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Molecular Biology and Genetics |
Department of Dermatology, Kyoto University, Graduate School of Medicine, Kyoto 606-8397, Japan [E. O.]; Department of Radiation Genetics, Kyoto 606-8501, Japan [T. Y.]; Radioisotope Center, Nara Medical University, Nara 634-8521, Japan [T. M.]; Division of Radiation Biology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa 920-0934, Japan [T. M., O. N.]; and Radiation Biology Center, Kyoto University, Kyoto 606-8501, Japan [E. O., S-T K., K. H., M. I., T. T.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Genetic analysis and direct DNA sequencing of mutated target genes suggest that both CPDs and 6–4PPs could be premutagenic lesions (7, 8, 9, 10) . Several studies have focused on the specific question of which of these two major UV-induced DNA lesions is the mutagenic one. An approach to this question is to compare the frequency of each UV lesion with mutation frequency. Except for some hot spots, the observed spectrum of UV-induced mutations correlates well with the frequency of UV-induced 6–4PPs rather than CPDs (5, 6, 7 , 11) , suggesting that the 6–4PPs are more efficient premutagenic lesions than the CPDs. However, certain mutation hot spots do not correlate with the frequency of CPD/6–4PP damage (7 , 11) , raising the possibility that "minor" photoproducts also play a specific role as the premutagenic lesion at some hot spots. More direct evidence that 6–4PPs may play a dominant role in UV-induced mutagenesis has been obtained from Escherichia coli (12, 13, 14) . Single-stand M13-based vectors containing a 6–4PP or CPD were introduced into E. coli cells, and the sequences of the recovered M13 phage DNA were determined. 6–4PPs at TT sites (TT 64PP) are highly mutagenic, causing 91% mutation, whereas the CPDs at TT sites (CPD TT) are not very mutagenic (7% mutation). UV irradiation caused primary base substitutions, and the G:C-A:T transition was the most dominant (1) . Thus, photoproducts formed at dipyrimidine sites containing C are very mutagenic. However, M13 vectors containing only TT 64PP, TC 64PP, or TT CPD were used. As a result, the mutagenic properties of photoproducts induced at C-containing sites are unclear, and more studies are needed. Another approach was to take advantage of the photoreversibility of CPD to provide evidence of the premutagenic role of these lesions (8 , 15, 16, 17, 18) . In E. coli, photoreactivation of CPDs was more effective in the rescue of lethality rather than in the reduction of mutation (15 , 16) . On the other hand, in mammalian cells, the selective removal of CPDs from a UV-irradiated shuttle vector by CPD photolyase resulted in a marked reduction of mutation frequency. However, the mutational hot spot did not change drastically, indicating that mutagenic photoproducts were still present after photoreactivation (8 , 17 , 18) . These results indicate that both CPDs and 6–4PPs are premutagenic lesions in both bacteria and mammalian cells, although 6–4PPs are more efficient premutagenic lesions in E. coli, whereas CPDs are more efficient premutagenic lesions in mammalian cells. Furthermore, the possibility that "minor" photoproducts play a significant role in UV-induced mutation cannot be ruled out because CPDs were the only substrate for CPD photolyase.
In 1993, a light-dependent DNA repair activity specific for 6–4PPs was found in Drosophila melanogaster, and the enzyme was named 6–4(6–4)photolyase (19) . Presently, the cDNAs of 6–4(6–4)photolyase have been cloned from Drosophila, Xenopus laevis, and Arabidopsis thaliana (20, 21, 22) . It was believed that 6–4PPs could not be photorepaired to their original form by a simple photochemical splitting as in the case of CPD repair by CPD photolyase because of the structural differences between 6–4PPs and CPDs (23 , 24) . However, 6–4(6–4)photolyase repairs 6–4PPs to the original nondamaged configuration, and those repaired products do not induce mutation (25, 26, 27, 28) . This permits the use of 6–4(6–4)photolyase to investigate the role of 6–4PPs as premutagenic lesions. Furthermore, the removal of both two major UV lesions by two types of photolyase may uncover the role of "minor" photoproducts as premutagenic lesions.
In this report, we removed CPDs and/or 6–4PPs from UV-irradiated shuttle vector plasmid pMY189 carrying the supF gene as the target for mutation by in vitro photoreactivation with CPD photolyase and/or 6–4(6–4)photolyase. Photoreactivated plasmid DNA was transfected into a repair-deficient XP-A cell line where the plasmids could be replicated by the human cell polymerase(s) without any effects of NER. The progeny plasmids were analyzed for the frequency of supF mutants, and the types and distributions of mutations were determined. We found an increase in the yield of replicated plasmids and a corresponding decrease in the frequency of supF mutants by photoreactivation of either UV lesion. Sequence analysis of the supF gene of mutant plasmids indicated that removal of either UV lesion does not alter the mutation spectrum drastically, and G:C-A:T transitions predominate. A notable difference between the two forms of photoreactivation was that A:T-G:C transitions were not recovered after the removal of 6–4PPs. Removal of both UV lesions resulted in the disappearance of mutational hot spots and a random distribution of mutations as was found in unirradiated controls. However, a high proportion of A:T-T:A transversions was recovered after the removal of both types of UV lesions.
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MATERIALS AND METHODS |
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Plasmids and E. coli Strains.
The shuttle vector pMY189 (30)
, which was derived from
pZ189 (31)
, was used in this study. pZ189/pMY189 contains
the replication origin and the early region (coding for the large T
antigen) of the SV40 virus, allowing its replication in human cells. It
also contains sequences for its replication, maintenance, and selection
(ampicillin resistance gene) in E. coli. The tyrosine amber
suppressor tRNA supF gene was used as a target gene for mutations. The
supF gene is able to abolish the effects of an amber mutation in the
LacZ and gyrA genes of E. coli strain KS40/pKY241
(32)
. pMY189 was constructed from pZ189 by inserting an
M13 universal primer sequence at just upstream of the supF gene for the
convenience of sequencing.
KS40 is a nalidixic acid-resistant (gyrA) derivative of MBM7070 [lacZ (am) CA7070 lacY1 HsdR HsdM D(araABC-leu)7679 galU galK rpsL thi; Ref. 32 ]. Plasmid pKY241 contains a chloramphenicol-resistant marker and a gyrA (amber) gene. E. coli KS40/pKY241 cells carrying the active supF gene are sensitive to nalidixic acid, whereas cells carrying the mutated supF form colonies on plates containing nalidixic acid. Thus, when KS40/pKY241 cells are transformed with pMY189, mutations in supF can be selected on plates containing nalidixic acid, chloramphenicol, and ampicillin. To ensure the selection of the mutated supF gene, isopropyl-b-D-thiogalactoside and 5-bromo-4-chloro-3-indolyl-b-D-galactoside were added to the selection plates. E. coli cells containing the active supF gene produce blue colonies, whereas cells having a mutated supF gene produce white colonies.
Preparation of DNA Photolyase and Photoreactivation of
UV-irradiated Plasmid.
Salmonella typhimurium CPD photolyase (33)
and
Xenopus laevis 6–4(6–4)photolyase (21)
were used
in this study. S. typhimurium photolyase and
Xenopus photolyase were overexpressed in E. coli
and purified as described by Li et al. (33)
and
Todo et al. (21)
, respectively.
pMY189 plasmid DNA was irradiated with 1 kJ/m2 of 254 nm of UV light using a germicidal lamp as described previously (21) . Concentrated buffer was added to give a solution of 0.03 mg of DNA/ml in 10 mM Tris-HCl (pH 7.4), 5 mM DTT. One hundred thirty mg of purified CPD photolyase or 130 mg of purified 6–4(6–4)photolyase were added, and samples were illuminated with photoreactivating light for 3 h at room temperature as described previously (21) . After photoreactivation, the samples were treated with proteinase K (final concentration of 0.1 mg/ml) in the presence of SDS (final concentration of 0.4%) at 500°C for 90 min. The samples were extracted with phenol and chloroform, and plasmid DNA was recovered by ethanol precipitation.
Measurement of CPDs and 6–4(6–4)Photoproducts by ELISA.
Direct binding of monoclonal antibodies to CPDs and 6–4(6–4)photoproducts
was measured by ELISA as described (34
, 35)
.
Transfection of Human Cells.
Human cells were trypsinized, washed, and suspended in Dulbecco’s PBS
solution (pH 7.5). Cells (2 x 107) plus 20 mg of pMY189 in PBS solution (0.2
ml) were placed in an electroporation chamber (electrodes 0.3-cm apart;
PDS Inc., Madison, WI), and the cells were transfected with the
plasmids by electric pulses (600 V, five times). The cells were
plated in five 10-cm dishes and incubated at 370°C for 72 h in a
CO2 incubator.
Plasmid Recovery, Selection of Mutated supF, and
DNA Sequencing.
The extrachromosomal plasmid DNA was recovered using a small-scale
alkaline lysis procedure and digested by the restriction endonuclease
Dpn I (TAKARA, Kyoto, Japan) to eliminate nonreplicated input plasmids
retaining the bacterial methylation pattern.
E. coli KS40/pKY241 was transformed by the plasmid. For the selection of mutated supF, the transformed bacterial cells were plated on Luria-Bertani agar containing nalidixic acid, chloramphenicol, ampicillin, isopropyl-b-D-thiogalactoside, and 5-bromo-4-chloro-3-indolyl-b-D-galactoside. A fraction of the cells was plated on Luria-Bertani agar containing chloramphenicol and ampicillin to measure the total number of transformants.
Mutated plasmids were purified from overnight cultures, and the nucleotide sequences of the supF gene of the plasmid were determined with the -21 M13 primer and Big Dye Terminator cycle Sequencing Kit using a 310 automatic DNA sequencer (Applied Biosystems). To obtain independent mutant clones, fewer than five mutants were isolated from each independent transfection.
Statistics.
Statistical comparisons were performed with Fisher’s exact test for
difference in proportions (36)
. Ps for a
one-tailed test are presented.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Plasmid Survival.
After UV irradiation and photoreactivation, plasmids were transfected
into XP-A cells and incubated for 3 days to permit replication of the
plasmid DNA and fixation of mutations. The progeny plasmids were
purified from the transfected human cells and used to transform
indicator bacteria. The survival of the plasmid was determined as the
relative yield of bacterial colonies obtained after transformation with
the purified progeny plasmids. The recovery of UV-irradiated plasmid
from the XP-A cell line was reduced to 0.29% compared to unirradiated
plasmid at a dose of 1 kJ/m2 (Table 1)
. Removal of either CPDs or 6–4PPs by DNA photolyase resulted in a
slight increase in plasmid recovery, 11.6% and 2.3%, respectively. On
the other hand, the removal of both types of UV damage drastically
enhanced plasmid recovery to 94.3% of that of unirradiated plasmid.
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. The background plasmid mutation frequency was 1.6 x 10-4 and increased 260-fold (to 4.2 x 10-2) following the treatment of
plasmids with 1 kJ/m2 UV. Removal of either or
both types of UV damage by DNA photolyase reduced the mutation
frequency. The mutation frequency, when either CPDs or 64PPs or both
were removed, was 4.2 x 10-3,
1.8 x 10-2, and 1.2 x 10-3, respectively (Table 2)
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, base sequence changes in plasmids were classified as single base
substitutions, tandem base substitutions, multiple base substitutions
(base substitutions more than three bases apart), frameshifts (single
base insertions or deletions), and large deletions.
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The mutant plasmids without deletions contained base substitutions and frameshifts. Whereas base substitutions were most frequent (66.7–100%), frameshifts were scarce (0-4%). Consistent with previous studies (1 , 8, 9, 10, 11) , a high frequency of tandem base substitutions was observed with UV-irradiated plasmid (37.7%), whereas in unirradiated plasmid (4.4%), they were rare.
Types of single and tandem base substitution mutations in supF mutant
plasmids are shown in Table 4
. A significantly smaller portion of mutant plasmids had transitions
(40.7%), with a greater portion having transversions (59.3%) in the
unirradiated plasmids. In contrast, 85.6% of single or tandem base
substitutions observed in the UV-irradiated plasmids were G:C-A:T
transitions, and the portion of mutant plasmids with transversions was
small (14.4%). Such a high proportion of transitions was also observed
after removal of either CPDs or 6–4PPs (73.7% and 80.9%,
respectively), but it was reduced to 44.5% when both forms of UV
damage were removed.
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. All mutations were found between bp 56 and 198. The point mutations
observed in UV-irradiated plasmids were not distributed randomly in the
supF gene but appeared preferentially at certain sites. About half the
G:C-A:T transitions were found in two strong hotspots at positions 123
and 168. Lesser hot spots were seen at positions 108, 156, and 169.
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were therefore expected. In Tables 5
and 6
, each type of base substitution mutation was classified with the
expected sites of UV-induced mutations.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. ELISA showed that
photoreactivation eliminates each type of photolesion completely within
the limit of detection. Consistent with the results of ELISA,
photoreactivation of both types of photolesion eliminated the effects
of UV irradiation on the recovery of transfected plasmid (Table 1)
and
reduced the frequency of mutations (Table 2)
. Furthermore, the types
and spectra of mutations in the plasmids after photoreactivation of
both types of photolesion (-CPD/-64PP plasmid) became similar to those
from unirradiated control plasmids; i. e., (a) a high
frequency of deletion mutations compared to CPD and
6–4PP-containing plasmids, (18.2% in -CPD/-64 plasmid and 31.8%
in control plasmid, whereas CPD and 6–4PP-containing plasmids had
0–2%; Table 3
); (b) the portion of mutant plasmids having
transversions (55.5%) was larger than that with transitions (44.5%;
Table 4
); (c) mutational hot spots disappeared from the
-CPD/-64PP plasmids (Fig. 2)
. These results indicate that
photoreactivation with each type of DNA photolyase selectively
eliminated either type of photolesion almost completely and that
the photoreactivated plasmids specifically manifested the effects of
the remaining photolesion. In the case of the removal of CPDs or
6–4PPs, the mutagenic effects of 6–4PPs or CPDs plus other minor
photoproducts were manifested selectively, and in the case of the
removal of both types of lesion, the mutagenic effects of the remaining
minor photoproducts alone became apparent.
A characteristic feature of UV mutagenesis is the high frequency of
G:C-A:T transitions at dipyrimidine sites including CC-TT double base
mutations (8, 9, 10, 11
, 37
, 38)
. This feature unique to UV was
also observed in the UV-induced mutation of plasmid DNA (82.7% of base
substitutions are G:C-A:T transitions and 24% of base substitutions
are CC-TT tandem substitutions). Although the mutagenicity was
substantially reduced by the removal of each UV lesion, the tendency
toward having a larger portion of G:C-A:T transitions was preserved
(63.2% in -CPD plasmid and 80.9% in -64PP plasmid), and the position
of mutational hot spots did not change dramatically (Table 4
and Fig. 2
). These findings indicate that both CPDs and 6–4PPs are premutagenic
lesions for G:C-A:T transitions. On the other hand, the frequency of
CC-TT tandem substitution was significantly reduced by the removal of
either photoproduct. In particular, removal of CPDs dramatically
reduced the frequency of CC-TT tandem substitution (7% in -CPD
plasmid, P = 0.007; 13% in -64PP plasmid),
indicating that although both CPDs and 6–4PPs are premutagenic lesions
for CC-TT tandem substitutions, CPDs induce this type of mutation more
effectively than 6–4PPs.
Although A:T-G:C transitions occurred less frequently than G:C-A:T
transitions, they represent a significant component of UV-induced
mutation. In contrast to G:C-A:T transitions, the frequency of A:T-G:C
transitions showed an apparent difference between the contribution of
CPDs and 6–4PPs. In the -CPD plasmids, 14.3% of transitions (8 of 56)
were the A:T-G:C type, whereas in the -64PP plasmids, all transitions
obtained (38 sites) were the G:C-A:T type, and no mutant having the
A:T-G:C transition was recovered (P = 0.02).
Thus, A:T-G:C transitions are induced at 6–4PPs specifically. As shown
in Table 5
, almost all of 6–4PP-specific A:T-G:C transitions were
formed at TT sites. In fact, in COS cells, TT 6–4PP is more mutagenic
than TT CPD and primarily elicits 3'T-C substitutions
(39)
. 6–4PP-specific A:T-G:C transitions might be due to
the following two reasons. First, duplexes with G opposite the 3'T of
the 6–4PPs are thermodynamically more stable than that with A
(40)
. The second reason is the properties of mammalian DNA
polymerases. Recently, a new type of DNA polymerase, DNA polymerase
, which is defective in XP-variant cells, was reported
(41, 42, 43)
. DNA polymerase h replicates CPD-containing, but
not 6–4PP-containing DNA templates. Furthermore, DNA polymerase
incorporates dATP at the site opposite to TT CPDs, indicating that DNA
polymerase h can bypass TT-CPDs without inducing mutation. This might
be the reason why A:T-G:C transitions were not recovered from the
plasmid containing CPDs. 6–4PP-specific A:T-G:C transitions might
reflect the absence of a DNA polymerase that bypasses TT-6–4PPs in
human cells.
When both types of photolesions were removed from UV-irradiated
plasmids, the hot spots disappeared and the mutations became randomly
distributed. However, the distribution of mutants in the -CPD/-64PP
plasmids differ significantly from that in the unirradiated plasmids.
In fact, A:T-G:C transitions and A:T-T:A transversions occurred in the
-CPD/-64PP plasmids at significant levels (6.2% and 11.1%,
respectively) but were rare in unirradiated samples (Table 4
and 5)
. The premutagenic lesions for these mutations were not CPDs and
6–4PPs, but they may have been other minor UV-induced
photoproducts.
As minor UV-induced photoproducts, thymine glycol (44)
,
pyrimidine hydrate (45)
, 8,8-adenine dehydrate (46
, 47)
, and thymidylyl-(3'-5')-deoxyadenosine (TA*; Ref.
48
) have been described. Of these, TA* is the most
probable candidate for causing A:T-T:A transversions. Although TA* was
produced by 254-nm irradiation of DNA with a quantum yield of 10–100
less than dipyrimidine products, it is highly mutable in
SOS-induced E. coli (82% of the recovered phages
were mutants; Refs. 48
and 49
). The most
abundant mutation was a 3'A-T substitution (49)
. Five of
eight A:T-T:A transversions observed in -CPD/-64PP plasmids were
induced at TA sequences and constituted a weak hot spot (positions
134–135, the predicted A:T-T:A transversions were indicated in white
letters in Fig. 2
; In Table 5
, they are classified as TT and
CT sites because Table 5
was based on the assumption that
mutations are originated from the dipyrimidine sites). Thus, it is
reasonable to conclude that TA* is the premutagenic lesion for A:T-T:A
transversions. Of course, we cannot exclude the possibility that
A:T-T:A transversions were made by other unknown photoproducts.
Furthermore, it is not proven clearly whether NER defects in XP-A cells
are defective for the excisions of all of the minor photoproducts.
Further studies of the minor UV photoproducts are needed.
Carcinogenesis is believed to be etiologically related to somatic cell mutations arising from unrepaired lesions. XP patients have a deficiency in NER of UV lesions, and unrepaired UV lesions result in a high level of skin tumors. On the other hand, trichothiodystrophy and Cockayne syndrome patients, other disorders having a defect in NER, do not develop skin tumors in body exposed to UV light (1, 2, 3) . The high mutation frequency due to a defect in NER in these cells was primarily caused by CPDs (50 , 51) . These results suggest that carcinogenic effects of 6–4PPs are different from those of CPDs and that unrepaired 6–4PPs resulting in a high mutagenic potential may play an important role in the molecular events leading to skin cancer development in XP patients (50) . As shown in this paper, our system is useful for determining the distinctive roles of CPDs, 6–4PPs, and minor photoproducts. Our system, when applied to the trichothiodystrophy, Cockayne syndrome, and XP-variant cells, would be able to clarify the role of each type of UV lesion on carcinogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a grant-in-aid for Scientific
Research (09308020, 11480140, and 11878093) and on Priority Area
(08280101) from Ministry of Education, Science, Sports and Culture of
Japan. 
2 To whom requests for reprints should be
addressed, at Radiation Biology Center, Kyoto University,
Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan. Phone:
81-75-753-7560; Fax: 81-75-753-7564; E-mail: Todo{at}house.rbc.kyoto-u.ac.jp
3 The abbreviations used are: CPD, cyclobutane
pyrimidine dimer; XP, xeroderma pigmentosum; XP-A, XP
complementation group A; 6–4PP, pyrimidine (6–4) pyrimidone
photoproduct; NER, nucleotide excision repair; kJ, kilojoule.
Received 9/24/99. Accepted 1/20/00.
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REFERENCES |
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