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1 The Cockayne syndrome group B gene product is involved in cellular repair of 8 -hydroxyadenine in DNA Jingsheng Tuo 1 , Pawel Jaruga 2 , Henry Rodriguez 2 , Miral Dizdaroglu 2 , and Vilhelm A. Bohr 1 * 1 Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224 2 Chemical Science and Technology Laboratory, National Institute of Standards and Technol- ogy, Gaithersburg, MD 20899-8311 *: To whom correspondence should be addressed: Phone: 410-558-8223 FAX: 410-558-8157 Email: [email protected] RUNNING TITLE: Cockayne syndrome group B gene product in 8-hydroxyadenine repair ABBREVIATIONS: CS: Cockayne syndrome; CS-B: Cockayne syndrome group B; CSB: CSB protein; CSB: CSB gene; BER: base-excision repair; NER: nucleotide-excision repair; TCR: transcription-coupled repair; 8-OH-Ade: 8-hydroxyadenine; 8-OH-dAdo: 8-hydroxy-2'- deoxyadenosine; 8-OH-Gua: 8-hydroxyguanine; 8-OH-dGuo: 8-hydroxy-2'-deoxyguanosine; WCE: whole cell extract; LC/MS: liquid chromatography/mass spectrometry; IDMS: isotope- dilution MS; SIM: selected-ion monitoring; hOGG1: 8-OH-Gua glycosylase/apurinic lyase; 4- NQO: 4-nitroquinoline-1-oxide. JBC Papers in Press. Published on June 11, 2002 as Manuscript M204814200 by guest on April 12, 2018 http://www.jbc.org/ Downloaded from

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The Cockayne syndrome group B gene product is involved in cellular

repair of 8-hydroxyadenine in DNA

Jingsheng Tuo1, Pawel Jaruga2, Henry Rodriguez2, Miral Dizdaroglu2, and Vilhelm A. Bohr1*

1 Laboratory of Molecular Gerontology, National Institute on Aging,

National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224

2 Chemical Science and Technology Laboratory, National Institute of Standards and Technol-

ogy, Gaithersburg, MD 20899-8311

*: To whom correspondence should be addressed:

Phone: 410-558-8223

FAX: 410-558-8157

Email: [email protected]

RUNNING TITLE: Cockayne syndrome group B gene product in 8-hydroxyadenine repair

ABBREVIATIONS: CS: Cockayne syndrome; CS-B: Cockayne syndrome group B; CSB: CSB

protein; CSB: CSB gene; BER: base-excision repair; NER: nucleotide-excision repair; TCR:

transcription-coupled repair; 8-OH-Ade: 8-hydroxyadenine; 8-OH-dAdo: 8-hydroxy-2'-

deoxyadenosine; 8-OH-Gua: 8-hydroxyguanine; 8-OH-dGuo: 8-hydroxy-2'-deoxyguanosine;

WCE: whole cell extract; LC/MS: liquid chromatography/mass spectrometry; IDMS: isotope-

dilution MS; SIM: selected-ion monitoring; hOGG1: 8-OH-Gua glycosylase/apurinic lyase; 4-

NQO: 4-nitroquinoline-1-oxide.

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ABSTRACT

Cockayne syndrome (CS) is a human disease characterized by sensitivity to sunlight, severe

neurological abnormalities and accelerated aging. CS has two complementation groups, CS-A

and CS-B. The CSB gene encodes the CSB protein with 1493 amino acids. We previously re-

ported that the CSB protein is involved in cellular repair of 8-hydroxyguanine, an abundant le-

sion in oxidatively damaged DNA, and that the putative helicase motif V/VI of the CSB may play

a role in this process. The present study investigated the role of the CSB protein in cellular re-

pair of 8-hydroxyadenine, another abundant lesion in oxidatively damaged DNA. Extracts of

CS-B null cells and mutant cells with site-directed mutation in the motif VI of the putative heli-

case domain incised 8-hydroxyadenine in vitro less efficiently than wild type cells. Furthermore,

CS-B null and motif VI mutant cells accumulated more 8-hydroxyadenine in their genomic DNA

than wild type cells after exposure to γ-radiation at doses of 2 Gy or 5 Gy. These results suggest

that the CSB protein contributes to cellular repair of 8-OH-Ade, and that the motif VI of the puta-

tive helicase domain of CSB is required for this activity.

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Cockayne syndrome (CS) is a human genetic disorder with diverse clinical symptoms (1). There

are two complementation groups of CS, CS-A and CS-B. The CSB gene (CSB) encodes the

CSB protein (CSB) with1493 amino acids and a molecular mass of 168 kDa (2;3). An important

clinical feature of CS-B is accelerated aging. Thus, CS-B may be a useful model for studying the

aging process in humans. Fibroblasts from CS-B patients are hypersensitive to ultraviolet (UV)

radiation and show delayed recovery of RNA synthesis after exposure (1). CS-B cells have a

pronounced defect in repair of UV radiation-induced DNA damage in actively transcribed genes

(4). UV radiation-induced DNA damages are mostly bulky helix-distorting lesions that are re-

moved by nucleotide-excision repair (NER). The transcription-coupled repair (TCR) pathway

specifically removes these lesions in actively transcribed genes (5). CS-B cells are deficient in

TCR and this is thought to be the molecular basis of some features of the CS phenotype (6),

especially the increased sensitivity to UV radiation. However, it is unlikely that the TCR defect

and the resulting UV radiation-sensitivity of CS-B cells account for other clinical features of CS

such as progressive neurodegeneration. Thus, CS-B cells may be deficient in other processes

besides TCR of UV radiation-induced DNA damage.

All organisms are continuously exposed to reactive oxygen species, which cause cellular dam-

age and are thought to contribute to the aging process (7). Reactive oxygen species damage

DNA and generate numerous DNA lesions such as modified bases and sugar moieties, strand

breaks and DNA-protein crosslinks (reviewed in (8)). 8-hydroxy-7,8-dihydroguanine (8-OH-Gua)

is a ubiquitous lesion in oxidatively damaged DNA that is mutagenic, causing G→T transver-

sions in vitro and in vivo (9;10). 8-hydroxy-7,8-dihydroadenine (8-OH-Ade) is another common

lesion formed in DNA exposed to oxidizing agents such as hydroxyl radicals in vitro and in vivo

(reviewed in (8) and structure in Fig. 1). 8-OH-Ade is premutagenic and induces A→G and

A→C mutations in mammalian cells, although it leads to a lower mutation frequency than 8-OH-

Gua (11-16). Nevertheless, the biological consequences of 8-OH-Ade could be significant, be-

cause it is abundant in oxidatively damaged DNA (17). 8-OH-Ade is repaired in mammalian

cells (18), but the mechanism by which it is repaired is not known (19).

Studies suggest that oxidative damage to DNA may contribute to the premature aging pheno-

type associated with progeroid syndromes (20). Thus, it is possible that the DNA repair defect

in CS plays an important role in the course and symptoms of this disorder. Whole cell extracts

(WCEs) from primary CS-B cells incise 8-OH-Gua at a lower rate than normal cell lines and this

deficiency is complemented by transfection of the cells with the wild type CSB (21). In addition,

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CS-B null cells and CS-B motif V/VI mutants are more sensitive to γ-radiation than wild type

cells (22). WCEs from transfected CS-B null cells and motif V/VI mutants also incise 8-OH-

Gua-containing oligonucleotides less efficiently than wild type cells. Furthermore, 8-OH-Gua ac-

cumulates to a greater level in CS-B null cells and motif VI mutants following γ-irradiation than in

wild type cells (22).

In this study, we investigated the role of CSB in cellular repair of 8-OH-Ade. CSB mutants were

expressed in CS-B null cells and in vitro incision of 8-OH-Ade was analyzed using WCEs from

these cell lines. In addition, the nucleoside form of 8-OH-Ade, 8-hydroxy-2'-deoxyadenosine (8-

OH-dAdo) was measured in genomic DNA of these cells by means of a recently developed as-

say that uses liquid chromatography/mass spectrometry (LC/MS) with the isotope-dilution tech-

nique (17).

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EXPERIMENTAL PROCEDURES

Cell Lines and Culture Conditions Cell lines were derived from CS1AN.S3.G2, a SV40-

transformed human fibroblast in which CSB is disrupted. These cells were described previously

(23). CS1AN.S3.G2 was transfected with pcDNA3.1 carrying the wild type CSB, mutant CSB al-

tered in the putative helicase motif VI (Q942E and R946A) or the vector pcDNA3.1 (pc3.1) (Invi-

trogen) (Fig. 2). The reason we applied Q942E and R946A is that these cell lines are the most

deficient ones in repair of 8-OH-Gua among the 8 stably transfected cell lines established in this

laboratory with site-direct mutation(s), which are distributed in various motifs of the helicase

domain (22). Construction of the mutants and cell lines were described previously (22;24).

Exposure was carried out as follows. Cells attached on 10-cm2 dishes were washed with PBS

and then irradiated at the indicated doses with Gammacell 40 Exactor 137Cs γ-source (Nordian

International Inc., Kanata, Ontario, Canada) followed by incubation in the complete medium at

37°C for 30 min. Genomic DNA was extracted as described (22).

In Vitro Incision of an 8-OH-Ade-Containing Oligonucleotide An oligonucleotide with a single

8-OH-Ade at position 11 was the substrate for the incision assay. The sequences of the oli-

gonucleotide used in the incision assay were listed in table I. The oligonucleotide with the lesion

was 32P-5’-end labeled and annealed with a complementary strand containing a T opposite 8-

OH-Ade. We also used a double stranded substrate containing C opposite the lesion because

this structure is the optimal substrate for nick-forming activity at position of 8-OH-Ade (25;26).

Incision reactions were carried out in a volume of 20 µL containing 100 fmol of oligonucleotide

duplex, 1 µg poly dIdC competitor, 20 mM Hepes-KOH, pH 7.8, 100 mM KCl, 5 mM DTT, 5 mM

EDTA, 2 mM MgCl2, and 40 µg WCE protein prepared as described (22;27). Reactions were

incubated at 37°C for 3 h, terminated by the addition of 0.8 µL 10% SDS and 0.8 µL 5 mg/mL

proteinase K and incubated for 10 min at 55°C. DNA was precipitated with 2 µL 5 mg/mL gly-

cogen (Ambion), 4 µL 11 M ammonium acetate and 70 µL cold ethanol overnight at -20 °C.

Samples were centrifuged for 1 h at 4 °C, washed with 200 µL 70% ethanol, collected by cen-

trifugation at 12,000 x g for 10 min. The pellet was dried in speed-vacuum and resuspended in

10 µL formamide loading dye (5% EDTA, 0.02% bromophenol blue, 0.02% xylene cyanol in

95% formamide). Samples were separated on a denaturing 20% polyacrylamide gel (containing

7 M urea, 89 mM Tris-borate pH 8.0 and 2 mM EDTA). The reaction products were visualized

by autoradiography and quantified on a phosphorimager (Molecular Dynamics). Oligonucleo-

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tides containing single uracil or 5-hydroxycytocine, which were known to have no differences in

incision activity of our CS-B wild type, null or mutant cell lines, were used for WCE control (22).

LC/MS and Preparation of the Stable Isotope-Labeled Analog of 8-OH-dAdo The measurement of

8-OH-dAdo in enzymic hydrolysates of DNA samples was performed by liquid chromatogra-

phy/isotope-dilution mass spectrometry (LC/IDMS) as previously described except for the stable

isotope-labeled internal standard, which was used for quantification by IDMS (17). In the present

work, a stable isotope-labeled analog of 8-OH-dAdo was prepared and isolated in pure form.

Commercially available dATP-15N5 (Medical Isotopes, Inc. Pelham, NH) was used as the starting

material. An aqueous solution of dATP-15N5 (5 mg/100 mL) was bubbled with N2O and exposed

to ionizing radiation in a 60Co γ-source at a dose of 400 Gray (Gy) (30 Gy/min). This treatment

was expected to produce 8-OH-dATP-15N5 from dATP-15N5 among other products, because it is

well known that the exposure of aqueous solutions of dAdo to ionizing radiation generates 8-

OH-dAdo (reviewed in (28;29)). 8-OH-dATP-15N5 was dephosphorylated to 8-OH-dAdo-15N5 as

follows: an aliquot of 100 mL of the irradiated solution of dATP-15N5 was lyophilized to dryness,

dissolved in 1 mL of 10 mM phosphate buffer (pH 8.0) and incubated with alkaline phosphatase

(5 units) at 37oC for 24 h. The sample was filtered by centrifugation at 6000 x g for 30 min

through an ultra filtration membrane with a molecular mass cutoff of 5 kDa (Millipore Corpora-

tion, Bedford, MA). An aliquot (5 µL) of the filtered sample was analyzed by LC/MS and found to

contain 8-OH-dAdo-15N5 on the basis of the previously reported LC/MS analysis of 8-OH-Ado

(17). Semi-preparative LC was used to isolate 8-OH-dAdo-15N5 from an irradiated and dephos-

phorylated sample of dATP-15N5 using a Supelcosil LC-8 DB reversed-phase column (25 cm x 1

cm i.d., 5 µm particle size) (Supelco, Bellefonte, PA). The solvents and the elution gradient were as

previously described (17), except that a flow rate of 2 mL/min was used. The column was kept at

room temperature. Under the experimental conditions used, 8-OH-dAdo-15N5 eluting at 18.3 min

was completely separated from dAdo-15N5, which eluted at 17.3 min. This is the same elution

order previously described for the unlabeled analogues of these compounds using an analytical

LC column (17). The fractions corresponding to 8-OH-dAdo-15N5 were collected. At least 30 in-

jections of 100 µL were performed. Collected fractions were combined, dried in a SpeedVac un-

der vacuum and then dissolved in 200 µL of water. The absorption spectrum of the solution was

recorded between the wavelengths of 210 and 350 nm. The spectrum was identical to the ab-

sorption spectrum of authentic 8-OH-dAdo (30).

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Analytical LC/MS was carried out to confirm the identity of 8-OH-dAdo-15N5 and its purity. The

isolated compound was pure and did not contain any detectable unlabeled 8-OH-dAdo. The elu-

tion time of 8-OH-dAdo-15N5 was the same as that of 8-OH-dAdo and its mass spectrum was

similar to that of 8-OH-dAdo (17). As expected, however, the masses of the typical ions of 8-

OH-dAdo-15N5 were shifted by 5 Da to greater masses, i.e., m/z 157 [the protonated base ion

(BH2+)], 273 [the protonated molecular ion (MH +)] and 295 [the sodium adduct ion (MNa+)]. The

concentration of the solution of 8-OH-dAdo-15N5 was determined by UV spectrophotometer us-

ing the absorption coefficient of 12764 M-1 cm-1 at 270 nm (30), and by LC/MS using a 0.1 mM

solution of 8-OH-dAdo as an internal standard. Both measurements yielded essentially identical

results. The concentration of the solution of 8-OH-dAdo-15N5 was 0.037 ± 0.003 mM.

8-OH-dAdo was measured in DNA samples spiked with 2 pmol of 8-OH-dAdo-15N5 per 70 µg

DNA. The concentration of DNA samples was determined by UV spectrophotometer. DNA sam-

ples were hydrolyzed with nuclease P1, phosphodiesterase I and alkaline phosphatase as de-

scribed (17), and filtered by centrifugation at 6000 x g for 30 min using an ultra filtration mem-

brane with a molecular mass cutoff of 5 kDa. An aliquot of 20 µL of the filtered samples contain-

ing 20 µg of hydrolyzed DNA was injected on the LC column. Characteristic ions of 8-OH-dAdo

at m/z 152 (BH2+), 268 (MH+) and 290 (MNa+), and those of 8-OH-dAdo-15N5 at m/z 157 (BH2

+),

273 (MH+) and 295 (MNa+) were recorded during LC/MS analysis using the selected-ion moni-

toring (SIM) mode at the retention time period, when these compounds eluted.

Statistics Groups were compared using one-way ANOVA tests. Duncan´s multiple range test

was used for post hoc comparison of means. Differences were considered significant when

p<0.05.

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RESULTS

In this study, we investigated the possibility that CSB is involved in cellular repair of 8-OH-Ade,

a major lesion in oxidatively damaged DNA. 8-OH-Ade repair was assessed and compared in

wild type, CS-B null and putative CS-B helicase motif VI mutant cells. Glycosylase/apurinic

lyase activity in CS-B cell lines was quantified by measuring incision of an oligonucleotide with a

single 8-OH-Ade residue. Fig. 3A shows the incision activity of wild type, CS-B null and two CS-

B mutant cell lines and the results are summarized in Fig. 3B. Figures 4A and 4B are similar

results, the only difference being that we used the substrate with C opposite the 8-OH-Ade le-

sion to possibly generate maximal nick-forming activity (25;26). CS-B null and mutant cell lines

incise 8-OH-Ade less efficiently than wild type cells in both situations. The activity of CS-B null

cells was approximately 3-fold lower than wild type cells (p < 0.05) and the activity of motif VI

mutants (CSBQ942E and CSBR946A) was approximately 2-fold lower than wild type cells

(p < 0.05). There were no differences in uracil or 5-hydroxycytocine incision of WCE of the

tested cell lines (data not shown) (22).

The role of CSB in repairing 8-OH-Ade was also examined by measuring the level of 8-OH-Ade

in DNA of cells exposed to γ-radiation at doses of 2 Gy or 5 Gy. 8-OH-Ade was measured as its

nucleoside 8-OH-dAdo in wild type, CS-B null, motif VI mutant cells using LC/IDMS, a recently

developed assay for identification and quantification of oxidatively modified DNA nucleosides

(31). DNA samples isolated from cells were hydrolyzed to nucleosides by endo- and exonucle-

ases. Prior to hydrolysis, an aliquot of 8-OH-dAdo-15N5 was added as internal standard to the

DNA samples. LC/IDMS was carried out in SIM mode to monitor the characteristic ions of 8-OH-

dAdo and 8-OH-dAdo-15N5 at the appropriate retention time period, when these compounds

eluted. The BH2+, MH+ and MNa+ ions of both compounds were simultaneously recorded. As

expected, no difference between the retention times of these analogues was observed. Fig. 5

shows the ion-current profiles at m/z 152 (BH2+) and m/z 290 (MNa+) of 8-OH-dAdo, and at m/z

157 (BH2+) and m/z 295 (MNa+) of 8-OH-dAdo-15N5, recorded during the LC/IDMS-SIM analysis

of the enzymic hydrolysate of a DNA sample isolated from CSBQ942E cells following γ-radiation

at 2 Gy. The results showed unequivocal identification of 8-OH-dAdo in DNA from all cell lines

used in this study. The quantification was achieved by the integration of the signals of the moni-

tored ions such as those in Fig. 5 and the calculation of the level of 8-OH-dAdo on the basis of

the known amount of 8-OH-dAdo-15N5 added to the DNA samples as an internal standard prior

to enzymic hydrolysis.

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Fig. 6 shows the level of 8-OH-dAdo in cells following exposure to ionizing radiation. Cells were

irradiated and allowed 30 min to recover and repair radiation-induced DNA damage. The level of

8-OH-dAdo was similar (approximately 0.7 molecules/106 DNA nucleosides) in genomic DNA of

non-irradiated cells regardless of genotype. No change in the 8-OH-dAdo level was observed in

γ-irradiated wild type cells, indicating complete and rapid repair of this lesion. The time of com-

plete repair within 30 min is in agreement with the recently reported repair kinetics of 8-OH-Ade

in human cells (18). In contrast, significantly greater levels of 8-OH-dAdo were observed in γ-

irradiated CS-B null and motif VI mutant cells. In motif VI mutants, exposure to γ-radiation at

5 Gy followed by 30 min incubation resulted in a higher level of 8-OH-Ado than exposure to γ-

radiation at 2 Gy. These results demonstrate that 8-OH-dAdo accumulates in a dose-dependent

manner in irradiated mutant cells, but does not accumulate in irradiated wild type cells.

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DISCUSSION

Previous studies have shown that mutations in CSB cause a deficiency in cellular repair of 8-

OH-Gua (22). The present study shows that CS-B null and motif VI mutant cells are also defi-

cient in incision of 8-OH-Ade. Consistent with this observation, 8-OH-Ade accumulates more in

genomic DNA of CS-B null and motif VI mutant cells than in wild type cells following exposure of

cells to γ-radiation at low doses. These results suggest that CS-B null and motif VI mutant cells

are deficient in cellular repair of 8-OH-Ade. This in turn indicates that CSB plays an important

role in cellular repair of 8-OH-Ade and that the putative helicase motif VI of CSB is important for

this DNA repair function.

CSB is highly homologous to proteins of the SWI/SNF2 family (2;3;32). SWI/SNF proteins par-

ticipate in a wide variety of cellular functions including DNA repair, regulation of transcription,

maintenance of chromosome stability and chromatin remodeling (5;32-34). SWI/SNF2 proteins

contain seven highly conserved motifs for DNA or RNA helicase activity (2;33), but so far, none

has been shown to have this function. Previous experiments were carried out to characterize

the cellular response of various CS-B mutant cell lines to different challenges (Table II)

(22;24;35). We have also mapped the CSB putative helicase motifs and determined their role in

cellular repair of 8-OH-Gua (Table II). Those experiments showed that motifs V and VI are es-

sential for the cellular response to oxidative stress and for cellular repair of 8-OH-Gua in ge-

nomic DNA. The results presented here show that CSB motif VI also plays an important role in

cellular repair of 8-OH-Ade.

It is not clear what mechanism underlies the role of CSB in the repair of 8-OH-Gua and 8-OH-

Ade. The enzymes involved specifically in repair of 8-OH-Ade have not yet been identified (re-

viewed in (19)), but some evidence suggests that repair of 8-OH-Ade is mechanistically different

from repair of 8-OH-Gua. Among the bacterial and mammalian DNA glycosylases, which were

investigated for their activity on lesions in oxidatively damaged DNA, only E. coli formami-

dopyrimidine glycosylase (Fpg) exhibited a low activity on 8-OH-Ade in DNA containing multiple

modified bases (36;37). However, this activity was insignificant when compared with the activity

of Fpg on 8-OH-Gua, 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4,6-

diamino-5-formamidopyrimidine (FapyAde), which are the principal substrates of Fpg. The inac-

tivity of Fpg was suggested to result from the absence of a C6-keto group in 8-OH-Ade (38). A

recent study on the cellular repair of modified DNA bases showed that 8-OH-Ade is efficiently

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repaired in human cells with kinetics similar to the repair kinetics of pyrimidine-derived lesions

rather than to that of purine-derived lesions (18). This suggests that human cells possess en-

zyme(s) to repair 8-OH-Ade. However, it is not known whether this repair activity involves BER

or NER, or both. A yeast functional homolog of E. coli Fpg encoded by the OGG1 gene of Sac-

charomyces cerevisiae (yOgg1) was shown to excise 8-OH-Gua and FapyGua, but not Fap-

yAde or 8-OH-Ade (39;40). Human homologues of yOgg1 were recently isolated (reviewed in

(41)). Two polymorphic forms of hOgg1 namely α-hOgg1-Ser326 and α-hOgg1-Cys326 are pro-

duced in human cells. Similar to yOgg1, both enzymes were reported to efficiently excise 8-OH-

Gua and FapyGua, but not FapyAde or 8-OH-Ade from DNA with multiple lesions (42). Two

enzymes from Drosophila melanogaster also exhibited similar activities that efficiently excised

8-OH-Gua and FapyGua from DNA, but not FapyAde and 8-OH-Ade (43;44). In the same con-

text, it should be pointed out, that although 8-OH-Ade was repaired in human cells with kinetics

similar to repair kinetics of pyrimidine-derived lesions, none of the known pyrimidine lesion-

specific DNA glycosylases removed 8-OH-Ade from DNA, either (reviewed in (19)). These stud-

ies clearly show that there is a paucity of knowledge about enzymes that repair 8-OH-Ade in

cells or in vitro. On the other hand, our results confirm results from the previous study on the

cellular repair of 8-OH-Ade in terms of the ability of human cells to repair this lesion and in terms

of its repair kinetics (18). By studying the biochemical contribution and expression regulation of

CSB to cellular repair of 8-OH-Gua, we found that CSB itself participates in the catalytic process

of 8-OH-Gua incision in vitro, and CSB facilitates the expression of hOgg1 (unpublished data).

Further research is necessary to understand the mechanism by which CSB facilitates cellular

repair of 8-OH-Ade and to identify other proteins that might contribute directly or indirectly to cel-

lular repair of this lesion.

8-OH-Ade was identified as its nucleoside form 8-OH-dAdo, and its level and accumulation in

genomic DNA of CS-B mutant cells was quantified by means of a recently developed methodol-

ogy using LC/IDMS (45). The present study is the first application of this technique to the meas-

urement of the formation and repair of 8-OH-dAdo in living cells. The specificity and sensitivity

of LC/IDMS in the SIM mode permitted us to detect and quantify 8-OH-dAdo at a level of ap-

proximately 7 molecules/107 DNA nucleosides. Such a level of detection by LC/IDMS-SIM had

previously been reported (45). For quantification, we isolated a stable isotope-labeled analog of

8-OH-dAdo in pure form for the first time and used it as the internal standard. It should be

pointed out that the use of liquid chromatography/tandem mass spectrometry (LC/MS/MS) has

also been reported for the identification of 8-OH-dAdo (46-49). However, the isotope-dilution

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technique has been applied for quantification in two instances only (46;49). The sensitivity level

for 8-OH-dAdo of this technique in the multiple reaction-monitoring (MRM) mode is similar to

that of LC/IDMS-SIM (17). On the other hand, the application of LC/MS/MS-MRM to the meas-

urement of 8-OH-dAdo in living cells has not been reported.

In conclusion, the present study suggests that CSB plays a role in the cellular repair of 8-OH-

Ade and that the putative helicase motif VI of CSB may be important in this process. In addition,

8-OH-Ade and other lesions such as 8-OH-Gua might accumulate in the DNA of CS patients

and could potentially contribute to pathology associated with CS.

ACKNOWLEDGMENTS

We thank Dr. C. Chan for DNA extraction. Certain commercial equipment or materials are iden-

tified in this paper in order to specify adequately the experimental procedures. Such identifica-

tion does not imply recommendation or endorsement by the National Institute of Standards and

Technology, nor does it imply that the materials or equipment identified are necessarily the best

available for the purpose.

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Table I. The oligonucleotides used in incision assay

Oligonucleotide Specificities

5’-GCTCTAGGCC(8-OH-Ade)AGCTTGATCTGCCAGTT-3’ 8-OH-Ade containing substrate

5’-GCTCTAGGCCTAGCTTGATCTGCCAGTT-3’ Normal A-T pair for control

5’-AACTGGCAGATCAAGCTTGGCCTAGAGC-3’ Complement strand for 8-OH-Ade-T pair

5’-AACTGGCAGATCAAGCTCGGCCTAGAGC-3’ Complement strand for 8-OH-Ade-C pair

Oligonucleotides were prepared by Midland Certified Reagent Co., Midland, TX

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Table II. The biological consequences of mutation(s) of putative helicase motifs of CSB in various assays

Cellular sensitivities

(22, 24, 35)

Glycosylase/apurinic lyase activity in

WCE (22)

Mutated

motif

(Fig. 1) UV 4-NQO γ-Ray

RNA recov-

ery after UV

(24, 35) 8-OH-Gua 5-OH-Cyt Uracil

Levels of 8-OH-

dGuo in DNA

after γ-ray (22)

WT + + + +++ +++ +++ +++ +

Null +++ +++ +++ + + +++ +++ +++

Ia + + + +++ +++ +++ +++ NA

II +++ +++ NA + +++ NA NA NA

III + + + +++ +++ +++ +++ NA

V +++ +++ ++ + ++ +++ +++ NA

VI +++ +++ +++ + ++ +++ +++ +++

NTB + + + +++ +++ +++ +++ NA

+, ++ and +++ are the relative responses referring to the column titles.

NA: not analyzed

4-NQO: 4-nitroquinoline-1-oxide

5-OH-Cyt: 5-hydroxycytosine

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FIGURE LEGENDS

Fig. 1. Structure of 8-hydroxyadenine.

Fig. 2. Structure of CSB and the location of designed mutants. The protein contains the seven

conserved helicase motifs, a highly acidic region, two nuclear localization signals (NLS) and a

nucleotide binding domain (NTB). CSBQ942E is constructed by replacing a glutamine residue

highly conserved in the SNF2 family with a negatively charged glutamic acid. CSBR946A is

constructed by replacing an arginine residue highly conserved in the SNF2 family with alanine.

Fig. 3. Glycosylase/apurinic lyase activities on 8-OH-Ade in wild type and CSB mutant cells.

The substrate contained T paired with 8-OH-Ade. (A) Incubations were carried out for 3 h and

reaction products were analyzed by denaturing polyacrylamide gel electrophoresis. The sub-

strate is a 28 mer, and the product is 11 mer. (B) Quantification of the activity of nick-forming at

the position of 8-OH-Ade. The mean values (± standard deviations) from four independent ex-

periments are shown. Stars indicate values that are significantly different from CSBWT

(p<0.05).

Fig. 4. Glycosylase/apurinic lyase activities on 8-OH-Ade in wild type and CSB mutant cells.

The substrate contained C paired with 8-OH-Ade. (A) Incubations were carried out for 3 h and

reaction products were analyzed by denaturing polyacrylamide gel electrophoresis. The sub-

strate is a 28 mer, and the product is 11 mer. (B) Quantification of the activity of nick-forming at

the position of 8-OH-Ade. The mean values (± standard deviations) from four independent ex-

periments are shown. Stars indicate values that are significantly different from CSBWT

(p<0.05).

Fig. 5. Measurement of 8-OH-dAdo by LC/IDMS in genomic DNA of wild type and CSB mutant

cells. Selected ion-current profiles at m/z 152 (BH2+) and m/z 290 (MNa+) of 8-OH-dAdo, and at

m/z 157 (BH2+) and m/z 295 (MNa+) of 8-OH-dAdo-15N5, which were recorded during the

LC/IDMS-SIM analysis of the enzymic hydrolysate of a DNA sample isolated from CSBQ942E

cells following γ-irradiation at 2 Gy.

Fig. 6. Level of 8-OH-dAdo in genomic DNA of γ-irradiated wild type and CSB mutant cells.

Cells were exposed to γ-radiation at doses of 2 Gy or 5 Gy and subsequently incubated for 30

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min. DNA was isolated and analyzed for 8-OH-dAdo as described above. The data shown rep-

resent the mean values (± standard deviations) of four independent experiments. Stars indicate

values that are significantly different from CSBWT (p<0.05).

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NN

NN

NH2

OH

H

Fig. 1

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CSBWT: 937PSTDTQARERA947

CSBQ942E: PSTDTEARERA

CSBR946A: PSTDTQAREAA

COOHNH2 ACIDIC I IA II IIII IV V VINLS NLS NTB

COOHNH2 ACIDIC I IA II IIII IV V VINLS NLS NTB

Fig. 2

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PC

3.1

WT

Q94

2E

R94

6A

Substrate

Product

Fig. 3

B

A

Con

trol

WT

PC

3.1

Q94

2E

R94

6A

0

10

20

30

*

**

Cell lines

% 8

-OH

Ade-

T in

cisi

on

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PC

3.1

WT

Q94

2E

R94

6A

Substrate

Product

Fig. 4

B

A

WT

PC

3.1

Q94

2E

R94

6A

0

10

20

30

40

50

** *

Cell lines

% 8

-OH

Ade-

C in

cisi

on

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min12 13 14 15

1000

2000

3000

4000

5000

6000

7000

8000

MSD1 152, EIC=151.7:152.7 (JS2A89.D) API-ES, Pos, SIM, Frag: 100 (TT)MSD1 157, EIC=156.7:157.7 (JS2A89.D) API-ES, Pos, SIM, Frag: 100 (TT)MSD1 290, EIC=289.7:290.7 (JS2A89.D) API-ES, Pos, SIM, Frag: 100 (TT)MSD1 295, EIC=294.7:295.7 (JS2A89.D) API-ES, Pos, SIM, Frag: 100 (TT)

m/z 157

m/z 290

m/z 295

m/z 152

Fig. 5

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Fig. 6

0 2 50.0

0.5

1.0

1.5

2.0

2.5

CSBWTCSBR946A

CSBQ942ECSBPC3.1

**

***

*

γ-radiation dose (Gy)

Leve

l(8

-OH

-dA

do/1

06

Nuc

leos

ides

)

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Jingsheng Tuo, Pawel Jaruga, Henry Rodriguez, Miral Dizdaroglu and Vilhelm A. Bohr8-hydroxyadenine in DNA

The cockayne syndrome group B gene product is involved in cellular repair of

published online June 11, 2002J. Biol. Chem. 

  10.1074/jbc.M204814200Access the most updated version of this article at doi:

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