Vol. 268, No. 15, Issue of May 25, pp. 11143-11151,1993
Printed in U.S.A.
THEJOURNAL
OF BIOLOGICAL
CHEMISTRY
T h e American Society for Biochemistry and Molecular Biology, Inc,
Q 1993 by
Preparation and Characterization
of a Set of Deoxyoligonucleotide 49mers Containing Site-specific Cis-syn, Trans-syn-I,(6-4), and Dewar
Photoproducts of Thymidylyl(3’ + 5’)-thymidine*
(Received for publication, August 28, 1992, and in revised form, February 4,1993)
Colin A. Smith and John-Stephen Taylor$
From the Department of Chemistry, Washington University, St. Louis, Missouri, 63130-4899
Deoxyoligonucleotide 49-mers containing a central
cis-syn, trans-syn-I, (6-4), or Dewar photoproduct of
TpT were constructed for
use in repair and
replication
studies by ligation of shorter photoproduct-containing
oligonucleotides. A (6-4) product-containing6-mer
was prepared in 3.4% yield by 254 nm irradiation of
d(AATTAA)and convertedin nearly quantitativeyield
to the Dewar isomer by irradiation with Pyrex- and
Mylar-filteredmedium-pressuremercury
arc light.
Trans-Syl~l
d(CGAATTAAGC)containing a site-specific cis-syn or
trans-syn-I dimer was prepared via automated solidphase DNA synthesis utilizing photoproduct building
blocks. Thephotoproduct-containing49-merswere
characterized by their susceptibility to base cleavage
and a number of enzymes routinely used to map the
sites of DNA photoproduct formation. 1 M piperidine
at 90 OC cleaved the Dewar product fasterthan the(64) product, but didnot cleave the cyclobutane dimers.
The 3’4 5’ exonuclease activity of T4 DNA polymerase was completely blocked by all the lesions except
the (6-4) product,which it slowly bypassed. T4 endoDewar
nuclease V did not cleave the (6-4) or Dewar photoproduct, but unexpectedly cleaved the trans-syn-I diFIG. 1. Photoproducts of a thymidylyl(3‘ 4 5’)thymidine
mer at most 1%the rateof the cis-syn dimer in double- site.
stranded DNA. The trans-syn-I dimer was cleaved at
a 50-fold higherrate in double- than insingle-stranded
DNA. Escherichia coliphotolyase was found to be spe- (<0.02) than in single-stranded (0.14) DNA (5) because they
cific for the cis-syn dimer at low concentrations. Im- result from a conformation in which one of pyrimidines is in
plications of this work to methodology for mapping the syn glycosyl conformation, rather than the anti-conforandquantifying
DNA photoproducts are also dis- mation found inB DNA. Of the two possible trans-syn
isomers, the one in which the 5”pyrimidine is in the syn
cussed.
glycosyl conformation (trans-syn-I) (6) is more likely to predominate than that in which the 3’-pyrimidine is in the syn
conformation (trans-syn-11)’ based solely on base stacking
The misrepair and misreplication of DNA photolesions considerations. The (6-4) products are not stable in sunlight
induced by sunlight are the most likely causes of skin cancer but are converted to their Dewar valence isomers via absorp(for a review see Ref. 1).Most recently, evidence has been tion of light by their long wavelength absorption band cenpresented to implicate the UV in sunlight as the primary tered near 325 nm (7, 8). To unravel the precise structurecause of mutations found in the p53 gene of squamous cell activity relationships in sunlight-induced mutagenesis, pure,
carcinomas (2). The major photoproducts of duplex DNA well characterized, site-specific photoproduct-containing
directly induced by the UV portion of sunlight occur at DNA substratesare required for physical, enzymatic, and
&pyrimidine sites and arethe cis-syn cyclobutane dimers and biological studies (9).
the (6-4) products (illustratedfor a TpT site in Fig. 1) (3, 4).
Recently, deoxyoligonucleotide 11-mers containingsiteTrans-syn dimers are also formed by 254 nm light, but at a specific cis-syn (lo), trans-syn ( l l ) , (6-4), and Dewar (12)
much lower rate relative to cis-syn dimers in double-stranded products have been isolated and used for obtaining mutation
spectra in Escherichia coli. Because of the small amounts of
* This work was supported by Public Health Service Grant R01- photoproduct-containing11-mers isolated, and the limited
CA40463, awarded by the National Cancer Institute, Department of methods used to characterize the products (NMR was not
Health and Human Services. The Washington University NMR
Service Facility was funded in part through National Institutes of used), only the structural integrity and purity of the cis-syn
Health Biomedical Research Support SharedInstrument Grant1 SI0 dimer-containing product was well established. The stereoRR02004. The costs of publication of this article were defrayed in chemistry of the trans-syn isomer, i.e. whether it was the
part by the payment of page charges. This article must therefore be
hereby marked “advertisement” in accordance with 18U.S.C. Section
1734 solelyto indicate this fact.
4 To whom correspondence should be addressed.
‘We have recently isolated and characterized the trans-syn-I1
isomer of TpT andfind that itis formed at about 1/20 the rate of the
trans-syn-I isomer (S. Nadji and J.-S. Taylor, unpublished results).
11143
11144
5"3'
Photoproduct-containing
Site-specific
DNA
third dissolved in 30 ml of water, degassed, put in a 155 cm2 dish on
ice under argon in a sealed polyethylene pouch (Ziplock), and irradiAGCTACCATGCCTGCACGA~~MGCMTTCGTMTCATGGTCATAGCT
49-mer
ated between 120 and 150 min with 1 milliwatt/cm* 254 nm light
from a Spectroline XX-15 source fitted with two 15-watt low pressure
AGCTACCATGCCTGCACG
18-mer
mercury bulbs. The three batches were combined and reduced in
vacuo and then fractionated by preparative C-18 HPLC with a 60
MxyM
6-mer
min 10-25% gradient of MeOH in 75 mM K2HP04/KH2P04 aatflow
rate of 1 ml/min. Fractions with an absorption maximum near 325
GCMTTCGTMTCATGGTCATAGCT
2'5-mer
nm were combined and concentrated, then desalted on a short C-18
AGCTACCATGCCTGCA
16-mer
gravity column, concentrated, and repurified by preparative phenyl
HPLC with a 60 min 10-40% MeOH gradient in 75 mM K2HP04/
CGAAxyMGC
10-mer
KHzP04, pH 6.6, at a flow rate of 1 ml/min. The major peak was
collected and desalted as before to yield d(AAT[6-4]TAA) in 3.4%
MTTCGTMTCATGGTCATAGCT
23-mer
(219)
overall yield (223 nmol, 12.2 optical density units, 425 pg).
d(AAT[DewarlTAA)-Approximately
100 nmol (6 optical density
AGCTACCATGCCTGCACGMxyMGCMTTCGTMTCATGGTCATAGCTGA 51-mer
units, 200pg) of d(AAT[6-4]TAA) that had been evaporated to
dryness was dissolved in 700 pl of "100%" D,O (Merck) and trans3"5'
ferred to a 5-mm NMR tube. The sample was irradiated by PyrexGATGGTACGGACGTGCTTMTTCGTTAAGCATTAGTACCAGTATCGACT 49-mer
and Mylar-filtered light from a 450-watt medium pressure mercury
arc lamp at a distanceof 30 mm from the lamp center and periodically
GTACGGACGTGCTTMTTCGTTAAGCATTAGTAC
34-mer
monitored by 'H NMR for a total of 125 min to give d(AAT[Dewar]
FIG. 2. Oligonucleotide sequences used in this study. X Y
TAA) in an estimated 99% yield.
refers to either a cis-syn, trans-syn-I, (6-4), or Dewar photoproduct
d(CGAAT[c,slTAAGC) and d(CGAAT[t,s-I]TAAGC)-The dimerof TpT (see Fig. 1 for the structures) or the undamaged TT site. The containing decamers were prepared by solid-phase synthesis on a 0.2nucleotide position indicated above the 49-mer is relative to the 5'
pmol scale utilizing cis-syn (14) and trans-syn-I (15) DNA synthesis
end, and that in parentheses above the 51-mer is relative to the 3'- building blocks and purified by both anion-exchange and C-18 HPLC
end.
as described. The cis-syn 10-mer eluted at 13.2 min and the transsyn-I 10-mer at 18 min from the LiChroCart C-18 column with a 1
ml/min, 40 min 10-30% methanol ramp in 75 mM K,HP04/KH2P04,
trans-syn-I or trans-syn-I1 isomer (6), was not determined,
pH 6.6. For preparative chromatography, a 1ml/min, 40 min 10-20%
and two different preparationsof the same Dewar-containing methanol ramp was used.
11-mer were reported to lead to different mutation spectra.
Preparation of 5' End-labeled Photoproduct-containing 49-mer
These recent results underscore the need for better methods Substrates-Separately, 2 nmol of each 6- and 10-mer were phosphorylated with 27 units of T4 polynucleotide kinase (New England
for preparingandcharacterizingphotoproduct-containing
Biolabs) and 67 nmol of ATP in 14 pl of kinase buffer (50 mM Tris.
substrates.
Herein, we report the preparation and characterization of HCI pH 7.5, 10 mM MgCl,, 10 mM dithiothreitol) for 1.5 h at 37 "C.
Likewise, 12 nmol of 25-mer and 8 nmol of 23-mer were separately
the first complete set of 49-mers of identical sequence con- phosphorylated with 100 units of kinase and 270 nmol of ATP in 55
taining central cis-syn, trans-syn-I, (6-4), andDewar photo- pl of kinase buffer for 1.5 h at 37 "C. The phosphorylations were
products of >95% puritysuitable for use in comparative stopped by inactivating the kinase at 100 "C for 5 min. The 25- and
chemical, enzymatic, and biological studies. The photoprod- 23-mers were spiked with 2 pmol of 32P-labeled 25- and 23-mers,
uct-containing 49-mers were subjected to a number of enzy- respectively. Each kinased 6-mer solution was added with one-third
matic and chemical agents that have been used to locate and of the kinased 25-mer solution to a tube containing 4 nmol each of
dry 18- and 34-mers. Each kinased 10-mer solution was added with
quantify photoproductsat a sequencelevel in order to evaluatehalf of the kinased 23-mer solution to a tube containing 4nmol each
the specificity of these methods.
of dry 16- and 34-mers. The mixtures were annealed and brought to
75 p1 with more kinase buffer containing 1500 units of T4 ligase (New
EXPERIMENTALPROCEDURES
England Biolabs) and 126 nmol of ATP. The reactions were incubated
Enzymes,Reagents, and Equipment-Oligonucleotides (Fig. 2) overnight at 15 "C, lyophilized, dissolved in 95% aqueous formamide,
were synthesized on an AB1 380B synthesizer by standard 8-cyanoe- electrophoresed, excised, eluted, and dialyzed. The 49-mers (0.1pmol)
thy1 phosphoramidite chemistry and purified by anion-exchange were 5' end-labeled by incubation with 0.2-2 pmol of [Y-~'P]ATP
HPLC. Oligonucleotide concentrations were determined by absorb- (5000 Ci/mmol, Amersham Corp.) and 2 units of T4 kinase in total
ance at 260 nm using estimatedextinction coefficients (13). T4 volume of 5 p1 of kinase buffer at 37 "C for at least 45 min, and then
endonuclease V was a gift fromR.S.Lloyd (University of Texas, inactivated by heating at 100 "C for 5 min.
T4 denV Endonuclease V Cleavage of 5' End-labeled SubstratesGalveston) and E. coli photolyase was a gift from A. Sancar (University of North Carolina, Chapel Hill). Dideoxy sequencing was carried For each 5' end-labeled 49-mer, 2 pmol were diluted to 24 pl of 32
out with 1 unit of Sequenase Version 1.0 (USB) and 200 p M dNTPs mM KHPO,, pH 8.3, 100 mM NaCI, and 10 mM EDTA. Two-thirds
of each solution were incubated with 2 pl of T4 endonuclease V (180
(USB), with the eponymous dNTP consisting of a 1:3 mixof
ddNTP:dNTP. Anion-exchange HPLC was carried out on a Nucleo- ng/pl) for 40 min at 37 "C. The enzyme-treated portions were split in
gen DEAE 60-7 column. Analytical reverse-phase HPLC was carried two, and half was diluted to 100 pl of 1 M piperidine and heated at
out on a Dynamax C-18 or phenyl column (4.6 X 250 mm, 8-pm 100 "C for 20 min. The piperidine wasremoved by concentrating
particle size). Preparative reverse-phase HPLC' was carried out on a three times from water. All samples were diluted in 95% aqueous
Merck LiChroCart C-18 column (4 X 125 mm, 8-pm particle size) or formamide and electrophoresed. For kinetic studies, 1pmol of 5' endlabeled cis-syn or trans-syn-I dimer-containing 49-mer was diluted to
on the phenyl column. Bulk C-18 packing for desalting was purchased
from Waters. 300 MHz 'H NMR spectra were obtained on a Varian 40pl containing 32 mM Tris.HCI pH 7.5, 10 mM EDTA, 100 mM
Gemini instrument and 500 MHz 'H spectra on a Varian XR-500 NaC1, and 100 mg/ml bovine serum albumin. Duplex substrates were
instrument. The residual HOD resonance was assigned as 4.67 ppm prepared by annealing to the complementary 34-mer by slow cooling
and suppressed by saturation. Concentration of samples was carried from 95 to 37 "C. At zero time, 10 pl of a prewarmed solution
out in a Savant Speed Vac under vacuum. All electrophoresis was containing 360 ngof T4 endonuclease V in the same buffer was added
carried out on a 0.4mm thick, 375 mm long, 7 M urea, 1:19 cross- with thorough mixing. The reaction was incubated at 37 "C, and 4-rl
linked, 15% acrylamide gel at 1800 V. DNA fragments were visualized aliquots were removed and quenched in 50 pl of 1.08 M piperidine.
by autoradiography with Kodak XAR-5 film at -70 "C and quantified The aliquots were quenched at 0.25, 0.5,0.75, and 1 min for the
single- and double-stranded cis-syn dimer 49-mers, 4, 8, 16, 32, 64,
by densitometry on a Joyce-Loebl Chromoscan 3.
d(AAT[6-4]TAA)-d(AATTAA)
(6.4 pmol, 446 optical density and 128 min for the single-stranded trans-syn-I dimer 49-mer, and
units, 12 mg) was irradiated to absorb an estimated 3.3 kJ of 254 nm 0.25, 0.5, 1,2,4, and8 min for the double-stranded trans-syn-I dimer
light by the following procedure. The sample was split in thirds,each 49-mer. The piperidine-quenched reactions were heated at 100 "C for
20 min and lyophilized, dissolved in 20 pl of water, and lyophilized
The abbreviations used are: HPLC, high performance liquid chro- again. The residue was dissolved in 20 pl of 95% aqueous formamide,
electrophoresed, autoradiographed, and quantified by densitometry.
matography; ppm, parts per million.
11145
Site-specific Photoproduct-containingDNA
C18 COLUMN
The apparent first-order rate constantswere obtained as theslope of
PHENYL COLUMN
a linear leastsquares fit to -ln(substrate/substrate products) versus
time.
Base Cleavage of 5' End-labeled Substrates-Each 5' 32P-labeled6or 49-mer (2 pmol) was dissolved in 100 p1 of 1M piperidine. Aliquots
of 10 pl were placed in sealed tubes, heated at 90 "C for 0, 1, 5, 25,
and 125 min, and quenched by freezing on dry ice and lyophilizing.
The samples were then lyophilized three times from 20 pl of water,
dissolved in 95% aqueous formamide, electrophoresed, and autoradiographed. Hydroxide cleavage experiments were carried out by incubating with 0.1 N NaOH at 90 "C or 0.4 N NaOH at room temperature
and were quenched by addition to 2 volumes of 1M K~HPO,/KHZPO~,
pH 6.6.
Base and T4 Endonuclease V Cleavage of 3' End-labeled 51-mer
Substrates-Each 49-mer (2 pmol) was annealed to a complementary
49-mer (Fig. 2), creating unique, two-nucleotide, 5' overhanging ends.
The duplex 49-mers were then incubated for 3 min at room temperature with 0.5 unit of Klenow fragment (New England Biolabs), in
20 pl of 10 mM Tris. HCI, 5 mM MgC12, and 7.5 mM dithiothreitol
b) a + 2 5 4 m
(5000 Ci/mmol, Amersham), 30
that contained 0.2 p~ [cz-~'P]~ATP
p~ dCTP, 30 p~ dGTP, and30 p~ dTTP. Thereactions were stopped
by heating at 100 "C for 10 min. The resulting undamaged, cis-syn
and trans-syn-I duplex 51-mers (0.3 pmol each) were annealed and
treated with T4 endonuclease V (1.8 ng) for 30 min at 37 "C in 12 p1
of 32 mM Tris. HC1, pH 7.5, 10 mM EDTA, 100 mM NaCl, and 100
mg/ml bovine serum albumin. The reactions were stopped by the
I
.
.
.
.
.
addition of 15 pl of 95% aqueous formamide. The undamaged, (6-4)
202630364046
303640466056
and Dewar duplex 51-mers (0.3 pmol each) were treated with 1 M
MIN
MIN
piperidine at 100 "C for 60 min, lyophilized twice, dissolved in 27 pl
of 50% aqueous formamide, heat denatured, and electrophoresed.
FIG. 3. HPLC chromatograms of d(AATTAA) and its irra3' + 5' Exonucleolytic Cleavage by T4 DNA Polymerase-To each diation products. Analytical C-18 and phenyl chromatography
5' end-labeled 49-mer (0.3 pmol) in 12 pl of 50 mM Tris. HCl, pH 7.5, traces of approximately 150 pmol of each sample with a 1 ml/min,
10 mM MgC12, and 10 mM dithiothreitol was added 3.2 units of T4 60-min gradient of -0%
MeOH in 75 mM KzHPO~/KHZPO, and
DNA polymerase (Promega) in 2 p1 of the same buffer. Aliquots of UV absorbance detection at 260 nm.
2.5 pI were removed after 1,5, and25 min, quenched by dissolving in
12.5 p1 of 95% aqueous formamide, and electrophoresed. To characTABLE
I
terize the 3' ends resulting from termination at the sites of the cksyn and trans-syn-Idimers, 1pmol of 5' end-labeled single- or double' H NMR data (in ppm) for undamaged and (6-4) and Dewar
stranded samples of the 49-mers were treated with T4 DNA polymphotoproduct-containing dinucleotides and 6-mers
erase. Duplex substrates were prepared by annealing the 49-mers to
CHI
H6
H2'
the complementary 34-mer. After 30 min of incubation with the T4
5'
3'
5'
3'
3'
polymerase as above, the samples were inactivated by heating at
100 "C for 5 min, followed by lyophilization. The samples were
1.88
1.90
7.67 2.39
7.70
TPT"
dissolved in 300 p1 of 60% ethanol, divided, and one set was irradiated
1.4gb 1.59'
7.11'
7.1gb
d(AATTAA)
for 25 min with 300 microwatts/cmz 254 nm light (-4.5 kJ/m*). All
1.76
2.32
5.09
8.00
3.04
T(6-4)pTd
the samples were then concentrated and dissolved in 20 pl of 95%
d(AAT(6-4)TAA) 2.19 1.50
e 3.15 7.54
aqueous formamide, electrophoresed, and autoradiographed.
T(Dewar)pTd
2.28 5.33 4.73 2.10 1.57
E. coli Photolyase Substrate Specificity-One pmol of each of the
1.88
d(AAT(Dewar)TAA) 1.39
5' end-labeled duplex 49-mers was treated as for the T4DNA polymRef.
40.
erase study, except that beforehand half of each sample was irradiated
Assignment to the5' or 3' T is arbitrary and may be interchanged.
with 366 nm for 1h in the presence of equimolar photolyase (100 nM)
e Could not be assigned.
and theother half was not. Both sets of samples were then treated as
Ref. 39.
before with T4 DNA polymerase for 1,5, and25 min, electrophoresed,
and autoradiographed.
+
n
A
h
~
Pyrex- and Mylar-filtered medium pressure mercury arc light
for 125 min as indicated by'H NMR (Fig. 5c). 'H NMR
Preparation of d(AAT[6-4]TAA)-Irradiation
of d(AAT- spectra taken duringthe irradiation period indicated that the
TAA) with 254 nm of light gave numerous products (Fig. 3b) half-life of the reaction was less than 20 min and there was
of which the (6-4) 6-mer was estimated to be produced in a no evidence for the formation of any additional products. The
maximum yield of about 5% when the irradiation was carried UV spectrum of the Dewar 6-mer showed the expected about to about 75% conversion of starting material. The (6-4)- sence of the 325 nm band characteristic of the (6-4) product,
containing 6-mer was isolated by preparative HPLC on a C - and the presence of a slight absorption tail >300 nm charac18 column followed by a phenyl column in 3.4% overall yield teristic of the Dewar product of TpT (Fig. 4). Likewise, the
and was >96% pure as estimated by 'H NMR spectroscopy 'H NMR spectrum (Fig. 5c, Table I) showed the loss of the
(Fig. 5b). The (6-4) 6-mer has the characteristic absorption pTH6 signal characteristic of the (6-4) product and the exratio of 10.5, compared to a pected shift in the methyl signals. The (6-4) and Dewar 6peak at 325 nm with an A2M)/A325
calculated value (13, 16) of 11.4. The 'H NMR spectrum mers coelute under the C-18 chromatography conditions used
shows the characteristic pTH6 and methyl signals of the (6- (Fig. 3e) but could be partially resolved byphenyl chromatog4) product of TpT (Table I), and what must be the pTH2'
raphy.
signal. There also appear to be two small peaks at 1.39 and
Construction of the 49-mers and Electrophoretic Analysis of
1.88 ppm that could be attributed to approximately 2% of the Their Cleavage Products-The photoproduct-containing 49Dewar product.
mers were prepared by ligation of the photoproduct-containPreparation of d(AAT[Dewar]TAA)-The
(6-4) 6-mer was ing 6- and 10-mers to the appropriate oligonucleotides (Fig.
isomerized to the Dewar product in essentially quantitative 2) in the presence of a 34-mer ligation scaffold. The yields of
yield (<2% remaining (6-4) product) by irradiation with the 49-mers based on the photoproduct-containing hexamer
RESULTS
11146
Site-specific
Photoproduct-containing
DNA
1
0.8
I
o*6
0.4
0.2
0
220
240
260
2 0
3w
320
380 360 340
w m q , nm
FIG. 4. Relative UV absorption spectra of d(AATTAA), d(AAT[6-4]TAA), and d(AAT[Dewar]TAA) in H,O.
or decamer as the limiting reagent before isolation were 1560% for the undamaged product, above 90% for the cis-syn
and trans-syn-I dimers, 12-30% for the (6-4) product, and
15-40% for the Dewar product. The mobility of the bands
produced in the subsequent cleavage experiments on labeled
substrates were referenced to dideoxy sequencing bands (not
always shown) and to background Maxam Gilbert cleavage
bands at purines resulting from the base treatments (not
visible in the figures).
T4 denV Endonuclease V Cleauuge Specificity and Kinetics-As expected, the cis-syn 49-mer was cleaved by T4 endonuclease V, and the undamaged, (6-4), and Dewar 49-mers
DNA
Photoproduct-containing
Site-specific
were not (Fig. 6). Unexpectedly, a small amountof the transsyn-I 49-mer was also cleaved. Both the cis-syn and transsyn-I 49-mers were cleaved to several products, all of which
were converted by treatment with 1 M piperidine a t 100 “C
for 25 min to a single 5’ end-labeled product that had the
same mobility as a 20-mer with 5’- and 3”phosphates. The
uppermost band and the central pair
of bands prior to piperidinetreatment of the 5’ end-labeledcis-syn dimerhave
approximately the samemobility as 23- and 22-mers with 5’phosphates. Because only a small amount of the trans-syn-I
49-mer was cleaved, the kinetics of the reaction were investigated to determine whether the
cleavage was due to the
presence of contaminating cis-syn dimer, orwas adirect result
of trans-syn-I dimer cleavage. The cleavage of the doublestranded cis-syn 49-mer waseffectively over in 15 s, although
a small amount of uncleaved 49-mer remained (-1.5%). Because the amount of uncleaved product did not change with
time, itwas attributed toa non-cleavable impurity. Assuming
that 15 s of reaction corresponds to at least six half-lives, a
lower bound to the rate constant
could be estimated tobe 15
rnin”. Thefirst-orderrateconstantsforthethreeother
substrates were derived from least-squares analysis of a minimum of four time points for each
of three independent runs.
Time points beyond two half-lives or 150 min were not used
because of deviation from first-orderkinetics.Unlikethe
double-stranded cis-syn 49-mer, the double-stranded transsyn-I 49-mer was cleaved with a rate constant of 0.14 & 0.01
min”. The single-stranded substrates
were cleaved at a much
slower rate than the double-stranded ones, with the cis-syn
and trans-syn-I49-mers being cleaved with rate constantsof
1.3 & 0.2 rnin”, and 0.003 & 0.001 rnin”, respectively. Because
there was no detectable cleavage of the trans-syn-I49-mer in
the time in
which morethan half the cis-syn would be cleaved,
N
cs
TS
64
DW
nnnnn
K V P K V P K V P K V P K V P
.
.-_
.
.1.1,..
11147
the presence of contaminating cis-syn dimer was ruled out.
Cleavage of 3‘ end-labeled cis-syn and trans-syn-I 51-mers
5‘led to asingle product migrating as a 31-merwitha
phosphate (Fig. 7).
Base Cleavage Specificity and Kinetics-Hot piperidine
treatment cleaves both theDewar and (6-4) 49-mers(Figs. 79), whereas the undamaged cis-syn and trans-syn-I 49-mers
are only very slowly and non-specifically degraded (Fig. 8).
The overall half-life for cleavage measured for the 5’ endlabeled substrate was about 140 min forthe (6-4)49-mer and
3 min for the Dewar 49-mer. When the kineticsof the piperidine cleavage reactions were examined more carefully, biphasic kinetics anda striking length dependence on the rate
of cleavage was observed (Fig. 9B). The cleavage reactions
were not clean and led to multiple 5’ end-labeled products
(Figs. 8 and 9A). At approximately 55% reaction, the
(6-4) 6mer led to three major products with relative mobilities ( R L )
of 1.03 and 1.07 in 17 and 20% yields, respectively, whereas
the Dewar 6-mer led to one major product with an R L of 1.12
in 39% yield (Fig. 9A). The major cleavage band of the 5’labeled Dewar 49-mer at short reaction times migrated approximately as a 23-mer with a 5’-phosphate, and a t longer
times asa 22-mer with 5’- and 3”phosphates. Atlong reaction
times a minor product was also observed that migrated as a
20-mer with 5’- and 3”phosphates. In contrast, piperidine
hot
cleavage of 3’ end-labeled 51-mer led to only one labeled
product, which migrated as though it were a 29-mer with a
5’-phosphate (Fig. 7). Treatment of the 5’ end-labeled 49mers a t 90 “C with 0.1 N NaOH in place of 1 M piperidine,
conditions that have also been used to map (6-4) products
(17), led to similar product distributions (data not shown).
The major difference betweenthe use of NaOH andpiperidine
was in the temperaturedependence of the cleavage reactions.
At room temperature, cleavage of the (6-4) andDewar products is muchslower with 0.4 N NaOH andproceeds with halflives of 1600 and 140 min, respectively. In contrast, at 90 “C
cleavage of the (6-4) product is faster
with 0.1 N NaOH (halflife of 10 min),whereas cleavage of the Dewar product is the
same with either0.1 N NaOH or piperidine.
3’ + 5’ Exonucleolytic Cleavage Specificity by T4 Polymerase-The 3‘ + 5’ exonuclease of T 4 DNA polymerase was
N
n
CS
n
TS
n
64
n
DW
n
- E P - E - E - P - P
T2
T2:
FIG.6. Cleavage of 5’ end-labeled photoproduct-containing
49-mers with T4 endonuclease V. Autoradiogram of a denaturing
electrophoresis gelof single-stranded (ss) 49-merscontainingno
the cis-syn dimer (CS), trans-syn-I dimer ( T S ) ,(6-4)
damage (N),
that were 5’ end-labeled with
product ( 6 4 ) ,and Dewar product (DW)
polynucleotide kinase ( K ) , followed by 40 min of endonuclease V
treatment ( V ) ,and then 20 min of 1 M piperidine at 100 “C (P).
The
mark at the left indicates the mobility of a 20-mer with a 5’- and 3’phosphate.
FIG. 7. Cleavage of 3’ end-labeled photoproduct-containing
51-mers with T4 endonuclease V or piperidine. Autoradiogram
of a denaturing electrophoresis gel of double-stranded 49-mers containing no damage(N),
a cis-syn dimer (CS), trans-syn-I dimer( T S ) ,
(6-4) product ( 6 4 ) , and Dewar product ( D W ) after 3’ end-labeling
(-), followed by either a 30-min treatment with T4 endonuclease V
( E ) ,or by a 60-min treatment with 1 M piperidine at 100 “C. T21 and
T22 refer to the sites base or enzymatic cleavage which produces
bands that migrate as though they were 31- and 29-mers with 5’phosphates.
11148
""-
DNA
Photoproduct-containing
Site-specific
cs
N
_"
0 1
-
5 25125 0
r-
-
"
1
5 25125 0
TS
1
DW
64
5 25125 0
1
5 25 125 0
1
5 25125
I
.
FIG.8. Piperidine cleavage of 5'
end-labeled photoproduct-containing 49-mers. Autoradiogram of a denaturingelectrophoresis gel of singlestranded 49-mers containing no damage
(A'), the cis-syn dimer( C S ) ,trans-syn-I
dimer (7's).(6-4) product ( 6 4 ) .and Dewar product ( D W )
after treatment with
1 M piperidine a t 90 "C for the times
indicated in min. The mark to the right
of the gel indicates the mobility of a 20mer with 5 ' - and 3"phosphates.
A
---
-
DW
64
N
0 51
- 51
125
25125
-
51
25125
B
2
I
1.8
16
1.4
end-labeled termination band migrating as a 22-mer with a
5'-phosphate, whereas the cis-syn and trans-syn dimers initially led to a band migrating as
a 23-mer with a 5'-phosphate.
In the case of the cis-syn dimer, this initial band was converted to a band migrating as a 22-mer with a 5"phosphate
with apparent first-order kinetics and a half-life of 9 min,
whereas for the trans-syn-I dimer itwas converted to a band
migrating asa 22-mer with5'- and 3"phosphates with a halflife of 3 min. Photoreversion converted both ultimate termination products to bands migrating as a 21-mer with a 5 ' phosphate (Fig. 11).
E. coli Photolyase Substrate Specificity-The undamaged,
cis-syn, trans-syn-I, (6-4), and Dewar duplex 49-mers were
treated with equimolar E. coli photolyase (100 nM) and 366
nm light and then treated
with T4 polymerase. Only the
undamaged and cis-syn 49-mers were completely degraded by
this treatment, all other photoproducts led to the expected
termination products (data not shown).
DISCUSSION
0
20
40
60
80
100
120
140
lime p i n )
FIG.9. Piperidine cleavage of (6-4) and Dewar productcontaining oligonucleotides. A, autoradiogram of a denaturing
electrophoresis gel of single-stranded 6-mers containing no damage
( N ) ,the (6-4) product ( f i 4 ) , and the Dewar product ( D W ) , before
(-), and after treatment with 1 M piperidine a t 90 "C for the times
indicated in minutes. R, time course for the 90 "C 1 M piperidinecatalyzed cleavage of the (6-4) andDewar 6-mers and49-mers.
stopped completely by the cis-syn, trans-syn-I, and Dewar
lesions, but unexpectedly proceeded past the (6-4) product
with non-first-order kinetics and a half-life of about 3 min
(Fig. 10). Both the (6-4) and Dewar 49-mers led to one 5'
Preparation of Site-specific Photoproduct-containing DNACurrently, there are two basic strategies for preparing oligonucleotides containing DNA photodamage suitable for further
construction purposes: (i) thebuilding-block approach (9, 14,
15),and (ii) thedirect-modification approach (10-12,18). The
building-block approach is the most flexible because it places
no sequence or length restrictions ( 4 0 0 nucleotides) on the
oligonucleotide synthesized, whereas to be practical, the direct-modification approach requires an isolated target site in
a short oligonucleotide to facilitate the formation and isolation of the desired product by HPLC. We decided on the
sequence d(CGAATTAAGC) in which to incorporate thecissyn and trans-syn-Iisomers directly by solid-phase synthesis,
and the subsequence d(AATTAA) for preparation of the (64) and Dewar oligonucleotides by irradiation. A longer oligonucleotide was not chosen for direct irradiation because of
the difficulty expected in resolving the different photoproducts (12), and a shorter one was not chosen because of the
expected difficulty in ligating it to otheroligonucleotides.
Photochemistry of dfAATTAA)-In addition toforming the
Site-specificPhotoproduct-containingDNA
11149
expected cis-syn, trans-syn-I, and (6-4) products of the TpT Dewar 6-mers increased asa result of the inefficiency of the
site in the 6-mer, we expected that 254 nm of irradiation ligation step. Aside from photoproduct contaminants, there
would produce minor amounts of the trans-syn-I1 product,’ was at most 2% contamination of each 49-mer with 48-, 47the TpdA photoproduct (19, 20), and dApdA photoproducts and 46-mers, possibly resulting from ligation of failure se(21, 22). Reverse-phase HPLC of the mixture following 254 quences. From primer extension experiments: there is no
nm of irradiation of d(AATTAA) revealed that many products detectable (less than0.5%) contamination by the undamaged
were indeed formed in addition to the (6-4) product, which 49-mer in anyof the photoproduct-containing49-mers. In all,
>95% pure.
was only formed in a maximum yield of about 5% (Fig. 3b). we estimate all these substrates to be
T4 denV Endonuclease V Cleavage of Cis-syn and TransThe twomajor products are probably the cis-syn and the
trans-syn-I 6-mers, although no attemptwas made to isolate syn-I Dimers-T4 endonuclease V is known to cleave DNA
and identify them. The
low maximum yield of the (6-4) 6-mer at cyclobutanedimers (25, 26) although its specificity has
is somewhat surprising, considering that cyclobutane dimers never been rigorously examined because of a lack of pure,
well-characterized photoproduct-containingsubstrates
for
are reversibly formed under these conditions whereas (6-4)
products are not, a situation that should result in increasing study. Unexpectedly,T 4 endonuclease V was found to cleave
a t a significant rate,
amounts of (6-4) product with irradiation time. A yield of the trans-syn-I in doubled-stranded DNA
20% has been reported for the (6-4) product
of the dinucleo- although a t least 100 times slower than the cis-syn dimer.
tide T p T (23),andalthoughnot
specifically reported, we Cleavage of both the cis-syn and trans-syn-I 49-mers led to
estimate a yieldof 10% for thatof d(GCAAGTTGGAG1 based the same mixture of 5’ end-labeled products, and itwas only
were
on the HPLC trace shown (12). It is known that the rate of upon treatment with hot piperidine that these products
(6-4) formation is dependent on sequence context (24), and itall converted toa single product with greater electrophoretic
mobility. These results are in accord with a previous study
is possible that (6-4) products also undergo further sequencefrom
dependent photochemistry, thereby reducing their yield with that established that the 5‘ end-labeled product resulting
piperidine treatment comigrates with the Maxam-Gilbert
seincreasing irradiation time.
quencing reaction product corresponding to the 5”pyrimidine
ThephotoproductseluteddifferentlyontheC-18and
phenyl columns, and separation appeared to be better on theof the dimer (27). Also in accord with that study, T4 endolatter (Fig. 3e), suggesting that its higherresolving power nuclease V cleavage of the 51-mer led directly to a 3’ endlabeled productcomigratingwiththeMaxam-Gilbert
semight be due to %-stacking interactions between the DNA
bases and the phenyl groups of the column. The ‘H NMR quencing product corresponding to the 5“pyrimidine of the
spectrum of the isolated (6-4) 6-mer indicated that it
was dimer. In a more recent study, it hasbeen shown that photocontaminated with about 2% of the Dewar product. It is not reversal of the 3‘ end-labeled T4 endonuclease V product
known whether formation of the Dewar product was a direct leads to a band which comigrates with the Maxam-Gilbertresult of the 254-nm irradiation, ora result of the use of a low sequencing reaction product corresponding to the 3“pyrimipressure mercury arc lamp. These lamps are known to have dine of the dimer (29).
The observed cleavage chemistry is consistent with what is
-1% of their output at 313 nm, which could have isomerized
the (6-4) product to itsDewar valence isomer. The contami- known about the mechanism of T4 endonuclease V cleavage
nating Dewar product could not be removed by the HPLC of cis-syn dimers (26).T4 endonuclease V first hydrolyzes the
dimerandthen
systems used, as neither column was capable of base-line glycosidic bond of the 5’ T ofacis-syn
separation of the two products. As expected (7,16), irradiation catalyzes a p-eliminationreaction which resultsinstrand
of the (6-4) 6-mer with Pyrex- and Mylar-filtered
medium cleavage. Thesestepsresult
ina 5’ end-labeledfragment
pressure mercury arc light gave the Dewar valence isomer in terminating at the 3‘ endwith a trans a&-unsaturated aldenearly quantitative yield and with correspondingly high pu- hyde (28), and a 3”labeled product terminating in the 3’rity.
pyrimidine of the dimer which bears a 5”phosphate and is
Purity of the 49-mers-Highly pure photoproduct-contain- still cyclodimerized to the 5”pyrimidine of the dimer (see
ing substrates are required for studies
of the specificity of Ref. 29). Presumably, the initialaldehyde product is relatively
force
repair and replication systems, both in vitro and in uiuo, as stable and requires treatment with hot piperidine to
elimination of the attached 5”phosphate that results in the
well as methods for quantifying photoproduct distributions.
The determination of the purity of the 49-mer substrates is 5’ end-labeled fragment bearing a 3”phosphate. The three
made difficult due to a lack of suitable analytical techniques intermediate bands seen in the absenceof piperidine (Fig. 6)
for such a task, and in their absence, we can only estimate could be due to bothcis and trans isomersof the ol,p-unsatutheir purity by less direct means. From cleavage experiments rated product, and the hemi-acetal
of the cisisomer. The
with T 4 endonuclease V, the cis-syn 49-mer contains approx- proposal that enzymatic cleavage of the N-glycosidic linkage
imately 1.5% of a non-dimer impurity.Evidence for very little involves protonation of the C-2 carbonyl (26) may have to be
cross-contamination between cis-syn and trans-syn-I 49-mers reconsidered in view of the fact that theC-2 carbonyl of the
and for the high purity of both substrates comes from the 5”pyrimidine of a cis-syn dimerlies in the minor groove of B
exonucleolytic degradation experiments, where it was found DNA, and in themajor groove for the trans-syn-I dimer (30).
that the termination bands unique to the trans-syn-I isomer The C-4 carbonyl is a more likely candidate for protonation,
are not discernable (<0.5%) among those of the other and
as well as recognition, as it is in a common orientation for
vice versa (Figs. 10 and 11).Because of the synthetic route both the cis-syn and trans-syn-I dimers.
used to prepare the cyclobutane dimer-containing decamers
Base Cleavage of (6-4) and Dewar Products-A number of
(14, 15) there is nopossibility that the cis-syn and trans-syn- studies have used hot hydroxide or hot piperidine (24, 31) to
I 49-mers are contaminated with (6-4) or
Dewar products. locate and quantify (6-4) products in DNA, although it has
Together, the T4 endonuclease and piperidine
cleavage assays only been recently determined that the
cleavage reaction may
establish this to be the case, and further indicate that there
not be quantitative under the conditions
used, particularly at
is no discernable contamination of the (6-4) and Dewar 49- dCpT and TpT sites (32). On the other hand, it was found
mers with cis-syn or trans-syn dimers. There is no evidence that cleavage at (6-4) sitescould be dramatically increasedby
from the piperidine
cleavage or T 4 exonucleolytic degradation first converting the (6-4) products to their
Dewarvalence
assays of the 49-mers that the 2% cross-contaminants and
<2% unidentified products detectedby NMR in the (6-4) and
C. A. Smith and J. A. Taylor, unpublished results.
11150
Site-specific
""-
Photoproduct-containing
DNA
N
A C G T
cs
TS
64
DW
0 15 2 5 0 15 2 5 0 15 2 5 0 15 2 5 01 5 2 5
FIG.10. 3' + 5' exonucleolytic
degradation of 5' end-labeled photoproduct-containing 49-mers by
T4 DNA polymerase. Autoradiogram
of a denaturing electrophoresis gel of
single-stranded 49-mers containing no
damage ( N ) , the cis-syn dimer (CS),
trans-syn-I dimer (TS),(6-4) product
( 6 4 ) , and Dewarproduct (DW),
after
treatment with T4 polymerasefor the
times indicated. On the left are dideoxysequencing reactionproductsfor
the
complementary strand. The site of the
TT photoproducts is indicated on the
left.
3'
T
T
5'
16
cs
TS
d(GCAAGTTGGAG) are
not cleaved under
these or a number
of other conditions (12). Whether this relates to a sequence
ss
ds
ss
ds TSds
context effect or the structural integrity of their samples
n n n n
remains to be resolved, although the samples did have the
- + - + - + - + photophysical properties expected for (6-4) and Dewar 11mers. Second, the rate of disappearance of the Dewar 49-mer
was much faster than that of the 6-mer, whereas there was
no apparent difference in rate between the (6-4) 6- and 49mers. Similar length dependence effects on the cleavage of
RNA have also been observed (34), but its basis is not well
understood. Third, there did not appear to be a difference in
FIG.11. 254 nm irradiation of the T4polymerase digestion the rateof disappearance of the (6-4) and Dewar 6-mers, even
products of the cis-syn and trans-I dimer-containing 49-mers. though there was a large rate difference for the 49-mers.
Autoradiogram of a denaturing electrophoresis gel of T4 polymerase Fourth, piperidine treatment of the (6-4) 6- and 49-mers led
digestion products of single-stranded (ss) and double-stranded (ds)
49-mers containing a cis-syn (CS) or trans-syn-I (TS)dimer, before to a different distributionof 5' end-labeled products than did
the Dewar product-containing substrates (Figs. 8 and 9A)
(-) and after (+) 25 min of 254-nm irradiation. The right-most lane
contains a mixture of the cis-syn and trans-syn-I double-stranded 49- suggesting that different intermediateswere involved, or that
mer digestion products without 254 nm treatment.
the same intermediates were being formed but at different
rates. Fifth, the cleavage reactions showed biphasic kinetics
isomers (32). Our results confirm that hot piperidine treat- in which the slow phase appeared to be the same for both
photoproducts and lengths (Fig. 9B), suggesting the formation
ment does not quantitatively cleave the DNA a t a(6-4)
product of TpT under the standard conditions and that the of a common intermediate that is slowly cleaved.If correct, it
cleavage rate is greatly accelerated (about 50 X ) by conversion also appears that more of this common intermediate is formed
of the (6-4) product to its Dewar valence isomer. Hot piperi- in the Dewar 6-mer than in the Dewar 49-mer.
T4 Polymerase Exonuclease Termination Specificity-One
dine treatment of 5' end-labeled 49-mer led to a number of
bands, the fastest moving of which has the same mobility as method for quantifying DNA photoproducts at a sequence
the band resultingfrom a Maxam-Gilbertsequencing reaction level is based on theconclusion that cis-syn dimers and (6-4)
corresponding to the 5'T of the (6-4) or Dewar product. The products block 3' + 5' exonucleolytic degradation of DNA
other 5' end-labeled bands are slower moving and diffuse in by T4 polymerase (35). Whereas we find that the cis-syn,
accord with previous observations (33). In accord with pre- trans-syn-I, and Dewar products do indeed block exonucleovious results (17), piperidine cleavage of both the 3' end- lytic degradation the 49-mer, the (6-4) product does not, and
labeled (6-4) and Dewar 51-mers leads to the formation of a its relative amount would be underestimated by this techsingle band (Fig. 7) that comigrates with the Maxam-Gilbert nique, especially at long reaction times. Given the structural
sequencing band corresponding to the 3"pyrimidine of the similarity between the (6-4) and Dewar products, it is not
photoproduct (17). Collectively these resultsconfirm that base easy to understand why one is bypassed and the otheris not.
principally degrades the 3'-pyrimidine of the (6-4) and Dewar Kinetic analysis indicates that the rate of (6-4) bypass is
products leading to multiple intermediates and that some appreciable, and toquantify the amountof (6-4) photoproduct
subsequent degradation of the 5"pyrimidine also takes place. would necessitate short reaction times. Finding an optimal
When the kinetics of the disappearance of the (6-4) time for quantifying allthe photoproducts may be difficult or
impossible, however, as we also discovered that the cis-syn
and Dewar 6-mers and 49-mers were investigated, a number of unusual features were observed. First, the (6-4) and and trans-syn-Idimers lead to two termination products, one
Dewar 6-mers were cleaved under the standard conditions of which converts to the other only at long reaction times.
used for mapping these photoproducts in much longer frag- The conversion was faster for the trans-syn-I product than
ments of DNA (Fig. 9A). Thiscontrasts with the report for the cis-syn product and is consistent with a previous study
that (6-4) and Dewar products of TpT flanked by Gs in of these products in a different sequence context (11).CuriCSds
Site-specific
11151
Photoproduct-containing
DNA
ously, we found that the ultimate 5' end-labeled termination
product of the trans-syn-I 49-mer migrated faster than the
corresponding products of the otherphotoproduct-containing
49-mers. The enhanced mobility of the trans-syn-I dimer
termination product suggested the presence of an additional
charge that might have resulted from enzymatic hydrolysis of
the intradimer phosphate linkage. Photoreversion of the terminationproducts with 254-nm radiation converted them
both to a band migrating as a 21-mer with a 5'-phosphate,
consistent with cleavage of the intradimer 03"P bond of both
the cis-syn and trans-syn-I dimers. We are unable, however,
to explain the origin of the mobility difference between what
would necessarily be the cis-syn and trans-syn-I22-mers with
cleaved intradimer phosphates.
Implications for Quantifying DNA Photoproducts at a Sequence Leuel-Highly specific and quantitative methods for
mapping DNA photoproducts at a sequence level are required
for accurate determination of damage spectra and the rates
ofphotoproduct repair. Whereas the T4endonuclease V repair
enzyme has been extensively used to quantify the production
and repair of cis-syn dimers, there are no known enzymes
with unique specificity for the trans-syn-I, (6-4) or Dewar
photoproducts. A variety of enzymatic approaches have been
developed to map (6-4) products, one of which involves discriminating between cis-syn and (6-4) products by combinations of T4 endonuclease V, E. coli photolyase, and theE. coli
uvr(A)BC excinuclease (36). Use has also been made of the
ability of photoproducts to arrestDNA exonucleases (35) and
polymerases (37). The most extensively used method for
mapping (6-4) products, however, is not enzymatic but chemical, and involves base-induced DNA strand cleavage at sites
of (6-4) products (17) or their Dewar valence isomers (32).
Recently, it has become possible to map (6-4) productsat the
nucleotide level in single copy mammalian genes by base
cleavage in conjunction with ligation-mediated polymerase
chain reaction (38).
Based on the studies presented in this paper, a number of
procedures can be recommended for best quantifying photoproduct formation at thesequence level. Cis-syn dimers would
be best quantified by treatment of 3' end-labeled doublestranded DNA with T4 endonuclease V for a long time period
and subtracting the amount of trans-syn-I dimer for each site.
The amount of trans-syn-I dimer could he quantified by first
photoreversing the cis-syn dimers with E. coli photolyase, and
then incubating with T4 endonuclease V for a long period of
time. The Dewar products could be quantified by subjecting
enzymatically photoreversed 5' end-labeled DNA to T4 polymerase for a sufficient period of time to completely degrade
all the (6-4)product-containing DNA and produce single
termination bands for the other products, and then subtracting the amount of trans-syn-I dimer. Care must be taken
during the photoreversal stepnot to expose the DNA to
wavelengths less than 360 nm whichwould convert(6-4)
products to their Dewar isomers (39). If the resolution of the
gel permits, termination productsdue to the trans-syn-I
dimer
could be distinguished from those due to the Dewar product
by their anomalous mobility, and by their increase in mobility
following subsequent photoreversal (Fig. 11). Otherwise, the
amount of trans-syn-I dimer determined by the T4 endonuclease V assay would have to be subtracted from the termination bands. (6-4) productscould be quantified by hot piperidine treatment of 3' end-labeled DNA following photoisomerization of the (6-4) products their Dewar isomers with 325
nm of light, andthensubtracting
the amount ofDewar
product originally present. Because all of the suggested procedures involve a series of steps and limited reaction times,
they may require further development and optimization. In
addition, the sensitivity of these methods may not allow the
precise quantification of minor photoproducts, such as the
trans-syn-I and Dewar products, or otherwise major products
which form in low yield at certain sites.
Acknowledgments-We thank R. S. Lloyd (University of Texas,
Galveston) for a generous gift of T4 endonuclease V and A. Sancar
(University of North Carolina, Chapel Hill)for a generous gift of E.
coli photolyase. We also thank Andre D'Avignon of the Washington
University NMR Facility for assistance with obtaining the NMR
spectra. A gift from the Monsanto Co. is gratefully acknowledged.
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