmodified DNA and a cisplatin diadduct. The purified
cisplatin-modified 14-mer was ligated to flanking sequences as
described above for AAF-modified DNA except that the splinting DNA in
ligation reactions was composed of the sequence,
5'-CACTGAAAGAGACCGAAGGACGTCGT-3'. The 53-mer product
(5'-TTTCCCAGTCACGACGTCCTTCGGTCTCTTTCAGTGAATTCGAGCTCGGAGCC-3', site of cisplatin modification underlined) was purified by
denaturing PAGE and identified similarly to the AAF-template.
An unmodified 53-mer oligonucleotide of the above sequence was used as
a wild-type control template. The unmodified and cisplatin-modified templates were annealed to the running start oligo, P1-PF-RS, as
described previously.
DNA Polymerase Assays--
Standard polymerase reactions (10 µl) were performed in 50 mM Tris-HCl (pH 7.0), 5 mM MgCl2, 1 mM dithiothreitol, 10 mM NaCl, 1% glycerol with 100 µM dNTPs, 2 nM GST/pol PCNA Experiments--
Human recombinant PCNA (provided by R. Wood, Imperial Cancer Research Fund, Clare Hall Laboratories, South
Mimms, Hertfordshire EN6 3LD, United Kingdom) and calf thymus pol Human DinB1 Protein Is a DNA Polymerase, pol
To test for DNA polymerase activity, various 5'-32P-end
labeled oligonucleotide primers were annealed to a 44-nucleotide
template and used as substrates. In the presence of dNTPs and
Mg2+, the Klenow fragment of E. coli DNA
polymerase I efficiently extended the primer to generate the expected
44-nucleotide product (Fig. 1B, lane 4). Purified GST/hDinB1
protein also extended the primer, demonstrating an intrinsic DNA
polymerase activity (Fig. 1B, lanes 2 and 3). We
therefore propose to rename the human DinB1 protein as DNA polymerase
As a control, a GST/hDinB1 mutant protein in which the conserved amino
acid residues Asp198 and Glu199 were
changed to alanine, was purified by the same procedure (Fig. 1A,
lane 2) and shown to be devoid of detectable DNA polymerase activity (Fig. 1C, lanes 3-6), indicating that the observed
polymerase activity is intrinsic to the human DinB1 protein. In
addition a truncated GST/hDinB1 fusion protein lacking 360 amino acids at the C terminus (GST/hDinB1
We performed a series of experiments to determine the optimal
conditions for pol
The range of incomplete extension products produced by GST/pol pol
Given the detectable levels of nucleotide misincorporation observed in
Fig. 4A, we tested GST/pol
To complete our preliminary characterization of GST/pol pol
In contrast to the results observed with cisplatin, GST/pol In this study we report that the product of the human
DINB1 gene is a DNA polymerase, pol The loss of DNA polymerase activity of pol To demonstrate DNA polymerase activity we used a simple primer
extension assay. Interestingly the largest extension product produced
by pol In most of the GST/pol As noted above, E. coli DNA pol IV is a specialized DNA
polymerase that is regulated by the SOS system, suggesting a role in
DNA replication and spontaneous mutagenesis in response to certain
cellular stress conditions. The apparent low fidelity and moderate
processivity of human DNA pol Abnormal templates might contain particular types of DNA damage or
possess aberrant structures. Efficient translesion synthesis by DNA pol
IV on templates containing an abasic site, thymine dimer, or 6-4 photoproduct has not been observed (7). However, the bacterial protein
can extend misaligned primer-templates resulting in single nucleotide
deletions (6). Similarly, a human GST/pol In this study we have shown that human pol In human cells pol. Human pol
lacks
detectable 3'
5' proofreading exonuclease activity and is not
stimulated by recombinant human proliferating cell nuclear
antigen in vitro. Between pH 6.5 and 8.5, human
pol
possesses optimal activity at 37 °C over the pH range
6.5-7.5, and is insensitive to inhibition by aphidicolin,
dideoxynucleotides, or NaCl up to 50 mM. Either
Mg2+ or Mn2+ can satisfy a metal cofactor
requirement for pol
activity, with Mg2+ being preferred.
Human pol
is unable to bypass a cisplatin adduct in the template.
However, pol
shows limited bypass of an 2-acetylaminofluorene lesion and can incorporate dCTP or dTTP across from this lesion, suggesting that the bypass is potentially mutagenic. These results are
consistent with a model in which pol
acts as a specialized DNA
polymerase whose possible role is to facilitate the replication of
templates containing abnormal bases, or possessing structurally aberrant replication forks that inhibit normal DNA synthesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
DNA introduced into the bacteria subsequent to irradiation (4).
Increased spontaneous mutagenesis is also observed following
overexpression of dinB in cells transfected with
F'lac plasmids, with the most prevalent mutations detected being
1 frameshifts (5). Recombinant E. coli DinB protein carrying a 6-histidine tag was purified and shown to be a DNA polymerase, designated DNA pol IV of E. coli, which is
devoid of detectable exonuclease activity (6). Consistent with its apparent ability to generate frameshift mutations in vivo,
DNA pol IV is able to extend a misaligned primer-template in
vitro, resulting in a
1 frameshift mutation (6). More recently,
DNA pol IV has been shown to be unable to efficiently bypass an abasic site, thymine dimer, or 6-4 photoproduct in vitro (7). Based on these observations, it has been suggested that DNA pol IV is a
specialized enzyme whose role is to negotiate sites of stalled or
arrested DNA replication caused by structurally abnormal replication forks, such as those caused by slippage at repeated sequences (2, 6,
7).
5' exonuclease activity and has optimal DNA
polymerase activity at 37 °C and pH 6.5-7.5 (over the range
6.5-8.5) in the presence of Mg2+ cations. The enzyme is
insensitive to inhibition by aphidicolin or dideoxynucleotides, and
retains optimal activity in the presence of NaCl at levels
50
mM. We designate this polymerase activity DNA pol
. Human
pol
is unable to bypass a cisplatin adduct in vitro, but
has limited ability to bypass an AAF lesion in an error-prone fashion
under the same conditions.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) were obtained
from New England Biolabs. Aphidicolin was from Sigma.
Dideoxynucleotides were from U. S. Biochemicals.
Glutathione-Sepharose was from Amersham Pharmacia Biotech. The protease
inhibitor mixture was purchased from Roche Molecular Biochemicals.
N-Acetoxy-2-acetylaminofluorene (AAAF) was obtained from the
National Cancer Institute. cis-Diamminedichloroplatinum(II) was purchased from Aldrich.
--
The human
DINB1 open reading frame was amplified by high-fidelity
polymerase chain reaction using HeLa cell cDNA as template with primers HDinB5'
(5'-GTGGATCCGCCATGGATAGCACAAAGGAGAAGTG-3') and
HDinB3'-His6
(5'-ATGGATCCGCGGTCGACTAATGGTGGTGATGATGGTGCTTAAAAAATATATCAAGGGTATG-3') that introduce BamHI restriction sites (underlined) on
both the 5' and 3' ends of the amplified fragment as well as six
histidine residues on the 3' end. The polymerase chain reaction product was cloned into pGEM-T Easy (Promega) to generate pHDINB1-6His and
sequenced to confirm the integrity of the coding region. The 2.6-kilobase BamHI fragment containing the human
DINB1 coding region was then cloned into the same site of
pAcG2T (Pharmingen) to generate an in-frame fusion with the glutathione
S-transferase gene, generating plasmid
pAcG2T/HDINB1-6His.
3'-6His (5'-ATGGATCCGCGGTCGACTAATGGTGGTGATGATGGTGAGATCTACCC-ATAAGCCTTAATCTCA-3') that introduce a BamHI restriction site (underlined)
and six histidine residues onto the 3' end of the amplified fragment.
The polymerase chain reaction product was cloned into pGEM-T Easy
(Promega) to give pHDINB1
C-6His. The D198A/E199A double
mutation was introduced into pHDINB1-6His using the Tranformer
site-directed mutagenesis kit (CLONTECH) and
primers GTE-MluI/HindIII
(5'-GAGCTCCCAAAGCTTTGGATGCAT-3') and HDinB-DE
AA
(5'-CCATGAGTCTTGCTGCAGCCTACTTG-3'), the latter introducing a PstI restriction site (underlined) to give
pHDINB1mut-6His. The BamHI fragments from pHDINB1
C-6His
and pHDINB1mut-6His were cloned into the same site of pAcG2T to give
pAcG2T/HDINB1
C-6His and pAcG2T/HDINB1mut-6His.
was assayed by immunoblotting with
anti-GST antisera. Two rounds of amplification produced a high titer
stock of recombinant virus expressing GST/pol
. The multiplicity of
infection yielding optimal expression of full-length fusion proteins
was determined empirically.
--
Approximately 1 × 108 virus-infected Sf9 cells were harvested 3 days
after infection and lysed in 20 ml of Lysis Buffer I (1% Triton
X-100, 10 mM Tris-HCl, pH 7.5, 10 mM
Na2HPO4, pH 7.5, 1 mM EDTA, 5 MM
-mercaptoethanol, 1 × protease inhibitors) by incubation on
ice for 10 min. Insoluble material was removed by centrifugation to
give the cytoplasmic extract. The pellet was resuspended in 20 ml of
Lysis Buffer I containing 500 mM NaCl, and incubated on ice
for 10 min. Insoluble material was removed by centrifugation to
generate nuclear extract. The nuclear extract was diluted 2-fold and
bound in batch to 500 µl of glutathione-agarose for 2 h at 4 °C. The resin was harvested by centrifugation and most of the supernatant removed. The resin was resuspended in the remaining supernatant and transferred to a 10-ml disposable column (Bio-Rad) to
collect the resin by gravity. The resin was washed with 5 ml of Lysis
Buffer I containing 250 mM NaCl, followed by 5 ml of Wash
Buffer II (10% glycerol, 100 mM NaCl, 20 mM
Tris-HCl, pH 7.5, 0.01% IPEPAL-630, 5 mM
-mercaptoethanol, 1 × protease inhibitors). Bound protein was
eluted with 3.5 ml of Wash Buffer II containing 10 mM
reduced glutathione, and collected in a total of 10 fractions of 350 µl each. GST/pol
-containing fractions (determined by SDS-PAGE and
immunoblotting) were aliquoted, frozen in liquid nitrogen, and stored
at
80 °C. GST/pol
DNA polymerase activity was stable to
multiple rounds of freezing and thawing. The mutant fusion proteins
were expressed and purified by the same procedure.
-32P]ATP and purified on Bio-Gel P2 (Bio-Rad) spun
columns equilibrated in STE (100 mM NaCl, 10 mM
Tris, pH 8.0, 1 mM EDTA). The various labeled primers (100 µl) were annealed to the template in a ratio of 1:1.5
(primer-template) by heating to ~95 °C for 5 min followed by slow
cooling to room temperature.
H+]
1 = 487.2), the expected lesion. The
AAF modification was assumed to be at C-8 of deoxyguanosine because
AAAF is known to modify DNA preferentially at this position (10-12).
No appreciable peaks were observed in the HPLC assay that could
potentially be attributed to modification at other positions of
deoxyguanosine or at other bases on the DNA oligo.
, and 5 nM primer-template for 5 min at 37 °C unless indicated otherwise. Reactions were terminated
by the addition of 1 µl of 0.5 M EDTA, concentrated under
vacuum, and resuspended in 5 µl of loading dye (90% deionized formamide, 0.1 × TBE, 0.03% bromphenol blue, 0.03% xylene
cyanole FF). Following denaturation at 95 °C for 2 min, products
were resolved by electrophoresis on 12% polyacrylamide gels containing 8 M urea. Gels were dried under vacuum and exposed to film
at room temperature.
(provided by U. Hübscher, Department of Veterinary Biochemisty,
University of Zurich-Irchel, CH-8057 Zurich, Switzerland) were prepared
as described (14, 15). DNA polymerase assays were performed using the
standard conditions described above unless indicated otherwise.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
To determine
whether the product of the human DINB1 gene is a DNA
polymerase, we expressed and purified recombinant human DinB1 protein.
Expression in both E. coli and the yeast
Schizosaccaromyces pombe consistently resulted in low yields
and/or degraded protein (data not shown). However, we were able to
express full-length hDinB1 protein fused to GST in insect cells
using a baculovirus expression system. The recombinant GST/hDinB1
protein was purified to near physical homogeneity from nuclear extracts
by affinity chromatography on glutathione-agarose (Fig.
1A, lane 1). The purified GST/hDinB1 fraction contained primarily full-length fusion protein; however, some degradation products, including free GST, were observed and confirmed by immunoblotting with anti-GST antisera (data not shown).
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Fig. 1.
Purification and DNA polymerase activity of
wild-type and mutant GST/hDinB1(pol ).
A, 50 ng of either wild-type (lane 1) or
D198A/E199A double mutant (lane 2). GST/pol
glutathione-agarose fractions were analyzed on a 7.5%
SDS-polyacrylamide gel and visualized by silver staining. Full-length
GST/pol
is indicated by an arrow. The positions of
molecular weight markers are indicated at the left. B,
GST/pol
has DNA polymerase activity. The indicated amounts of
GST/pol
were assayed for DNA polymerase activity at 30 °C on the
25/44 primer-template (lanes 1-3). A DNA polymerase
reaction using 5 units of the Klenow fragment of E. coli DNA
pol I (exo+) was performed as a positive control and is
shown in lane 4. The position of the expected full-length
product (44 nucleotides) is indicated by an arrow. C, the
indicated amounts of either wild-type (lane 2) or mutant
(lanes 3-6) were assayed for DNA polymerase activity as
described in B.
(pol
) and the gene encoding it, POLK, in accordance
with standard nomenclature for eukaryotic DNA polymerases (16, 17).
This designation has been approved by the Human Genome Organization
nomenclature committee.
C) did not demonstrate DNA polymerase activity, indicating that sequences within this less highly conserved portion of the protein are required for activity (data not shown).
DNA polymerase activity in vitro. As
shown in Fig. 2A, between pH
6.5 and 8.5 GST/pol
was most active over the range 6.5-7.5 (Fig.
2A, lanes 1-5), with reactions carried out at 37 °C
(Fig. 2A, lanes 6-9). To investigate the effect of ionic
strength on DNA synthesis, increasing amounts of NaCl were added to the
reactions (Fig. 2A, lanes 11-16). GST/pol
activity was
relatively insensitive to NaCl concentration up to 50 mM, but was significantly inhibited at salt concentrations of 100 mM or higher. As expected, a metal cofactor was required
for activity (Fig. 2B). Either Mg2+ or
Mn2+ could be utilized, with the former being preferred
(Fig. 2B, compare lanes 2 and 3).
Based on these observations, all standard DNA polymerase assays using
GST/pol
were performed at pH 7.0 and 37 °C in the presence of
Mg2+.
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Fig. 2.
Optimization of reaction conditions for
GST/pol activity. A, the
effect of pH (lanes 1-5), temperature (lanes
6-9), and NaCl concentration (lanes 11-16) on
GST/pol
activity was investigated. The pH, temperatures, and NaCl
concentrations tested are indicated at the top of the
figure. The pH and temperature effects were assayed on the 24/44
primer-template while the NaCl titration was performed using the 27/44
primer-template. All reactions contained 2 nM GST/pol
(except lane 10; NP, no protein) and were performed at
30 °C unless indicated otherwise. B, pol
activity in
the absence of a metal cofactor (lane 1), in the presence of
5 mM MgCl2 (lane 2) or 5 mM MnCl2 (lane 3). All reactions
contained 2 nM GST/pol
and the 25/44
primer-template.
in
the experiments described above suggested that human pol
is endowed
with limited processivity, as has also been observed for the E. coli DinB protein (6, 7). We therefore examined whether purified
human PCNA, a sliding clamp known to stimulate the processivity of the
replicative DNA polymerases pol
and pol
(18-20), increases the
extent of DNA synthesis by GST/pol
. The ability of PCNA to stimulate
the activity of pol
on short oligonucleotide-derived primer-templates has been observed previously (21). As shown in Fig.
3, addition of recombinant human PCNA had
no detectable effect on GST/pol
activity (Fig. 3, lanes
1-4) but could readily stimulate synthesis of full-length
products by purified pol
(Fig. 3, lanes 5-7) on a
slightly longer template. As was observed on the shorter template, no
effect of PCNA on pol
activity was observed on the longer template
nor did the PCNA possess polymerase activity of its own (data not
shown). In contrast to pol
, E. coli pol IV is stimulated
3000-fold by addition of the bacterial sliding clamp
,
complex
(7). A comprehensive study of the effects on replication factors RPA,
RFC, and PCNA on pol
is currently underway.
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Fig. 3.
The DNA polymerase activity of
GST/pol in vitro is not stimulated
by PCNA. The activity of 2 nM GST/pol
was assayed
in the absence (lane 2) or presence (lane 3) of
30 ng of recombinant human PCNA. The activity of PCNA alone is shown in
lane 4. Lane 1 contained no added protein. Stimulation of
pol
activity by either 30 ng (lane 6) or 60 ng
(lane 7) of PCNA. Lane 1 contained pol
but no
added PCNA. Reactions in lanes 1-4 were performed at
30 °C for 5 min on the 24/44 primer-template. Reactions in
lanes 5-7 contained 0.2 units of calf thymus pol
and
were performed for 10 min at 37 °C using the 20/54
primer-template.
Is a Template-directed DNA Polymerase Lacking 3'
5'
Proofreading Exonuclease Activity--
To demonstrate that GST/pol
is a template-directed DNA polymerase we performed polymerase assays in
the presence of single deoxyribonucleoside triphosphates (dNTPs) on 4 different primer-templates, each designed to test for the correct
incorporation of a particular dNTP. As shown in Fig.
4A, under the single set of
conditions tested GST/pol
preferentially incorporated the correct
nucleotide on each template. However, in all cases significant levels
of misincorporation were also observed. For example, on the 27/44 primer-template GST/pol
primarily catalyzed the accurate
incorporation of dGTP as the first nucleotide, but also supported
misincorporation of dATP and to a lesser extent dCTP (Fig. 4A,
lanes 7-11). It can also be observed from Fig. 4A that
the level of GST/pol
activity on the 24/44 substrate was
significantly and consistently lower than on the other
primer-templates. The cause of this phenomenon is presently unclear,
but is most likely related to the immediate sequence context and is
apparently exacerbated by decreasing the dNTP concentration from 100 µM to 10 µM (compare Fig. 3A, lane 2, and Fig. 4A, lane 6). Poor extension of this
primer-template by E. coli pol IV was also observed by
Wagner et al. (6).
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Fig. 4.
GST/pol is a
template-directed DNA polymerase lacking a 3'
5' proofreading exonuclease activity. A,
GST/pol
activity was assayed on the indicated templates in the
presence of either 10 µM individual dNTPs (lanes
2-5, 8-11, 14-17, and 20-23) or a 10 µM pool of all four dNTPs (lanes 6, 12, 18, and 24). Reactions lacking GST/pol
contained all four
dNTPs (lanes 1, 7, 13, and 19). B,
GST/pol
, Klenow (exo
), and Klenow (exo+)
were assayed on the 3' mispaired 31/44 primer-template in the absence
(lanes 2-4) or presence of dNTPs (lanes 6-8).
Reactions contained 2 nM GST/pol
or 5 units of Klenow
where indicated. Lanes 1 and 5 contained no added
protein. The partial sequence of each primer-template is shown at the
top of each panel. For panel B, the size of the
expected full-length product (44 nucleotides) produced by Klenow
(exo+) is indicated by an arrow.
for 3'
5' proofreading exonuclease activity. Using a substrate in which the 3' nucleotide of
the primer was not base paired with the template, no shortening of the
primer by GST/pol
or Klenow (exo
) enzyme was observed
in the absence of dNTPs (Fig. 4B, lanes 2 and 3).
In contrast, Klenow (exo+) enzyme readily cleaved the
primer (Fig. 4B, lane 4). In the presence of dNTPs, the
primer could be efficiently extended by Klenow (exo+)
enzyme only following cleavage of the mispaired base (Fig. 4B, lane 8). Limited extension by GST/pol
was also observed from the 3' mispaired primer (Fig. 4B, lane 6). The low level of
primer extended by Klenow (exo
) enzyme yielded a product
45 nucleotides in length due to incorporation of an additional dATP in
a template-independent fashion (22). This nucleotide is normally
removed by the 3'
5' exonuclease activity of Klenow
(exo+) enzyme. The detectable levels of misincorporation
together with the observed lack of a proofreading exonuclease activity
suggest that pol
is endowed with a low level of fidelity during
synthesis of DNA.
we tested
the sensitivity of the enzyme to aphidicolin and dideoxynucleoside triphosphates (ddNTPs). Aphidicolin is an inhibitor of eukaryotic DNA
polymerases
,
, and
while pol
and -
are sensitive to ddTTP (23). In the presense of 25 µM dNTPs GST/pol
activity was not inhibited by either aphidicolin (50 ng/µl) or up to
an 8-fold molar excess of ddNTPs (data not shown). The lack of
sensitivity of pol
to aphidicolin and ddNTPs is similar to that
observed for human pol
(24).
Cannot Bypass a Cisplatin Adduct but Can Bypass an AAF
Lesion in a Potentially Error-prone Manner--
A number of the DNA
polymerases in the UmuC/DinB superfamily have been implicated in DNA
damage-induced mutagenesis and translesion synthesis (2). We asked
whether GST/pol
is able to bypass cisplatin (Fig.
5) or an AAF adduct (Fig.
6), lesions that have previously been
shown to strongly block other DNA polymerases (reviewed in Ref.
25). GST/pol
is unable to bypass
d[GpG-N7(1)-N7(2)] cisplatin intrastrand
cross-links, even when a 2-fold molar excess of enzyme is added to the
primer-template (Fig. 5, lanes 6-9). Klenow
(exo
) enzyme is also unable to bypass this lesion (Fig.
5, lane 10), unless high concentrations of enzyme are added
(data not shown). Both GST/pol
and Klenow (exo
) enzyme
arrest one nucleotide prior to the lesion, suggesting that neither
enzyme is able to efficiently insert nucleotides across from the
damaged bases.
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Fig. 5.
GST/pol is unable to
bypass a cisplatin adduct. Replication of 5 nM
wild-type (lanes 2-4) or cisplatin-containing (lanes
7-9) primer-templates was tested using the indicated
concentrations of GST/pol
. Reactions were performed for 10 min at
37 °C. 1 nM Klenow (exo
) enzyme was used
as a control (lanes 5 and 10). The position of
the cisplatin adduct is indicated at the right; the first G
of the adduct is located at the 30 nucleotide (nt) position.
The unextended running start primer (lanes 1 and
6) is 20 nucleotides and the full-length extension product
is 53 nucleotides in length.
View larger version (38K):
[in a new window]
Fig. 6.
GST/pol displays
inefficient, potentially error-prone bypass of an AAF lesion.
A, replication of 5 nM wild-type (lanes
2-4) or AAF-containing (lanes 7-9) primer-templates
was tested using the indicated concentrations of GST/pol
. Reactions
were for 10 min at 37 °C. 1 nM Klenow
(exo
) enzyme was used as a control (lanes 5 and 10). The position of the G-AAF adduct is indicated at
the right and is located at the 30 nucleotide
(nt) position. The unextended running start primer
(lanes 1 and 6) is 20 nucleotides and the
full-length extension product is 53 nucleotides in length.
B, specificity of nucleotide incorporation by GST/pol
across from undamaged G (left) or G-AAF (right).
Nucleotide incorporation was tested using 2.5 nM GST/pol
and either 100 µM individual dNTPs (lanes 2-5
and 8-11) or a 100 µM pool of all four dNTPs
(lanes 6 and 12) for 10 min at 37 °C.
Reactions lacking GST/pol
contained all four dNTPs (lanes
1 and 7). A standing start primer of 29 nucleotides was
used; the first nucleotide incorporated is directly across from the
G-AAF lesion (or undamaged control).
does
appear to have an intrinsic ability to bypass
N-(deoxyguanosin-8-yl)-N-2-acetylaminofluorene (G-AAF) adducts (Fig. 6A, lanes 6-9). The bypass appears to
be relatively inefficient under the conditions used in these
experiments, since relatively high enzyme concentrations are necessary
to achieve bypass. Furthermore, even under conditions where bypass
occurs, a large percentage of the primer extension products appear to terminate at the site of the AAF lesion. Since it is clear that the
GST/pol
enzyme is able to insert nucleotides across from the AAF
lesion, we were interested in determining which nucleotides could be
incorporated. As shown in Fig. 6B, GST/pol
primarily inserts dCTP across from an undamaged G residue (lanes
2-5). In contrast, GST/pol
appears equally able to incorporate
either dTTP or dCTP across from the G-AAF lesion (Fig. 6B, lanes
8-11). A darker exposure of this experiment shows that dATP can
also be incorporated across from the G-AAF lesion at much lower
frequency (data not shown). These results suggest that pol
may play
a role in bypass of AAF lesions in vivo and that such bypass
could potentially be mutagenic.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. While this work was
in progress, a GST/hDinB1 fusion protein purified from the yeast
Saccharomyces cerevisiae was reported to have DNA polymerase
activity (26). These authors designated human DinB1 protein as DNA
polymerase
(pol
). In view of the fact that this name has been
assigned to the product of another gene (27), we suggest that the DinB1 gene product be referred to as pol
, a designation approved by the
Human Genome Organization nomenclature committee.
associated with mutation
of two highly conserved residues known to be required for the activity
of both E. coli pol IV and yeast pol
(6, 28) eliminates
the possibility that the activity of our purified GST/pol
fraction
is due to the presence of a contaminant or interacting protein. We can
also conclude that fusion of pol
to GST does not abolish its DNA
polymerase activity. However, we cannot exclude the possibility that
the GST domain alters the DNA polymerase activity of pol
in a more
subtle way. Pol
represents the tenth eukaryotic polymerase reported,
and the seventh member of the UmuC/DinB superfamily of proteins endowed
with such activity. It is therefore likely that most if not all members
of this superfamily are DNA polymerases or nucleotidyl transferases.
is a single nucleotide shorter than the template. This
phenomenon was consistently observed on templates used in the present
studies regardless of the primer (e.g. Figs. 1B
and 4B). Similar observations have been made using other
templates of varying length and sequence
composition2 and were
observed independently by Johnson et al. (26). The incomplete extension products produced by pol
could be extended by
the Klenow fragment of E. coli DNA polymerase I,
demonstrating that failure to replicate precisely to the end of a
linear DNA template is likely an intrinsic property of pol
rather
than the result of template slippage.2 Conceivably the
enzyme requires interactions with sequences downstream to those located
at the active site in order to form a stable protein-DNA complex. The
ability of pol
to fill in short single-stranded gaps is currently
being investigated.
polymerase assays the reaction products
consisted of a ladder of primer extension products, ranging from the
addition of a single nucleotide to 1 nucleotide short of full-length. A
similar size range of products was observed at high GST/pol
dilutions where the primer-template was present in vast excess (29).
These results suggest that the processivity of pol
is variably low
to moderate, but not entirely distributive as is the case for E. coli DNA pol IV (6, 7). Similarly, the apparent high rate of
nucleotide misincorporation (Fig. 4A) and lack of 3'
5'
proofreading exonuclease activity (Fig. 4B) suggest that
pol
possesses low fidelity. These conclusions are supported by the
results of independent experiments that quantitatively measured the
fidelity and processivity of pol
in vitro (29).
are consistent with a similar role in
human cells. The demonstration that the human DINB1 gene
encodes pol
supports a model in which the role of this polymerase is
to facilitate the replication of abnormal templates which inhibit the
activity of replicative DNA polymerases pol
, pol
, and/or
pol
.
fusion protein is unable
to bypass abasic sites, cis-syn thymine-thymine dimers or
6-4 photoproducts in vitro (26).
is also unable to bypass
a cisplatin lesion. The enzyme is, however, able to inefficiently bypass an AAF-adduct and incorporates primarily either dTTP or dCTP
across from the lesion. This result suggests that pol
has the
potential to bypass AAF by an error-prone mechanism and therefore that
pol
might play a role in targeted mutagenesis. However, the
characterization of translesion synthesis using simple primer extension
assays in vitro requires caution. A number of experimental parameters may influence the outcome, including reaction conditions, enzyme concentration, nucleotide concentration, and template sequence context (25). The physiological relevance of the bypass of AAF lesions
by pol
is uncertain since it is observed only at high enzyme
concentrations, and even under these conditions a significant fraction
of the enzyme is arrested at the site of the lesion. However, it
remains a formal possibility that pol
can be utilized in human cells
for translesion synthesis of specific lesions (such as AAF or other
types of DNA damage which have not yet been tested), as may be the case
for pol
(30).
may function in additional or alternative modes
other than the replication of abnormal templates. For example, the
enzyme may be required to replicate specific normal template regions in
an abnormal (error-prone) manner, such as appears to be required during
somatic hypermutation of immunoglobulin genes (31). More precise
definition of the biological function(s) of this enzyme will require
further detailed biochemical and genetic studies, including the
phenotypic characterization of mammalian cells defective in this enzyme activity.
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ACKNOWLEDGEMENTS |
---|
We thank Rajnish Bharadwaj for providing SF9
insect cells, Lisa McDaniel for advice on preparing nuclear and
cytoplasmic extracts from insect cells, the laboratory of Clive
Slaughter for mass spectral analysis, Rick Wood for providing
recombinant human PCNA protein, and Ulrich Hübscher for providing
calf-thymus pol. We are particularly grateful to Tom Kunkel and
Haruo Ohmori for helpful discussions and communication of results prior
to publication.
![]() |
FOOTNOTES |
---|
* This work was supported in part by NCI, National Institutes of Health Grant CA69029 (to E. C. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Contributed equally to the results of this study.
§ Supported by NCI, National Institutes of Health Postdoctoral Fellowship CA75733.
¶ Supported by NCI, National Institutes of Health Postdoctoral Fellowship CA83314.
To whom correspondence should be addressed. Tel.:
214-648-4020; Fax: 214-648-4067; E-mail:
friedberg.errol@pathology.swmed.edu.
Published, JBC Papers in Press, October 6, 2000, DOI 10.1074/jbc.M004413200
2 V. L. Gerlach, W. J. Feaver, and E. C. Friedberg, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GST, glutathione S-transferase; AAAF, N-acetoxy-2-acetylaminofluorene; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; PCNA, proliferating cell nuclear antigen.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Gerlach, V. L.,
Aravind, L.,
Gotway, G.,
Schultz, R. A.,
Koonin, E. V.,
and Friedberg, E. C.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
11922-11927 |
2. | Friedberg, E. C., and Gerlach, V. L. (1999) Cell 98, 413-416[Medline] [Order article via Infotrieve] |
3. | Kenyon, C. J., and Walker, G. C. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 2819-2823[Abstract] |
4. | Brotcorne-Lannoye, A., and Maenhaut-Michel, G. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 3904-3908[Abstract] |
5. |
Kim, S. R.,
Maenhaut-Michel, G.,
Yamada, M.,
Yamamoto, Y.,
Matsui, K.,
Sofuni, T.,
Nohmi, T.,
and Ohmori, H.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
13792-13797 |
6. | Wagner, J., Gruz, P., Kim, S., Yamada, M., Matsui, K., Fuchs, R. P. P., and Nohmi, T. (1999) Mol. Cell 4, 281-286[Medline] [Order article via Infotrieve] |
7. | Tang, M., Pham, P., Shen, X., Taylor, J. S., O'Donnell, M., Woodgate, R., and Goodman, M. F. (2000) Nature 404, 1014-1018[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Ogi, T.,
Kato, T., Jr.,
Kato, T.,
and Ohmori, H.
(1999)
Genes Cell
4,
607-618 |
9. | Fischhaber, P. L., Reese, A. W., Nguyen, T., Kirchner, J. J., Hustedt, E. J., Robinson, B. H., and Hopkins, P. B. (1997) Nucleosides and Nucleotides 16, 365-377 |
10. | Kriek, E., Miller, J. A., Juhl, U., and Miller, E. C. (1967) Biochemistry 6, 177-182[Medline] [Order article via Infotrieve] |
11. | Kriek, E. (1969) Chemico-Biological Interact. 1, 3-17[Medline] [Order article via Infotrieve] |
12. | Kriek, E., and Reitsema, J. (1971) Chemico-Biological Interact. 3, 397-400[Medline] [Order article via Infotrieve] |
13. | Bruhn, S. L., Toney, J. H., and Lippard, S. J. (1990) Prog. Inorg. Chem. 38, 477-516 |
14. | Biggerstaff, M., and Wood, R. D. (1999) in DNA Repair Protocols: Eukaryotic Systems (Henderson, D. S., ed) , pp. 357-372, Humana Press, Totowa, NJ |
15. |
Schumacher, S. B.,
Stucki, M.,
and Hübscher, U.
(2000)
Nucleic Acids Res.
28,
620-625 |
16. | Weissbach, A., Baltimore, D., Bollum, F., Gallo, R., and Korn, D. (1975) Science 190, 4014 |
17. | Burgers, P. M., Bambara, R. A., Campbell, J. L., Chang, L. M., Downey, K. M., Hübscher, U., Lee, M. Y., Linn, S. M., So, A. G., and Spadari, S. (1990) Eur. J. Biochem. 191, 617-618[Medline] [Order article via Infotrieve] |
18. | Prelich, G., Tan, C. K., Kostura, M., Matthews, M. B., So, A. G., Downey, K. M., and Stillman, B. (1987) Nature 326, 517-520[CrossRef][Medline] [Order article via Infotrieve] |
19. | McConnell, M., Miller, H., Mozzherin, D. J., Quamina, A., Tan, C. K., Downey, K. M., and Fisher, P. A. (1996) Biochemistry 35, 8268-8274[CrossRef][Medline] [Order article via Infotrieve] |
20. | Hindges, R., and Hübscher, U. (1997) Biol. Chem. 378, 345-362[CrossRef] |
21. |
Mozzherin, D. J.,
Shibutani, S.,
Tan, C. K.,
Downey, K. M.,
and Fisher, P. A.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
6126-6131 |
22. | Clark, J. M., Joyce, C. M., and Beardsley, G. P. (1987) J. Mol. Biol. 198, 123-127[Medline] [Order article via Infotrieve] |
23. | Wang, T. S. F, Conger, K. L., Copeland, W. C., and Arroyo, M. P. (1999) in Eukaryotic DNA Replication: A Practical Approach (Cotterill, S., ed) , pp. 67-92, Oxford University Press, New York |
24. |
Masutani, C.,
Araki, M.,
Yamada, A.,
Kusumoto, R.,
Nogimori, T.,
Maekawa, T.,
Iwai, S.,
and Hanaoka, F.
(1999)
EMBO J.
18,
3491-3501 |
25. | Hatahet, Z., and Wallace, S. S. (1998) in DNA Damage and Repair, Vol. 1: DNA Repair in Prokaryotes and Lower Eukaryotes (Nickoloff, J. A. , and Hoekstra, M. F., eds) , pp. 229-262, Humana Press Inc., Totowa, NJ |
26. |
Johnson, R. E.,
Prakash, S.,
and Prakash, L.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
3838-3843 |
27. | Sharief, F. S., Vojta, P. J., Ropp, P. A., and Copeland, W. C. (1999) Genomics 59, 90-96[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Johnson, R. E.,
Prakash, S.,
and Prakash, L.
(1999)
J. Biol. Chem.
274,
15975-15977 |
29. | Ohashi, E., Bebenek, K., Matsuda, T., Feaver, W. J., Gerlach, V. L., Friedberg, E. C., Ohmori, H., and Kunkel, T. A. (2000) J. Biol. Chem. 275, in press |
30. |
Masutani, C.,
Kusumoto, R.,
Iwai, S.,
and Hanoaka, F.
(2000)
EMBO J.
19,
3100-3109 |
31. |
Friedberg, E. C.,
Feaver, W. J.,
and Gerlach, V. L.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
5681-5683 |