From the Radiation Biology Center, Kyoto University,
Kyoto 606-8501, § Biomolecular Engineering Research
Institute, Suita, Osaka 565-0874, ¶ Research Institute for
Microbial Diseases, Osaka University, Suita, Osaka 565-0871,
Institute for Molecular and Cellular Biology, Osaka University,
Suita, Osaka 565-0871, ** Mitsubishi Kasei Institute of Life Sciences,
Machida, Tokyo 194-8511, and
Institute for
Virus Research, Kyoto University, Kyoto 606-8507, Japan
Received for publication, October 27, 2000, and in revised form, January 10, 2001
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ABSTRACT |
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cDNA sequences were identified and isolated
that encode Drosophila homologues of human Rad30A and
Rad30B called drad30A and drad30B. Here we show
that the C-terminal-truncated forms of the drad30A and
drad30B gene products, designated dpol DNA is frequently damaged by environmental and endogenous
genotoxic agents. Although various mechanisms exist to ensure that the
majority of DNA damage is recognized and repaired and the integrity of
the DNA is faithfully restored, some DNA lesions escape repair and
persist in the cell. Unrepaired DNA
lesions can block the progress of the replication machinery. To help
resolve this problem, cells have specialized polymerases that carry out translesion DNA synthesis (TLS)1, which is a mechanism that
permits nucleotides to be incorporated opposite lesions. After TLS
bypasses the DNA damage, replication can continue with the normal
replication machinery downstream of the site of the damage.
Recently, a new family of DNA polymerases was identified called the
UmuC/DinB/Rev1/Rad30 superfamily (1-3). Several of these polymerases
participate in TLS (4-11), and members of this protein family exhibit
lower fidelity and processivity than replicative DNA polymerases (10,
12-15). The lower fidelity enables these polymerases to carry out TLS
and the lower processivity facilitates dissociation of these enzymes
after bypass of a lesion; this is important because it allows the
normal replication machinery, with high fidelity and processivity, to
preferentially carry out DNA synthesis distal to the lesion.
However, TLS is mutagenic when the base inserted opposite the DNA
lesion is different than the base normally inserted opposite an
undamaged base at that site. The chemical structure of the lesion is an
important determinant for mutagenic or nonmutagenic bypass during TLS.
Irradiation of DNA with UV light produces a variety of photoproducts
that can cause mutations (16). cis-syn-cyclobutane pyrimidine dimer (CPD) and (6-4)-photoproducts ((6-4)PP) are the two
major classes of UV-induced DNA photoproducts. In DNA irradiated with
UV, CPD is the most abundant lesion, and it is ~3-5-fold more
abundant than (6-4)PP. However, the mutagenic properties of these two
lesions are different, and the relative contributions of the two
lesions to the mutagenesis are not simply proportional to their
relative abundance. The mutagenic specificities of CPD and (6-4)PP
have been studied using well characterized site-specific UV
photoproducts in DNA substrates. The mutation frequency obtained either
with a site-specific TT-CPD or a TC-CPD is quite low (17-18), and in
comparison, (6-4)PP is more mutagenic. The (6-4)PP at the 5'-TT site
was highly mutagenic, and most of the mutations induced by this lesion
are T to C transitions at the 3'-T (19). However, this type of mutation
is not very common, because the (6-4)PP at the TT site is relatively
rare. On the other hand, the TC-(6-4)PP is less mutagenic than the
TT-(6-4)PP but more mutagenic than the TT-CPD. Of the mutations at
this site, 80% were C to T transitions at the 3' base (20). Since TC
to TT is frequently observed in DNA exposed to UV and (6-4)PP forms most frequently in the TC sequence, the TC-(6-4)PP could be a candidate for a strongly premutagenic lesion.
Human cells have three TLS polymerases that belong to the
UmuC/DinB/Rev1/Rad30 superfamily: pol The goal of this work was to identify and characterize the
Drosophila homologue(s) of the UmuC/DinB/Rev1/Rad30
superfamily. Two cDNAs were isolated that encode
Drosophila Rad30A and Rad30B, called drad30A and
drad30B. Truncated forms of dRAD30A and dRAD30B proteins,
designated dpol Isolation of Drosophila rad30A and rad30B cDNAs--
To
identify the genes encoding the UmuC/DinB/Rev1/Rad30 superfamily
proteins in Drosophila, we searched the data base of
expressed sequence tag (GenBankTM dbEST) for
Drosophila cDNAs that share homology with the proteins of this family. Several Drosophila cDNA sequences that
share significant homology with the proteins of the superfamily were
identified. Three such clones, SD05329, LD09220, and GH11153, were
obtained from Research Genetics (Huntsville, AL), and the entire insert of each clone was sequenced with an automated DNA sequence analyzer (Applied Biosystems PRISM310). Translation of the large continuous open
reading frame from each cDNA revealed that each translated protein
shares significant similarity to Rad30A, Rad30B, and Rev1 proteins,
respectively. Thus, the clones were designated as drad30A, drad30B, and drev1, respectively. The cDNAs
did not contain in-frame stop codons. Thus, the 5' ends of each clone
were amplified by 5'-rapid amplification of cDNA ends. The sequence
determination of the amplified 5' ends revealed the presence of the
in-frame stop codon. Screening of the dbEST indicated that
Drosophila seems to lack a true DinB ortholog, which was
confirmed by searching the recently completed sequence of the
Drosophila genome. The drad30A and
drad30B cDNAs each hybridized to Drosophila
polytene chromosomes at a single site corresponding to bands 3L-79BC
and 3R-84EF, respectively.
Overproduction of the dRAD30A and dRAD30B Proteins--
The
expression vector used in these studies was pGEX-4T (Amersham Pharmacia
Biotech). To overexpress the Drosophila dRAD30A and dRAD30B
proteins, each gene was cloned in-frame with the glutathione S-transferase gene. A chimeric GST-drad30A gene
was constructed by recloning the 3-kilobase pair
EcoRI/XhoI DNA fragment from the EST clone
SD05329 in EcoRI/XhoI-digested pGEX-4T-1, thus
generating plasmid pGEX-dRad30A. The C-terminal deletion mutant of the
drad30A gene was constructed by recloning the 1640-base pair
EcoRI/Eco52I DNA fragment, which contains the
N-terminal 545 residues, in EcoRI/NotI-digested pGEX-4T-1, thus generating pGEX-dRad30A
Mutant forms of the GST-rad30A and
GST-rad30B genes were constructed by utilizing the
Mutant-Super Express mutagenesis kit (Takara, Japan). The adjacent
residues, Asp126-Glu127 in drad30A
and D115-E116 in drad30B, were changed to alanine using
primers R30A-AA 5'-GCTTCCGTGGCAGCCGCGTACT -3' and R30B-AA 5'-CTAGGCTTCGCTGCAAACTTTA-3', respectively. The 400-base pair SphI fragment from pKF30A and the 800-base pair
EcoRI/BglII fragment from pKF30B were sequenced
to verify that the region only contained the desired mutation, and then
each fragment was subcloned into the similarly digested pGEXrad30A
E. coli JM109 harboring pGEX-dRad30A Purification of the dRAD30A and dRAD30B Protein--
To purify
the GST-tagged dRAD30A or dRAD30B, E. coli cells were
resuspended in phosphate-buffered saline and disrupted using a
sonicator before centrifugation at 10,000 × g. 10 mM Mg-ATP and 5 mg/ml casein were added to the extract,
which was then incubated at room temperature for 20 min. The extract
was then passed over a 5-ml glutathione-Sepharose 4B column (Amersham
Pharmacia Biotech) and washed with 10 column volumes of wash buffer (1 M NaCl and 50 mM Tris-HCl (pH 8.0)). The
GST-tagged protein was eluted from the GST column in a stepwise fashion
by a wash with five column volumes of elution buffer (50 mM
Tris-HCl (pH 8.0), 10 mM glutathione). The GST fusion
protein in each eluate was monitored by the GST activity according to
the manufacturer's protocol (Amersham Pharmacia Biotech), and the
eluates containing GST activity were pooled. The pooled eluates were
concentrated by Centriplus 50 ultrafiltration (Amicon) to an
approximate final volume of 1.0 ml. The concentrated eluate was applied
to a HiTrap Q column (Amersham Pharmacia Biotech) equilibrated with
buffer A (50 mM Tris (pH 8.0)). The column was subsequently
washed with 10 column volumes of buffer A, and the GST fusion protein
was eluted with a 0-1 M linear gradient of NaCl in buffer
A. The GST activity in each fraction was measured, and the positive
fractions were pooled. The pooled fractions were dialyzed against
buffer A and loaded on a HiTrap heparin column (Amersham Pharmacia
Biotech) equilibrated with buffer A. The column was washed with buffer
A, and the protein was eluted by a linear gradient of 0-0.5
M NaCl in buffer A. Glycerol was added to the GST-containing fraction (10% v/v) before storage at
The eluate from the glutathione-Sepharose 4B column contained a
contaminating DNA polymerase activity derived from E. coli because the eluate from the column of the cell extract from E. coli harboring the GST vector had a weak DNA polymerase activity. To remove the contaminating DNA polymerase activity, we tested two
kinds of antibodies, one specific for E. coli pol I and
another specific for pol II. The addition of the antibody for pol I to the eluate, but not the antibody for pol II, eliminated the DNA polymerase activity of the eluate from E. coli expressing
GST, indicative that the contaminating DNA polymerase is E. coli pol I. The pol I antibody also eliminated the DNA polymerase
activity in the eluate of the cell extract from E. coli
harboring the mutant plasmids, pGEXrad30A-DE126AA and
pGEXrad30B-DE115AA. Thus, the pol I antibody was added to the purified
protein in all experiments in this paper. However, after the addition
of pol I antibody, a slight 3'-exonuclease activity still remained.
DNA Polymerase Assays--
The 30-mer oligomer (non-damaged TT)
with the sequence 5'-CTCGTCAGCATCTTCATCATACAGTCAGTG-3' was used as the nondamaged template. The same 30-mer oligomers containing the CPD (29) and the (6-4)PP (30) at the underlined sites were
synthesized as described. The 30-mer oligomers, in which the underlined
TT sequence of the "nondamaged TT" oligomers was changed to TC,
were also used as nondamaged templates (designated nondamaged TC), and
the 30-mer oligomers containing (6-4)PP at the TC site were
synthesized as described (31). Various lengths (16-18-mer) of
oligomers with sequences complementary to the 30-mer template were
synthesized and used as primers. Each primer was labeled at the 5' end
using T4 polynucleotide kinase and [ Detection of Correctly Replicated Products Opposite
Lesions--
Two kinds of oligomers were used: 49-mers bearing the
centrally located (6-4)PP or CPD at the TT site (32) and 30-mers bearing the centrally located (6-4)PP at the TC site (31). The
substrate sequence is as follows (the introduced pyrimidine dimer is
underlined): 49-mer,
5'-AGCTACCATGCCTGCACGAATTAAGCAATTCGTAATCATGGTCATAGCT-3', and 30-mer, CCTGGTTAGTACTTCGATTTAGGTGACACT. The
photoproduct in the 49-base pair duplex DNA is at an MseI
site (MseI cleaves between the two thymines in the TTAA
sequence), and that in the 30-base pair duplex DNA is at a
TaqI site (TaqI cleaves between the two pyrimidines in the TCGA sequence). The assay measures the
susceptibility of the bypass product to MseI cleavage. A
20-mer oligomer (5'-ACCATGATTACGAATTGCTT-3') and a 15-mer oligomer
(5'-AGTGTCACCTAAATC-3') with sequences complementary to the 49- and
30-mer damage-containing strands, respectively, were synthesized and
used as primers. After labeling with [ Isolation of Drosophila drad30A and drad30B cDNAs--
Two
Drosophila cDNA clones were identified in the
GenBankTM dbEST data base, which had significant homology
to human Rad30A and Rad30B protein, and designated drad30A
and drad30B. DNA sequence analysis of the full-length
cDNAs revealed that drad30A encodes a protein of 885 amino acids, with 31% identity to the human XPV/RAD30A protein
(hpol
The amino acid sequences were aligned for all the members of the
UmuC/DinB/Rev1/Rad30 protein family, and the alignment was used to
generate a phylogenetic tree (Fig. 1C). The tree reveals several distinct subgroups of proteins; the validity of these subgroups
is convincingly supported by the bootstrap test. Since the Rad30B
subfamily is equally distant from the RAD30A and DinB subfamilies, the
superfamily can be classified into five subgroups; UmuC, DinB, Rev1,
Rad30A, and Rad30B. The Drosophila genes drad30A and drad30B belong to the Rad30A and Rad30B subfamilies,
respectively. A Drosophila homologue of human Rev1 was also
identified, which belongs to the Rev1 subfamily as shown in Fig.
1C; however, no Drosophila homologue of the human
DINB1 protein (hpol Overproduction and Purification of dRAD30A and dRAD30B
Proteins--
The proteins encoded by drad30A and
drad30B (dRAD30A and dRAD30B, respectively) were purified as
recombinant fusion proteins with a GST tag at the N terminus.
Initially, the entire coding sequences of the drad30A and
drad30B genes were fused with the GST gene;
however, these constructs produced a very low yield of the desired
recombinant proteins in E. coli. However, when a mutation
was introduced in each gene that creates a stop codon in the C-terminal
region, the resulting C-terminal-truncated proteins were expressed at a
higher level in E. coli. These truncated forms of the
drad30A and drad30B gene products are similar in
size to the truncated forms of hpol DNA Polymerase Activities of dRAD30A
If the observed DNA polymerase activity is intrinsic to the purified
protein, then site-directed mutagenesis of amino acids in the enzyme
active site should create a mutant deficient in DNA polymerase
activity. This prediction was tested by carrying out site-directed
mutagenesis on adjacent Asp and Glu residues within motif III that are
believed to be critical for the catalytic activity of the
UmuC/DinB/Rad30 family proteins (4, 9, 10, 22, 28, 35, 36). The highly
conserved Asp and Glu residues are present at positions 126 and 127 and
at positions 115 and 116 in dRAD30A and dRAD30B, respectively (Fig.
1B). In the mutants generated for this study, these residues
were changed to alanine. The mutant proteins displayed the same
chromatographic properties as the wild type protein, but they lacked
DNA polymerase activity (Figs. 2, B and C,
lanes 2-4). Therefore, the observed DNA polymerase activity
is intrinsic to the dRAD30A and dRAD30B proteins. We therefore propose
that dRAD30A be renamed dpol
The nucleotide selectivity of dpol Translesion Synthesis by dpol Nucleotide Selectivity of dpol
It should be noted that on the template containing no damage, both
dpol Influence of Base-pairing at the Lesion on Elongation Past the
Lesion by dpol
Next, such primers were annealed to TT-(6-4)PP or TC-(6-4)PP to
examine whether they could be extended by dpol Direct Analysis of Bypass Products Synthesized by dpol Damage-induced mutagenesis is an important biological process
whose molecular mechanism is not yet understood and which is the
subject of much current research. Recently, the proteins of the
UmuC/DinB/Rev1/Rad30 superfamily have been isolated and characterized biochemically, and it was shown that these proteins have a
nonprocessive DNA polymerase activity that can bypass DNA lesions.
Thus, these enzymes are central to the process of damage-induced
mutagenesis. In this study, Drosophila homologues of this
polymerase superfamily, dpol Saccharomyces cerevisiae and human pol However, evidence also indicates that dpol Despite the biological relevance of this DNA lesion, the tertiary
structures of oligonucleotide duplexes, which include TC-(6-4)PP, have
not been well characterized. Fujiwara and Iwai (42) show that the
duplex containing G opposite the 3'-C of TC-(6-4)PP is thermodynamically more stable than another duplex (42). Horsfall and
Lawrence (20) studied the mutagenicity of TC-(6-4)PP in E. coli cells and found that 66% of the bypass products have the correct TC sequence, 28% of the mutants having the TT sequence (20).
Our observation that both G and A are incorporated opposite the 3'-C of
TC-(6-4)PP (Fig. 4B) fit well with these results. On the
other hand, the nucleotide incorporated opposite the 5'-T of
TC-(6-4)PP is not consistent with the mutation spectrum. Although the
in vivo mutation spectrum indicates that A is incorporated preferentially opposite the 5'-T of TC-(6-4)PP, our in
vitro results indicated that dpol The mutagenic properties of dpol In the Rad30 protein family, dpol In this study, TT (6-4)PP was bypassed by dpolC and
dpol
C, respectively, exhibit DNA polymerase activity. dpol
C
and dpol
C efficiently bypass a cis-syn-cyclobutane
thymine-thymine (TT) dimer in a mostly error-free manner. dpol
C
shows limited ability to bypass a 6-4-photoproduct ((6-4)PP) at
thymine-thymine (TT-(6-4)PP) or at thymine-cytosine (TC-(6-4)PP) in
an error-prone manner. dpol
C scarcely bypasses these lesions.
Thus, the fidelity of translesion synthesis depends on the identity of
the lesion and on the polymerase. The human XPV gene
product, hpol
, bypasses cis-syn-cyclobutane
thymine-thymine dimer efficiently in a mostly error-free manner but
does not bypass TT-(6-4)PP, whereas Escherichia coli DNA
polymerase V (UmuD'2C complex) bypasses both lesions, especially TT-(6-4)PP, in an error-prone manner (Tang, M., Pham, P.,
Shen, X., Taylor, J. S., O'Donnell, M., Woodgate, R., and Goodman, M. F. (2000) Nature 404, 1014-1018). Both
dpol
C and DNA polymerase V preferentially incorporate GA opposite
TT-(6-4)PP. The chemical structure of the lesions and the similarity
in the nucleotides incorporated suggest that structural information in the altered bases contribute to nucleotide selection during
incorporation opposite these lesions by these polymerases.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(encoded by the
XPV/RAD30A gene (21, 22)), pol
(encoded by
RAD30B (10, 23)), and pol
(encoded by DINB1
(24)). In addition, the human protein hRev1 is a homologue of
yeast REV1 (25). Pol
, pol
, and pol
bypass UV-induced damage
with different efficiencies. Pol
bypasses TT-CPD efficiently and
inserts AA opposite the lesion; however, it adds only one base opposite
the 3'-T of TT-(6-4)PP (5). Pol
bypasses TT-CPD and TT-(6-4)PP
with low efficiency, adding one or a few bases opposite these lesions
(26, 27). Pol
stops DNA synthesis and is blocked one base before
TT-CPD and TT-(6-4)PP (9). Thus, in human cells, none of these
polymerases bypass (6-4)PP. In contrast, Escherichia coli
DNA polymerase V (UmuD'2C (28)) efficiently bypasses both
lesions, inserting AA or GA opposite TT-CPD or TT-(6-4)PP,
respectively (8). Thus, the chemical structure and the properties of
each TLS polymerase determine which base is inserted and the likelihood
of a mutation at a specific lesion.
C and dpol
C, respectively, have been purified, and their properties were studied, including the mechanism of
UV-induced mutagenesis and the products of lesion bypass in reactions
with these enzymes. The bypass templates used in this study include
TC-(6-4)PP, TT-CPD, or TT-(6-4)PP; a template with TC-(6-4)PP was
studied because of the biological relevance of this lesion. The results
indicate that both polymerases bypass the TT-CPD in an error-free
manner. Furthermore, dpol
C bypasses TT-(6-4)PP and TC-(6-4)PP
in a highly error-prone manner.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
C. The chimeric
GST-drad30B gene was constructed as follows. The
EcoRI restriction site was generated just upstream of the
start codon by polymerase chain reaction using the primer
30B5'Eco, 5'- CGGAATTCATGGACTTCGCTAGCGTAC-3', and the 3-kilobase pair
EcoRI/XhoI DNA fragment from the EST clone LD09220 was then cloned in-frame with GST in the
EcoRI/XhoI-digested pGEX4T-1, thus generating
plasmid pGEX-dRad30B. The C-terminal deletion mutant of the
GST-rad30B gene was constructed by first synthesizing the
oligomer 30Bdel3'Xho, 5'- CCGGCTCGAGTGGGACTTGTGAGACTC-3'. Two
polymerase chain reaction primers, 30B5'Eco and 30Bdel3'Xho, were used
to amplify a 1.36-kilobase pair DNA fragment that contained the
N-terminal 448 residues of the drad30B gene. The polymerase chain reaction products were digested with EcoRI and
XhoI, and the resultant 1.35-kilobase pair fragment was
cloned in the EcoRI/XhoI-digested pGEX-4T-1, thus
generating pGEX-dRad30B
C.
C
and pGEXrad30B
C to create pGEXrad30A-DE126AA and pGEXrad30B-DE115AA, respectively.
C, pGEX-dRad30B
C,
pGEX-dRad30A-DE126AA, or pGEX-drad30B-DE115AA was grown in 1 liter of
LB medium containing 150 mg/liter ampicillin at 25 °C until an
A600 of 0.9-1.0 was reached. Isopropyl
-D-thiogalactopyranoside (0.1 mM) was added
to induce expression of the chimeric gene. After incubation at 25 °C
for 12 h, the cells were harvested by centrifugation and were
stored at
80 °C.
80 °C.
-32P]ATP and was
annealed with the template at a molar ratio of 1:1. Standard reactions
(10 µl) contained 40 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 100 µM each of the four
dNTPs, 10 mM dithiothreitol, 250 µg/ml bovine serum
albumin, 60 mM KCl, 2.5% glycerol, 40 nM primer-template, and the indicated amount of enzyme. After incubation at 37 °C for 15 min, the reactions were terminated by the addition of 10 µl of formamide followed by boiling. The products were
subjected to 20% polyacrylamide, 7 M urea gel
electrophoresis followed by autoradiography.
-32P]ATP, the
primer was annealed with the template and was used as the substrate for
DNA synthesis. DNA synthesis was carried out as described in the DNA
polymerase assay. After DNA synthesis, the replication products were
denatured by incubation at 95 °C for 5 min and then annealed with a
10-fold excess of the 49-mer nondamaged oligonucleotide (400 nM). The annealed duplex DNA was digested with one unit of
MseI or TaqI for 90 min at 37 or 65 °C,
respectively. The digested DNA was denatured at 95 °C for 5 min in
the presence of 50% formamide and was then separated on a 10%
polyacrylamide sequencing gel. The MseI or
TaqI-susceptible and -resistant fractions were quantified by
scanning the gels with a Fuji Bioimage analyzer (BAS-2000, Fuji Film,
Japan), and the ratio was calculated as follows. The amounts of the
undigested bypass products (Counts-bp) were obtained by measuring the
radioactivity of the areas corresponding to the 24-42-base fragments
or the 19-30-base fragments in each lane for the template containing no damage, TT-CPD and TT-(6-4)PP, or TC-(6-4)PP, respectively. The radioactivity of the same area in the control sample, which did not
contain enzyme, was used as the background for bypass products
(Counts-bp-bg). The amounts of the bypass products susceptible to each
enzyme were determined by measuring the radioactivity of the 19-base or
14-base fragments (Count-dig). The radioactivity of the same area of
the undigested reaction was measured and used as the background of the
digested products (Counts-dig-bg). The % restriction enzyme-sensitive
bypass products in Table I was calculated
as (Count-dig)
(Counts-dig-bg)/(Counts-bp)
(Counts-bp-bg) × 100.
Ratio of bypass products with normal sequences
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
), and drad30B encodes a protein of 737 amino acids, with 30% identity to the human RAD30B protein (hpol
). Previous studies show that the proteins in the UmuC/DinB/Rev1/Rad30 superfamily are most highly conserved in their N-terminal portion, which includes five discrete conserved regions (1, 33). As shown in Fig. 1, A and B, these
regions (I-V) are conserved in the N-terminal portion of all the
RAD30-like proteins including the Drosophila proteins
encoded by drad30A and drad30B.
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Fig. 1.
Deduced amino acid sequences of the
dRAD30A and dRAD30B proteins. A, schematic
representation of the conserved regions in the dRAD30A and dRAD30B
proteins. The conserved regions I-V are indicated. Schematic
representations of the C-terminal deletion mutants are also shown, and
the sites of the conserved DE sequences where the mutations were
introduced are indicated by arrows. aa, amino
acids. B, alignment of the region III amino acid sequences
of the Rad30A proteins from Homo sapiens (XPV),
Arabidopsis thaliana (AtRad30), Caenorhabditis
elegans (CeRad30), S. cerevisiae (ScRad30),
Schizosaccharomyces pombe (SpRad30), and Drosophila
melanogaster (DRad30A), and the Rad30B proteins from H. sapiens (hRad30B) and D. melanogaster (DRad30B). The
alignment was performed using the MegAlign program (DNA star Inc.,
Madison, WI). The exact locations of the amino acids in each protein
are indicated at the left-hand side of the figure. Residues identical
to dRAD30A or dRAD30B are indicated in black. The conserved
DE sequence within which mutations are produced is marked with
asterisks below the sequence. C, possible
phylogenetic tree of the DinB/UmuC/Rev1/Rad30 family proteins. The
accession numbers in the Swiss-Prot and GenBankTM for the
sequences and the species names are as follows EcUMUC (E. coli, P04152), AtRev1 (A. thaliana, T19K24.14 in
AC02342), CeRev1 (C. elegans, ZK675.2 in Z46812), ScREV1
(S. cerevisiae, P12689), SpREV1 (S. pombe,
SPBC1347.01c in AC035548), HsRev1 (H. sapiens, AF151538),
MmREV1 (Mus musculus, AF179302), DmRev1 (D. melanogaster, AB049435), EcDINB (E. coli, Q47155),
HsDINB1 (H. sapiens,
AA576919), MmDINB1 (M. musculus,
AB027563), SpDINBh (S. pombe, SPCC553.07c), CeDINBh
(C. elegans, F22B7.6), AtRAD30 h (A. thaliana,
T19K24.15 in AC02342), CeRAD30h (C. elegans, F53A3.2 in
AF025460), HsRAD30A (XPV) (H. sapiens, AB024313 and
AF158185), MmRAD30A (M. musculus, AB027128), DmRAD30A
(D. melanogaster, AB049433), ScRAD30 (S. cerevisiae, S69702), SpRAD30 (S. pombe, SPBC16A3.11 in
AL021748), HsRAD30B (H. sapiens, AF140501), MmRAD30B
(M. musculus, AF151691), DmRAD30B (D. melanogaster, AB049434).
) was identified in the cDNA libraries that
were searched. In addition, the sequence of the Drosophila
genome (34) does not include a homologue of pol
.
and hpol
proteins and include
all of the N-terminal conserved regions of the UmuC/DinB/Rev1/Rad30 superfamily; thus, they were expected to be active polymerases despite
the missing C-terminal region (5, 9, 14, 21). The truncated forms of
dRAD30A (dRAD30A
C) and dRAD30B(dRAD30B
C) include the N-terminal
545 residues and 445 residues of each polypeptide, respectively. They
were purified by GST-Sepharose affinity chromatography (see
"Experimental Procedures") and characterized enzymatically as
described below (Fig. 2).
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Fig. 2.
Detection of DNA polymerase activity.
A, an SDS-polyacrylamide gel electrophoresis of the purified
dRAD30A C and dRAD30B
C protein is shown. Lane 1,
molecular weight marker. 0.5 µg each of GST-dRAD30A
C (lane
2), GST-dRAD30A-DE126AA (lane 3), GST-dRAD30B
C
(lane 4), and GST-dRAD30B-DE115AA (lane 5) were
applied. The dRAD30A
C and dRAD30B
C proteins are indicated by the
arrow. B and C, DNA polymerase
activities of dRAD30A
C (B) and dRAD30B
C
(C). Three or five different amounts of dRAD30A
C,
dRAD30A-DE126AA, dRAD30B
C, and dRAD30B-DE115AA (10, 100, and 500 fmol in lanes 2-4, respectively, and 0.1, 1, 10, 100, and
500 fmol in lanes 5-9) were added in the reaction mixture.
Control reactions with no enzyme are shown in lane 1.
C and
dRAD30B
C--
The dRAD30A
C and dRAD30B
C proteins were assayed
for DNA polymerase activity using a primer extension assay with a
5'-end-labeled 16-mer primer annealed to a 30-mer template. As shown in
Figs. 2, B and C, both dRAD30A
C (Fig.
2B, lanes 5-9) and dRAD30B
C (Fig.
2C, lanes 5-9) synthesize DNA in a
template-dependent fashion, and the size of the replication
products gradually increases with increasing enzyme concentration in
the reaction. The polymerase activity of dRAD30A
C is 10 times higher
than that of dRAD30B
C.
and dRAD30B be renamed dpol
. In the
following text, dpol
C will be used to refer to the truncated
protein dRAD30A
C, and dpol
C will be used to refer to
dRAD30B
C.
C and dpol
C was analyzed
in DNA synthesis reactions using a nondamaged DNA template in the
presence of only one deoxyribonucleotide (i.e. dA, dG, dC,
or T). Incorporation of the correct complementary nucleotides and, to a
less extent incorrect nucleotides opposite each template, occurred
(data not shown), suggesting that both dpol
C and dpol
C
have relatively low fidelity.
C and dpol
C--
To
determine whether dpol
C and dpol
C bypass DNA lesions,
polymerase assays were carried out using templates with TT-CPD or
TT-(6-4)PP lesions. These templates were described in previous studies
of hpol
(5 7), hpol
(26), and hpol
(9). In addition, a
template with TC-(6-4)PP in the same sequence context as the above two
templates was constructed and used in this study. A 16-mer primer was
annealed to the template-containing lesion, which
terminates just before the lesion site (Fig.
3). dpol
C and dpol
C
efficiently bypassed TT-CPD (Fig. 3A), and the efficiencies of DNA synthesis on the lesion-containing template were almost the same
as on the nondamaged template. dpol
C showed some ability to
bypass TT-(6-4)PP (Fig. 3B, lanes 2-4) and
TC-(6-4)PP (Fig. 3C, lanes 2-4) but in most
cases added one or two bases opposite the lesions before arresting DNA
synthesis. dpol
C added one base opposite the 3'-T of TT-(6-4)PP
(Fig. 3B, lanes 6-8) or the 3'-C of TC-(6-4)PP
(Fig. 3C, lanes 6-8); in addition, it bypassed TC-(6-4)PP at a very low level when the enzyme was present at a high
concentration in the reaction (Fig. 3C, lane 8).
As reported previously (7), hpol
did not bypass TT-(6-4)PP,
arresting DNA synthesis after the addition of one base opposite the
3'-T of the lesion. Therefore, the ability to bypass TT- and
TC-(6-4)PP is a remarkable feature of dpol
C, which distinguishes
it from other RAD30-like polymerases.
View larger version (55K):
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Fig. 3.
Translesion synthesis by
dpol C and
dpol
C. Increasing amounts
of dpol
C (10, 100, and 500 fmol in lanes 2-4,
respectively) and dpol
C (30, 300, and 1500 fmol in lanes
6-8, respectively) were incubated with the
5'-32P-labeled primer-template indicated above the
panel for 15 min at 37 °C in the standard reaction
mixture. The products were subjected to polyacrylamide gel
electrophoresis, and the autoradiograms of the gel are shown. The DNA
lesions on the templates are TT-CPD (A), TT-(6-4)PP
(B), and TC-(6-4)PP (C). Control reactions with
no enzyme are shown in lanes 1 and 5.
C and dpol
C during
Incorporation Opposite Lesions--
Polymerization reactions by
dpol
C and dpol
C were performed with the lesion-containing
template-primer substrates described above in the presence of a single
deoxynucleotide. The reaction products were analyzed and are shown in
Fig. 4. On a template with TT-CPD,
dpol
C and dpol
C preferentially incorporated dAMP and
arrested synthesis after adding four dAMPs opposite TT-CPD and the
following CT residues (Fig. 4A, CPD). dpol
C
and dpol
C preferentially incorporated dAMP opposite the 3'T of
TT-(6-4)PP, incorporating dGMP at a significant level (Fig.
4A, TT-(6-4)PP). Both enzymes incorporated dGMP, dAMP, and
dTMP opposite the 3'-C of TC-(6-4)PP (Fig. 4B,
TC-(6-4)PP). Thus, the selectivity of the correct nucleotide on the
template containing (6-4)PP was lower than on the template containing
CPD.
View larger version (72K):
[in a new window]
Fig. 4.
Selectivity of
dpol C and
dpol
C nucleotide incorporation
opposite the lesions. dpol
C (20 fmol; upper
panels) and dpol
C (100 fmol; lower panels) were
incubated with a 30-mer template containing either a TT-(6-4)PP
(A) or a TC-(6-4)PP (B) annealed to
5'-32P-labeled 16-mer primer with one of the indicated
dNTPs. The sequence of the template and the primer are the same as that
used in Fig. 3, and the partial sequences of the 3' ends of each primer
and template are shown above each panel. No damage (TT) or
(TC) are undamaged controls of TT-CPD and TT-(6-4)PP or TC-(6-4)PP,
respectively.
C and dpol
C incorporated not only A but also G opposite
T (Fig. 4A, no damage). Misincorporation of G
opposite T is a common feature of the pol
and pol
family, and
especially, hpol
incorporates G opposite T more efficiently than A
opposite T (10, 13, 27, 37). However, dpol
C misincorporates G opposite T less often than it correctly incorporates A opposite T.
C and dpol
C--
The results shown in Fig. 4
indicate that dpol
C and dpol
C can insert a variety of bases
opposite the 3' base of TT-CPD, TT-(6-4)PP, or TC-(6-4)PP. However,
it is possible that the misincorporated base may not be extended as
efficiency as the correctly incorporated base is, as shown in the case
of hpol
for the bypass of various lesions (7). To explore this
question, primers were synthesized in which each of the four bases
would be correctly or incorrectly paired with the 3' or 5' base of the
above three lesions. First, the ability of dpol
C to elongate such
primers annealed to TT-CPD was tested, and the results are shown in
Fig. 5. dpol
C elongated DNA chains
more efficiently from the correctly base-paired primers, the 17-mer
with a 3'-terminal A opposite 3'-T of TT-CPD (Fig. 5B) and
the 18-mer with AA opposite the lesion (Fig. 5D). Similar results were obtained with dpol
C (data not shown).
View larger version (52K):
[in a new window]
Fig. 5.
Ability of
dpol C to elongate DNA chains
past TT-CPD. Sets of 5'-32P-labeled 17-mer oligomers
(N17-mer, A and B) or 18-mer (AN18mer,
C and D) primers, which contain different
sequences at their 3' ends (indicated by N, where
N is A, C, G or T, as seen beneath each
panel), were annealed to the 30-mer template.
Increasing amounts of dpol
C were incubated with these primed
templates. The results of a set of three reactions for each A, C, G,
and T primer are shown. Each set contained three reactions with
different amounts of enzyme; the one shown on the left (
) contained
no enzyme, and the middle and right ones contained increasing amounts
of enzyme; 1 and 5 fmol (A and C) and 2.5 and
12.5 fmol (B and D). The template containing no
damage (A and C) or TT-CPD (B and
D), respectively. The autoradiograms of the gels are
shown.
C. On a template with TT-(6-4)PP and a 17-mer primer whose 3'-terminal nucleotide pairs
with the 3'-T of the lesion, dpol
C elongated the DNA chain more
efficiently from a primer with a 3'-terminal G (G17) than from a primer
with a 3'-terminal A (A17) (Fig.
6A). This makes a sharp
contrast with the above result that in the case of TT-CPD, dpol
C
carried out the elongation more efficiently from the A17 primer than
from the G17 primer (Fig. 5B). To examine which base is incorporated after the G residue opposed the 3'-T of TT-(6-4)PP, 18-mer oligomers with GN (N is any base) opposite the lesion were synthesized and examined for the extension by dpol
C. The result showed that the enzyme elongated the DNA chain most efficiently from
the primer with GA opposite the lesion (Fig. 6B). However, on a template with TC-(6-4)PP, dpol
did not show selective
elongation from a specific 3' end; it elongated the DNA chain with
equal efficiency from 17-mer oligomers with a 3'-terminal A or G
opposite the 3'-C of TC-(6-4)PP and from 18-mer oligomers terminating
with AA, AG, GA, or GG opposite the lesion (data not shown). Thus, the
ability of dpol
to recognize the correctly inserted base opposite a
lesion site is lost more severely in TT-(6-4)PP and TC-(6-4)PP than
in TT-CPD.
View larger version (37K):
[in a new window]
Fig. 6.
Ability of
dpol C to elongate DNA chains
past TT-(6-4)PP. Sets of 5'-32P-labeled17-mers
(N17-mer, A) or 18-mers (GN18-mer, B) were
annealed to the 30-mer template. In GN18-mer, G is opposite the 3'-T of
(6-4)PP, and N is opposite the 5'-T of (6-4)PP. Conditions are the
same as in Fig. 5, except for the amount of enzyme; middle
and right lanes contain 50 and 250 fmol, respectively.
C and
dpol
C--
The above results suggest that dpol
C
incorporates a significant level of incorrect nucleotides during bypass
of (6-4)PP; and in contrast, correct nucleotides are selectively
incorporated opposite CPD by dpol
C and dpol
C. To know
whether dpol
C and dpol
C the incorporate correct nucleotide
opposite each damage, the bypass products were analyzed directly (Fig.
7). For this experiment, three kinds of
oligonucleotides containing TT-CPD, TT-(6-4)PP, or TC-(6-4)PP were
synthesized. TT-CPD and TT-(6-4)PP were positioned at the TT of the
TTAA sequence, and TC-(6-4)PP was positioned at the TC of the TCGA
sequence; these are the recognition sequences for the restriction
enzymes MseI and TaqI, respectively. If the
bypass products by dpol
C or dpol
C have the correct AA or GA
sequence, then they should be cleaved by MseI or
TaqI after rehybridization with a nondamaged
oligonucleotide; the product of this cleavage reaction can be readily
detected as a 19- or 14-base fragment, respectively (Fig.
7A). Almost all of the bypass products of TT-CPD by
dpol
C or dpol
C were cleaved by MseI (Fig.
7B, lanes 9-12); more than 90% of the bypass
products have the AA sequence (Table I). On the other hand, only some
of the bypass products of either TT-(6-4)PP or TC-(6-4)PP were
susceptible to restriction enzyme digestion (Fig. 7B,
lanes 15, 16, 19, and 20). The ratio
of the correct base incorporated opposite (6-4)PP is much lower than
for the CPD. Of the bypass products of TT-(6-4)PP by dpol
C,
22.6% are susceptible to MseI digestion, and 36% of the
TC-(6-4)PP bypass products are susceptible to TaqI
digestion (Table I), indicating that bypass of (6-4)PP by dpol
is
highly error prone.
View larger version (34K):
[in a new window]
Fig. 7.
MseI or TaqI
cleavage assay of the TLS bypass products by
dpol C and
dpol
C. A, a
schematic drawing of the experimental protocol of the MseI
cleavage assay is shown. In TaqI assay, a 30-mer substrate
was used, and a cleavage of the substrate produced a 14-base fragment.
B, the 49-mer oligomer with no lesion (lanes
1-6), a TT-CPD (lanes 7-12), a TT-(6-4)PP
(lanes 13-16), or the 30-mer oligomer with TC-(6-4)PP
(lanes 17-20) were annealed to a 5'-32P-labeled
primer and then were mixed with Klenow fragment (2 units in
lanes 1, 2, 7, 8,
13, 14, 17, and 18),
dpol
C (500 fmol in lanes 3, 4,
9, 10, 15, and 16 or 1 nmol
in lanes 19 and 20), or dpol
C (500 fmol in
lanes 5, 6, 11, and 12).
The reaction products were denatured by heating at 95 °C for 5 min,
and then excess amounts of the 49-mer oligonucleotide (lanes
1-16) or the 30-mer oligonucleotide (lanes 17-20),
which does not contain a DNA lesion, were added and annealed to the
newly synthesized DNA. The resultant duplex DNA was cleaved with
MseI or TaqI, respectively, denatured in the
presence of formamide, and then electrophoresed on a sequencing gel.
The autoradiograms of the gels are shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and dpol
, were identified and
characterized with respect to their ability to bypass UV-induced DNA lesions.
are considered to
bypass TT-CPD in a mostly error-free manner (7, 15, 21). Similarly,
dpol
C and dpol
C incorporate AA opposite TT-CPD more
efficiently than other nucleotides (Fig. 4A), and the
elongation starts selectively from a primer ending in A opposite the
lesion (Fig. 5, B and D). These results suggest
that dpol
C and dpol
C bypass TT-CPD in a error-free manner
as in the case of hpol
. In addition, direct analysis of the products
of lesion bypass also indicates that dpol
C and dpol
C carry
out error-free bypass of TT-CPD (Fig. 7). Thus, for these enzymes,
TT-CPD maintains a structure that directs correct Watson-Crick
base-pairing despite the base modification. In fact, Lawrence et
al. (38) argue that the CPD must be an instructive lesion by
virtue of its high coding specificity in E. coli, and
structural studies show that the Watson-Crick base pair is still intact
at the CPD site (39-41). Therefore, the TLS polymerases bypass this
lesion by incorporating the correct bases on this instructional lesion.
C does not carry out
error-free bypass of (6-4)PP lesions. On templates with TT-(6-4)PP,
dpol
C did not show selective elongation from the correct base
paired primer/template (Figs. 6A). Furthermore, the bypass
products of TT-(6-4)PP by dpol
C showed high levels of misincorporation (Fig. 7B). dpol
C elongated DNA
efficiently from the 17-mer end with a mismatched G opposite the 3'-T
of TT-(6-4)PP (Fig. 6A) and preferentially from the 18-mer
with GA opposite the lesion (Fig. 6C). The favored
elongation from the mismatched GA/TT-(6-4)PP terminus is consistent
with structural studies. An NMR study of a duplex DNA with (6-4)PP
revealed that TT-(6-4)PP greatly distorts the DNA structure and
prevents base-pairing with A in the complementary strand (41). However,
a mismatched base pair between the 3'-T of TT-(6-4)PP and G forms
hydrogen bonds and stabilizes the helix (42, 43). Furthermore, the G/T
mispair at the 3'-T of TT (6-4)PP retains the normal Watson-Crick-type base-pairing between the 5'-T of the TT-(6-4)PP and an opposed A, and
the resultant GA/TT-(6-4)PP duplex can assume the typical B-form-DNA
conformation (44). Our results also fit well with the specificity of
base alteration of UV-induced mutations in which a G residue is
preferentially inserted opposite the 3'-T of the 6-4-photoproduct
during TLS in E. coli (19). These results indicate that the
TT-(6-4) photoproduct can be classified as a mis-instructional lesion.
C elongated the DNA chain
equally well from the 18-mer primer with A or G opposite the 5'-T (data
not shown). It will be necessary to determine the sequence of the
bypass products directly or perform steady-state kinetics to clarify
this point.
C and dpol
C fit well with
the mutation spectrum obtained using a defined photoproduct introduced
into SOS-induced E. coli (19, 38). The mutation spectrum
reported in those studies reflects the mutagenic properties of pol V
(UmuD·2C complex). Thus, the mutagenic properties of dpol
C on TT-CPD and TT-(6-4)PP are very similar to those of pol
V. Both enzymes bypass TT-CPD in an error-free manner and incorporate
GA opposite TT-(6-4)PP cells (Ref. 8 and this study). The similarity
in the nucleotides incorporated suggests that structural information in
the altered bases contribute to nucleotide selection during
incorporation opposite these lesions by these polymerases.
C seems to be an unusual member
of the Rad30 family in having the ability to bypass (6-4)PP, although
we cannot rule out a possibility that having an N-terminal GST tag and
a C-terminal truncation have affected the TLS specificity and activity;
we also have to keep in mind that we cannot draw a quantitatively
definitive conclusion since the observations present in this study are
qualitative. It should also be noted that the DNA repair capacity of
Drosophila cells is somewhat different from that of human
cells. The most striking difference is that Drosophila cells
have a 6-4-photolyase, which specifically repairs (6-4)PP in a
light-dependent manner, but human cells lack this enzyme
(45). The (6-4)PP lesions are removed rapidly by nucleotide excision
repair. However, a small amount of the lesion might remain that would
be toxic because (6-4)PP is highly mutagenic. The remaining (6-4)PP
lesions can be removed by the 6-4-photolyase in Drosophila
cells, and thus, the error-prone bypass activity of dpol
might not
be so deleterious. On the other hand, human cells lack the
6-4-photolyase, and thus, more lesions might remain, so that
error-prone bypass of this lesion would have a deleterious effect.
Thus, hpol
might have lost the ability to bypass (6-4)PP because
that loss enhances genetic stability. Paradoxically, we have recently
shown that in human cells, mutations are produced at the (6-4)PP site,
although the frequency is low (46). Thus, in human cells, some DNA
polymerases bypass the (6-4)PP in an error-prone manner. Another
enzyme, for example hpol
, might bypass (6-4)PP in vivo,
in conjunction with pol
(27).
C but not by
dpol
C. The TLS activity of dpol
C and dpol
C was also
examined on chemically modified damage, and all of the other lesions
tested were bypassed by both enzymes (data not shown). Thus, at present we cannot assign any specific biological function to dpol
. Further analyses of the substrate specificity of these polymerases by screening
various types of DNA lesions may reveal differences in their substrate
specificity. Another possibility is that each polymerase interacts with
other protein differently in vivo. The recombinant protein
used in this study has a C-terminal deletion. The deleted C-terminal
portion might have an important biological interaction with other
proteins in vivo, whereas it is not required for polymerase
activity in vitro. It is also possible that dpol
and
dpol
have different in vivo functions in
Drosophila that have not been detected by in
vitro studies carried out to date. One enzyme may be important for
the defense against DNA damage, and another may be involved in some
biological process that requires DNA replication on a primer-template
complex containing an unusual mismatch. Detailed analyses of the
expression pattern of each gene at the tissue and cellular levels will
provide clues to the function of each gene. Furthermore, the phenotypes
of transgenic flies that overexpress these genes and mutant flies in
which each gene is disrupted by an insertion or repressed by RNA
interference (RNAi) will clarify the biological roles of these
proteins in Drosophila.
![]() |
ACKNOWLEDGEMENT |
---|
We thank E. Ohashi for technical assistance and for kindly providing oligonucleotides used as primer and template and T. Ogi for the phylogenetic analysis to construct the tree (Fig. 1C).
![]() |
FOOTNOTES |
---|
* This work was supported by Grants-in-aid from the ministry of Education, Science, Sports, and Culture of Japan 09308020, 11146206, 11480140, 11878093 and by the REIMEI Research Resources of Japan Atomic Energy Research Institute.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB049435 (DmRev1), AB049433 (DmRAD30A), and AB049434 (DmRAD30B).
§§ To whom correspondence should be addressed: Radiation Biology Center, Kyoto University, Yoshidakonoe-cho, Sakyoku, Kyoto 606-8501, Japan. Tel.: 81-75-753-7555; Fax: 81-75-753-7564; E-mail: todo@house.rbc.kyoto-u.ac.jp.
Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.M009822200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: TLS, translesion DNA synthesis CPD, cis-syn-cyclobutane pyrimidine dimer; (6-4)PP, pyrimidine-pyrimidone 6-4-photoproduct; GST, glutathione S-transferase; TT, thymine-thymine; CT, thymine-cytosine; pol, polymerase.
![]() |
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