Proc. Nati. Acad. Sci. USAVol. 89, pp. 10772-10776, November 1992Biochemistry

An intrastrand d(GpG) platinum crosslink in duplex M13 DNA isrefractory to repair by human cell extracts

(dsplatin/DNA repair/DNA polymerase)

DAVID E. SZYMKOWSKI*, KEVIN YAREMAt, JOHN M. ESSIGMANNt, STEPHEN J. LIPPARDt,AND RICHARD D. WOOD*t*Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts EN6 3LD, United Kingdom; and tDepartment of Chemistry, MassachusettsInstitute of Technology, Cambridge, MA 02139

Contributed by Stephen J. Lippard, July 21, 1992

ABSTRACT We have examined the ability of human cellextracts to repair the most frequent DNA adduct caused by thecancer chemotherapeutic agent cis-diamminedichloroplati-num(ll). A circular DNA duplex with an intrastrand d(GpG)crosslink positioned at a specific site was synthesized. Humancell extracts were unable to induce repair synthesis in a29-base-pair region encompassing the adduct or in adjacentregions. The same extracts could repair a single defined2-acetylaminofluorene lesion in a similar location. When mol-ecules containing the platinum adduct were cleaved by Esch-erichia coli UvrABC enzyme, human cell extracts could per-form repair synthesis at the damaged site, suggesting thathuman enzymes fail to make incisions near the d(GpG)crosslink but can complete repair once incisions are made. Thisresult indicates that most repair synthesis in DNA damagedwith multiple cis-diamminedichloroplatinum(ll) adducts takesplace at lesions other than the predominant d(GpG) crosslink.These data support the idea that the clinical effectiveness ofcis-dlamminedichloroplatinum(II) may be explained by theinefficient repair of the major DNA adduct caused by this drug.

The drug cis-diamminedichloroplatinum(II) (cis-DDP or cis-platin) is useful for the treatment of various tumors, partic-ularly testicular and ovarian carcinomas (1). The cytotoxiceffect is thought to be mediated by platinum-DNA adductsthat block replication or inhibit transcription (2-4). The mostcommon adducts formed in vitro are 1,2-intrastrand d(GpG)crosslinks (65%) and d(ApG) crosslinks (25%), with minoramounts of 1,3-d(GpNpG) crosslinks, interstrand crosslinks,and monoadducts (2, 5, 6). The relative contribution totoxicity and clinical effectiveness of these different adductsis not known, although a growing body of literature impli-cates the GG and AG adducts as blocks to DNA and RNAsynthesis (4, 7, 8).Mammalian cells are capable of repairing at least a fraction

of the DNA damage caused by cis-DDP, but it is noteworthythat a substantial fraction of adducts is never removed fromDNA (9-12). Indeed, it has been suggested that the reason forthe biological effectiveness ofcis-DDP is that the majorDNAadducts formed by the drug may not be well-repaired (13).Conversely, the cells of some cisplatin-resistant tumors andcell lines are suspected to owe their resistant phenotype to anenhanced rate of repair of cytotoxic adducts (9, 14-16).To study DNA nucleotide excision repair in vitro, we have

examined repair DNA synthesis carried out by mammaliancell extracts. These extracts remove lesions caused by anumber of damaging agents, including N-acetoxy-2-acetylaminofluorene (N-Ac-AAF) and UV light (17-19). Cellextracts also remove some platinum lesions induced by

treatment of DNA with cis-DDP or its clinically inactiveisomer trans-DDP (4, 19-22). These initial studies ofcis-DDP-induced repair measured synthesis in DNA containing the fullspectrum of platinum adducts, and it was not possible todetermine whether different lesions were repaired with vary-ing efficiencies. To investigate the identity of the lesionsresponsible for repair synthesis induced by platinum damage,we have applied the in vitro system to study repair ofthe mostabundant lesion induced by cis-DDP, the d(GpG) intrastrandcrosslink.

MATERIALS AND METHODSDNA Damaged with Multiple cis-DDP, UV, or N-Ac-AAF

Lesions. To examine repair of globally platinated DNA,closed circular M13mp18GG duplexDNA [7.3 kilobases (kb)]was purified on CsCl gradients and then incubated (15 hr,370C) at a nucleotide concentration of 3 mM with a fresh 15!LM solution of cis-DDP (Sigma) as described (20). Thecis-DDP-treated DNA contained =%5.3 platinum adducts perkilobase pair (kbp) as determined by atomic absorptionspectroscopy of plasmid DNA platinated using identicalconditions (20). Plasmid pBluescript KS+ (3.0 kbp, Strata-gene) was irradiated with UV light (450 J/m2) to give =4.1photoproducts per kbp (23). Alternatively, pBluescript KS+was treated with N-Ac-AAF (by R. P. P. Fuchs, Institut deBiologie Moleculaire et Cellulaire, Strasbourg) to give --4AAF lesions per kbp, of which >90% are the C8-G-AAFlesion (24). Treated and mock-treated DNAs were purified onsucrose gradients to remove nicked circular forms and usedin repair assays (25).Double-Stranded M13 DNA Containing One GG-Pt

Crosslink. Closed circular DNA was produced by priming 75,ug of single-stranded (+) M13mp18GG (at 200 ng/ILd) with a3-fold molar excess of either platinated oligonucleotide 24Pt(Fig. 2) or control unplatinated oligonucleotide 24U andincubating with T4 DNA polymerase holoenzyme, single-stranded DNA binding protein (Stratech Scientific, Luton,U.K.), and T4 DNA ligase as described (18, 26). DNAproducts primed with 24Pt were then incubated with Stu I tolinearize any duplex M13 not containing platinum. The closedcircular products were purified on CsCl gradients for use inrepair reactions.The M13mpl8 derivative M13mpl8G-AAF, containing a

single 2-acetylaminofluorene adduct, was constructed asdescribed (18) and was a generous gift from M. Munn and D.

Abbreviations: DDP, diamminedichloroplatinum(II); N-Ac-AAF,N-acetoxy-2-acetylaminofluorene; M13mp18GG, M13 constructwith a priming site for oligonucleotides 24U and 24Pt;M13mpl8GG-Pt and -U, constructs with or without a single d(GpG)crosslink, respectively; M13mpl8G, M13 construct with a primingsite for N-Ac-AAF-treated oligonucleotides; M13mpl8G-AAF and-U, constructs with or without a single AAF lesion, respectively.tTo whom reprint requests should be addressed.

10772

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 89 (1992) 10773

Rupp (Yale University). The lesion was inserted into thesame region of M13mpl8 as the platinum adduct, using aprimer of sequence 5'-CACCACACCGACCAACCCTA-3'containing a single C8-AAF guanine adduct.In Vitro Excision Repair of Damaged DNA. Extracts from

HeLa cells and GM2250 xeroderma pigmentosum comple-mentation group A cells were prepared as described (17) andhad a protein concentration of 10-20 mg/ml. In vitro repairassays (50sul) using single-lesion substrates contained 100 ngof platinum- or AAF-adducted DNA in reaction buffer (17)including 10 AM (each) dGTP, dCTP, dTTP, and dATP, 10ACi (each) of [a-32P]dATP and [a-32P]dCTP (3000 Ci/mmol;1 Ci = 31 GBq), and 200 ,ug of cell extract protein. The useof smaller amounts of protein led to higher relative levels ofdamage-independent DNA synthesis and unreliable results.Reaction mixtures were incubated at 30'C for 3 hr beforeDNA was isolated (17). To measure repair synthesis in theentire molecules, multiply damaged and control undamagedDNAs were linearized with HindIII and electrophoresed on0.8% agarose gels. DNAs containing single lesions werecleaved with restriction enzymes as indicated and the prod-ucts were resolved on 12% polyacrylamide gels. Fixed anddried gels were exposed to x-ray film without intensifyingscreens. Data were quantified with the aid of a MolecularDynamics computing densitometer to determine relative syn-thesis from autoradiographs and by liquid scintillation spec-troscopy of excised bands.

RESULTSRepair by Human Cell Extracts of Multiply Platinated DNA.

In this study, M13 vectors were used for the construction ofsite-specifically damaged DNA substrates. We first con-firmed that at least a portion of the DNA damage caused bycis-DDP treatment of M13mp18GG DNA was repaired byHeLa cell extracts. Fig. 1 shows a comparison of repairsynthesis inDNA multiply damaged by cis-DDP, UV light, orN-Ac-AAF. A fraction of the damage caused by each agentleads to repair synthesis by HeLa cell extracts (Fig. 1A), butthe different lesions induce different levels of synthesis (Fig.1B). Taking into account the number of lesions on eachsubstrate (four to five adducts per kbp), overall platinumdamage provokes repair synthesis ofapproximately one-thirdthe level ofAAF adducts. An extract from a repair-deficientxeroderma pigmentosum cell line has a 5- to 10-fold reducedcapacity to repair damage induced by N-Ac-AAF, UV light,and cis-DDP (Fig. 1). Therefore, the repair ofglobal platinumdamage by extracts is accomplished by a nucleotide excisionrepair mechanism as is damage induced by UV and N-Ac-AAF (17, 21, 23).

Synthesis of Duplex DNA Containing a Single cis-DDPCrosslink. To investigate the identity of the adduct(s) causingthe repair synthesis in nonspecifically platinated M13 DNA,we synthesized duplex DNA circles, each containing ad(GpG)-platinum intrastrand crosslink in a specific sequence.Digestion with BstNI cuts the 519 bp around the platinumadduct into six fragments (Fig. 2). The lesion is in the centerofa 29-bp fragment and is flanked by 68- and 99-bp fragmentson the 5' side and 3' side, respectively. The remaining 93%of the M13mp18GG DNA molecule consists of three BstNIrestriction fragments that are well-separated from the site ofdamage, and the damage-independent DNA synthesis medi-ated by cell extracts is primarily confined to these fragments.Single-stranded (+) M13mp18GG DNA was primed with theplatinated oligonucleotide 24Pt or 24U, an unplatinated con-trol oligonucleotide, and the second strand was synthesizedwith T4 DNA polymerase holoenzyme in the presence of T4DNA ligase to yield closed circular products with almost100%1 efficiency (Fig. 3). The use of other DNA polymerases

AXP-A HeLa XP-A HeLa

-soT--UvF

Ui ++U~~~~-U12 - +3 Pt

1 2 3 4 1 2 3 4

B2.0O

o0s-4

1.2-

0.8

0.4.

IEv -

Pt Mock UV AAF

HeLa (normal)

* + damageo - damage_

Pt Mock UV AAF

GM2250 (XP-A)

FIG. 1. Repair of damaged DNA by HeLa and GM2250 xero-derma pigmentosum complementation group A (XP-A) extracts. (A)Damaged (+) and undamaged (-) linearized DNAs (100 ng per lane,from reactions that contained 250 ng of each plasmid) were resolvedby electrophoresis after incubation with cell extracts in repair assay.The 3.7-kbp plasmid pHM14 (23) served as the undamaged control inall lanes. To the left is the ethidium bromide-stained gel used toquantify DNA recovery; to the right is the autoradiogram of the gel.DNA in lanes 1 and 2 was incubated with GM2250 XP-A extract andin lanes 3 and 4 with HeLa extract. For the AAF and UV panels,lanes 1, 2 and 3, 4 are duplicate reactions. In the Pt panel, theM13mp18GG DNA in lanes 1 and 3 was cis-DDP-treated, whereasthe DNA in lanes 2 and 4 was mock-treated. (B) Repair synthesisnormalized to DNA recovery. The UV and AAF results are theaverage of the duplicate reactions in A.

and shorter (12-mer) primers failed to provide an acceptableproduct.The single cis-DDP intrastrand crosslink falls within the

only Stu I site of M13mpl8GG and the inability of Stu I tocleave the DNA is diagnostic of the correct location of theadduct (27). Fig. 3 shows a digestion of the platinated andunplatinated substrates with Stu I or Sal I (which recognizesa site 15 bases 5' to the adduct). The substrate constructedwith an unplatinated primer (M13mp18GG-U) was linearizedby both enzymes; in contrast, no linear DNA was formed byStu I digestion of the platinated construct (M13mp18GG-Pt),although Sal I linearized this plasmid. This result confirmsthat the purified material is homogeneous closed circularDNA with one platinum crosslink in the expected location.The presence of the single platinum crosslink and the AAFadduct (used as a positive control for repair) was furtherestablished by annealing an upstream primer to the singlyadducted DNA strand and determining the site at which

Biochemistry: Szymkowski et al.

10774 Biochemistry: Szymkowski et al.

11,N N-H1

GGj

mI--§I II1 2- 68 29 99

A AAF P-t_ + _ r

:3

1'39tZ

1809 j

397r5

FIG. 2. Construction ofM13mpl8GG-Pt, an M13mpl8 derivativecontaining one d(GpG) platinum crosslink. An oligonucleotide ofsequence 5'-TCTTCTTCTAGGCCTTCTTCTTCT-3' (designated24U) was treated with cis-DDP and the singly platinated product,designated 24Pt, was purified as described (27, 28). HPLC analysisconfirmed the presence of one cis-[Pt(NH3)2{d(GpG)}] intrastrandadduct per oligonucleotide. The vector M13mpl8GG containing apriming site for the platinated oligonucleotide was constructed byreplacing the 33-bp EcoRI - Sal I fragment of M13mpl8 with afragment formed by annealing the two oligonucleotides 5'-TCGACCAGGTCTTCTTCTAGGCCTTCTTCTTCTCCAGG-3'and 5'-AATTCCTGGAGAAGAAGAAGGCCTAGAAGAA-GACCTGG-3'. The new construct contained on the (+) strand asequence complementary to 24Pt, with the crosslinked GG located ina Stu I site flanked by BstNI sites. Fragment sizes shown are forBstNI digestion.

synthesis by Klenow polymerase was blocked (8). Primerextension was interrupted by the platinum and AAF adducts(but not by undamaged control substrates) at the nucleotideimmediately before the adduct (data not shown).

Repair of Defined Adducts by Human Cell Extracts. Theability of HeLa cell extracts to repair the single d(GpG)crosslink and AAF adduct was tested in vitro. After repairincubations, the DNA was cleaved with restriction enzymesand electrophoresed on a polyacrylamide gel. Fig. 4A showsthat HeLa extracts caused repair synthesis in the 31-bpfragment containing AAF damage, whereas the identicalundamaged 31-bp fragment incorporated no label. Damage-dependent synthesis was confined primarily to the fiagmentcontaining the adduct (Fig. 4B), with a small amount ofadditional synthesis seen in the 5' and 3' fragments (51 and

3975 bp -1809 bp -952 bp

139 bp -127bp >

3' 116 bp

dL-f do

m7y bp - NoS. 51 bp -- of

031 bp --

13 1.01

9nSO

Relati eRepairSvntlhelsiS

3:

410

"Ie

;20

I

- 3975 bp- 1804Pp- 952 bp

- L39 Pp12-7bPp99 bp T

- 68 bp :t!- m.

-29 bp (DI.

-1 -111-I-- :"

4 I,.-

I1.- -....I-A E

1k'' 29 ..(I !-fall

M KPlatinum_+

nicked/CirculaT

linear

_- closedcircular

:; = _ : -,.-4~ "

ci urfcr r-,cruil

T.. C AGC TGGTCCAG ;AAOTC CMG-Sall St-l

FIG. 3. Confirmation of the correct location of the d(GpG)crosslink and homogeneity ofDNA. Platinated (+) and unplatinated(-) M13mp18GG DNAs (100 ng) were digested with Stu I or Sal I.

The trace of nicked circular DNA seen after digestion of theplatinated circle with an 80-fold excess of Stu I may be due to aninefficient cleavage of the DNA strand opposite the platinum adduct.M, covalently closed circular M13mpl8GG marker; A, BstNI digestof A DNA.

FIG. 4. Repair of single-lesion GG platinum crosslinks and AAFadducts vs. undamaged controls. (A) Single-lesion DNA (Fig. 2) wasincubated with HeLa extracts and digested with BstNI (platinum andcontrol) or BstNI, BamHI, and Sph I (AAF and control). Repairsynthesis was detected after gel electrophoresis and autoradiogra-phy. (B) Label incorporation in the six fiagments around the singlelesions, normalized to fragment size and base composition. For Pt,bars from left to right in each group represent the 127-, 68-, 29-, 99-,139-, and 57-bp fragments; see Fig. 2. For AAF, fragment sizes leftto right are 127, 51, 31, 116, 139, and 57 bp (18).

116 bp, respectively). This result confirms that the AAFadduct is repaired by these extracts with a patch size of -30nucleotides, as noted in previous studies on repair of thislesion (18, 30). In striking contrast, the substrate containingthe single cis-DDP intrastrand crosslink showed almost noDNA synthesis in the 29-bp fragment containing the damage.The control and the platinated substrate show only a back-ground level of synthesis in all fragments, proportional tofragment size and base composition (Fig. 4B). This observa-tion has been repeated with additional separate preparationsof platinated and unplatinated substrates and with indepen-

IF-:

Proc. Natl. Acad Sci. USA 89 (1992)

Proc. Natl. Acad. Sci. USA 89 (1992) 10775

dently isolated extracts from different human and hamsterrepair-proficient cell lines with the same qualitative results(data not shown). The experiments were also repeated with[a-32P]dGTP in the reaction mixture, in case the GG adductwas repaired with a patch size of only two nucleotides, but no

repair synthesis was detected. This result suggests that therepair synthesis seen in DNA globally treated with cis-DDPis not due to repair of the d(GpG) intrastrand adduct.

Previously we noted that when cis-DDP-treated plasmidDNA was incubated in successive repair reactions withnormal human cell extract, about the same amount of dam-age-dependent repair synthesis took place in the secondreaction as in the first (20). It was suggested that a smallfraction of a major adduct was repaired during each reaction.Only -3% of total cis-DDP adducts are removed during asingle in vitro incubation, however (20). If repairable lesionscomprise significantly >3% of the total, sufficient lesions willremain to stimulate similar levels of synthesis in the secondincubation.

Stimulation of Repair Synthesis in Molecules Containing thed(GpG) Platinum Lesion by Preincubation with E. coliUvrABC Enzyme. Nucleotide excision repair involves pro-teins that (i) recognize and incise damaged DNA and (ii)incorporate new nucleotides and ligate the DNA to completethe repair process. To investigate the step at which the humancell extracts failed to process the GG crosslink, the UvrABCrepair proteins from E. coli were used first to specifically nickthe DNA near the site of the crosslink (Fig. 5). This procedureyields a double-stranded DNA molecule with one incision oneither side of the adduct (31). We previously showed thatwhen DNA containing adducts is pretreated with UvrABC,cell extracts (including xeroderma pigmentosum cell extractsdeficient in the initial damage recognition-incision steps ofrepair) can remove the oligonucleotide containing the adductand synthesize the repair patch (21). This treatment withUvrABC bypasses the human incision-excision system byproducing incisions 12 or 13 nucleotides apart, whereas thenormal human nucleotide excision repair system excisesdamage in a fragment of -27-29 nucleotides (32). UvrABCpretreatment stimulated repair synthesis specifically in the29-bp fragment containing the platinum adduct but had no

effect on the unplatinated control (Fig. 5). This result sug-

gests that normal human cell extracts do not make the initialincisions in DNA containing a cis-DDP-induced d(GpG)crosslink. Once incisions are made, however, proteins in theextract can remove the damaged oligonucleotide and com-

plete DNA synthesis to form a repair patch.Factors (including proteins in the high mobility group

family) that can bind to the structural distortion caused bycis-DDP-induced GG and AG adducts are present in humancell extracts (29, 33-37). It has been suggested that binding ofsuch proteins to cis-DDP lesions might inhibit their repair(29). In preliminary experiments similar to those in Fig. 5, thesubstrates containing a single cis-DDP-induced d(GpG)crosslink or AAF adduct were incubated with cell extractbefore addition of UvrABC protein. For both lesions,UvrABC stimulated synthesis at the site of damage. Formolecules containing either the cis-DDP adduct or the AAFadduct the degree of stimulation by UvrABC was lower in thepresence of cell extract than when UvrABC was first per-mitted to preincise the damaged DNA (data not shown). Areduced efficiency ofUvrABC in the presence of cell extracthas previously been noted for UV-irradiated DNA (21, 38).From these data it is not possible to decide whether proteinsthat bind to cis-DDP-induced DNA damage play a role in themodulation ofrepair. A more definitive analysis ofa potentialrepair role for platinum-DNA binding proteins may be madewhen it is possible to deplete the binding proteins from cellextracts or when purified human incision proteins becomeavailable to test in the presence or absence ofplatinum-DNA

APlatinum - - + +

UvrABC - + - +

3975 bp1809 bp952 bp

139 bp127 bp

3' 99 bp

M

2

40

doM

5' 68 bp57bp

(5) 29bp

B 100-

90

80 -

70 -

Le -

w 60-

C 50-

40

CZl0. 30-

c, 20-

Cd 10-I~~~~~~~~~~~~C)X4 0

X+ damage- damage

il I

Extract +UvrABC

10 .....fnili

Cell extract only5 I U 3'

127 68 29 99 139 57

Pt

FIG. 5. Treatment of single-lesion platinated and undamagedDNA with E. coli UvrABC. (A) M13mpl8GG-Pt and -U wereincubated with E. coli UvrABC proteins prior to addition of HeLaextract. DNA (100 ng) was incubated in repair reaction buffer for 15min at 300C with 36nM UvrA and 29 nM UvrB. UvrC was then addedto 3.4 nM and incubation was continued for 15 min (21). Cell extractprotein (200 Ag) was added and the reaction mixtures were incubatedfor 3 hr at 300C. M, as in Fig. 3. (B) Label incorporation normalizedto size and base composition of the six fragments surrounding thedamage (as in Fig. 4B).

binding proteins. Alternatively, it is possible that the cellextracts lack a factor that could increase the efficiency ofrepair ofthe d(GpG) crosslink. For example, no transcriptiontakes place in the in vitro system used here, and there isevidence that damage caused by cis-DDP may be removed=2-fold more rapidly in transcribed genes (12).

DISCUSSIONIt is well-established that a portion ofplatinum-DNA lesionsformed in vivo are repaired by mammalian cells. For exam-

V Fj I I I .Fj ___nl

Biochemistry: Szymkowski et A

I

i....

is,2..4040 Mi410

10776 Biochemistry: Szymkowski etal.Pc.Ni.Aa.S.UA89(92

pie, studies that have used atomic absorption spectroscopy to

examine removal of cis-DDP adducts from chromosomal

DNA indicate that only about 40% of the total adducts are

repaired, even after prolonged incubation periods of 1 3

days (10-12). In vivo experiments that have specificallyexamined removal of the d(GpG) intrastrand crosslink sug-

gest that this adduct is poorly repaired in mamlan cells.

Very limited removal of GG or AG adducts in human cell

lines has been detected by using antibodies directed against

these lesions (39, 40). Murine L1210/0 cells were able to

repair only -30% of GG adducts as detected by HPLC

analysis, whereas some cis-DDP-resistant variants could

remove a greater fraction of the lesions (9). Data in the

literature are therefore generally consistent with the idea that

the 1,2-d(GpG) intrastrand crosslink is poorly repaired in

mammalian cells. This adduct is apparently also poorly

repaired by purified E. coli UvrABC'enzyme. The relative

ability ofUvrABC to cleave diferent specific platinum-DNAlesions has been examined, and the d(GpG) intrastrand

crosslink was the least well-incised adduct of those tested

(41), possibly because it causes a lower degree of local duplex

unwinding (42).

Consistent with studies on intact cells, the in vitro assay

detects repair of DNA that has been treated globally with

cis-DDP to form a number of different platinum adducts. In

this study, however, we found that a cis-DDP-induced

d(GpG) crosslink situated at a defined site is very poorly

repaired by human cell extracts. The sequence environment

studied here is not generally refractory to repair, as evi-

denced by the observation that a single AAF lesion at a G in

nearly the same sequence is repaired with at least 10-fold

greater efficiency (Fig. 4). We have also observed repair of

single UV photoproducts located at this position in M13 DNA

(D.E.S. and R.D.W., unpublished). These results suggest

that an adduct other than the d(GpG) crosslink is responsible

for the repair synthesis observed in cis-DDP-treated DNA.

Further supporting this idea, DNA damaged with the clini-

cally ineffective isomer trans-DDP stimulates repair synthe-

sis in vitro as or more efficiently than DNA damaged with

cis-DDP (4, 20). The trans isomer does not form 1,2-

intrastrand platinum crosslinks but instead forms interstrand

and 1,3-intrastrand crosslinks and monoadducts (43). Classes

of adducts formed by both cis- and trans-DDP, such as

interstrand or 1,3-crosslinks, may thus be responsible for the

repair synthesis observed in globally modified DNA. It will

be important to construct molecules containing single ad-

ducts representing other cis-DDP-induced DNA lesions and

to examine their repair by cell extracts.

Although cis-DDP and related drugs have been recognized

for >20 years as effective agents in chemotherapy, the bais

for the therapeutic effectiveness of these compounds is not

fully understood. As noted previously, poor repair of the

major adducts could increase cellular toxicity (13). The

studies reported here indicate that the ma~jor d(GpG) DNA

adduct produced by cisplatin is poorly repaired by tehman

nucleotide excision repair system and we suggest that this

inefficient repair may make a significant contribution to the

efficacy of the drug.

We are grateful to M. Munn and W. D. Rupp for the substrate

containing a single AAF lesion and for helpful advice. We thank L.

Grossman for UvrABC proteins, Mi. Biggerstaff for preparing some

ofthe plasmid substrates, and R. P. P. Fuchs for assistance. Valuable

technical support was provided by lain Goldsmith's Imperial Cancer

Research Fund Oligonucleotide Synthesis Laboratory and by the

Imperial Cancer Research Fund Cell Production Unit. This work was

funded by the Imperial Cancer Research Fund and by U.S. Public

Health Service Grants CA52127 to J.M.E. and CA34992 to S.J.L.

1. Loehrer, P. J. & Einhorn, L. H. (1984) Ann. Intern. Med. 100,

704-713.

2. Pinto, A. L. & Lippard, S. J. (1985) Proc. Nati. Acad. Sci. USA 82,4616-4619.

3. Viliani, G., Hilbscher, U. & Butour, J.-L. (1988) Nucleic Acids Res.-16, 4407-4418.

4. Heiger-Bernays, W. J., Essigmann, J. M. & Lippard, S. J. (1990)Biochemistry 29, 8461-8466.

5. Fichtinger-Shepman, A. M. J., van der Veer, J. L., den Hartog,J. H. J., Lohman, P. H. M. & Reedik, J. (1985) Biochemistry 24,707-713.

6. Eastman, A. (1986) Biochemistry 25, 3912-3915.7. Corda, Y., Job, C., Anin, M.-F., Leng, M. & Job, D. (1991)

Biochemistry 30, 222-230.8. Comess, K. M., Burstyn, J. N., Essigmann, J. M. & Lippard, S. J.

(1992) Biochemistry 31, 3975-3990.9. Eastman, A. & Schulte, N. (1988) Biochemistry 27, 4730-4734.

10. Fraval, H. N. & Roberts, J. J. (1979) Cancer Res. 39, 1793-1797.11. Gibbons, G. R., Page, J. D., Mauldin, S. K., Husain, 1. & Chancy,

S. G. (1990) Cancer Res. 50, 6497-6501.12. Jones, J. C., Zhen, W. P., Reed, E., Parker, R. J., Sancar, A. &

Bohr, V. A. (1991) J. Biol. Chem. 266, 7101-7107.13. Ciccarelli, R. B., Solomon, M. J., Varshavsky, A. & Lippard, S. J.

(1985) Biochemistry 24, 7533-7540.14. Chao, C. C.-K., Lee, Y.-L., Cheng, P. W. & Lin-Chao, S. (1991)

Cancer Res. 51, 601-605.15. Dijt, F. J., Fichtinger-Schepman, A. M. J., Berends, F. & Reedjk,

J. (1988) Cancer Res. 48, 6058-6062.16. Masuda, H., Ozols, R. F., Lai, 0. M., Fojo, A., Rothenberg, M. &

Hamilton, T. C. (1988) Cancer Res. 48, 5713-5716.17. Wood, R. D., Robins, P. & LidhT. (1988) Cell 53, 97-106.18. Hansson, J., Munn, M., Rupp, W. D., Kahn, R. & Wood, R. D.

(1989) J. Biol. Chem. 264, 21788-21792.19. Sibghat-Ullah, Husain, I., Carlton, W. & Sancar, A. (1989) Nucleic

Acids Res. 17, 4471-4484.20. Hansson, J. & Wood, R. D. (1989) Nucleic Acids Res. 17, 8073-

8091.21. Hansson, J., Grossman, L., Lindahl, T. & Wood, R. D. (1990)

Nucleic Acids Res. 18, 35-40.22. Hansson, J., Keyse, S. M., Lindahl, T. & Wood, R. D. (1991)

Cancer Res. 51, 3384-3390.23. Robins, P., Jones, C. J., Biggerstaff, M., Lindahi, T. & Wood,

R. D. (1991) EMBO J. 10, 3913-3921.24. Koffel-Schwartz, N., Maenhaut-Michel, G. & Fuchs, R. P. P.

(1987) J. Mol. Biol. 193, 651-659.25. Biggerstaff, M., Robins, P., Coverley, D. & Wood, R. D. (1991)

Mutat. Res. 254, 217-224.26. Kodadek, T. & Gamper, H. (1988) Biochemistry 27, 3210-3215.27. Naser, L., Pinto, A., Lippard, S. & EssimnJ. (1988) Biochem-

istry 27, 4357-4367.28. Bellon, S. F. & Lippard, S. J. (1990) Biophys. Chem. 35, 179-188.29. Donahue, B. A., Augot, M., Beilon, S. F., Treiber, D. K., Toney,

J. H., Lippard, S. J. & Essigmann, J. M. (1990) Biochemistry 29,5872-5880.

30. Coverley, D., Kenny, M. K., Munn, M., Rupp, W. D., Lane, D. P.& Wood, R. D. (1991) Nature (London) 349, 538-541.

31. Beck, D. J., Popoff, S., Sancar, A. & Rupp, W. D. (1985) NucleicAcids Res. 13, 7395-7-412.

32. Huang, J. C., Svoboda, D. L., Reardon, J. T. & Sancar, A. (1992)Proc. NatI. Acad. Sci. USA 89, 3664-3668.

33. Chu, 0. & Chang, E. (1988) Science 242, 564-567.34. Toney, J. H., Donahue, B. A., Keilett, P. J., Bnihn, S. L., Essig-

mann, J. M. & Lippard, S. J.- (1989) Proc. NatI. Acad. Sci. USA 86,8328-8332.

35. Chao, C. C.-K., Huang, S. L., Lee, Y.-L. & Lin-Chao, S. (1991)Biochem. J. 277, 875-878.

36. Bruhn, S. L., NR, P. Mi., Essigmann, J. M., Housman, D. E. &Lippard, S. J. (1992) Proc. Natl. Acad. Sci. USA 89, 2307-2311.

37. Pi, P. M. & Lippard, S. J. (1992) Science 256, 234-237.38. Sibghat-Ullah & Saancar, A. (1990) Biochemistry 29, 5711-5718.39. Bedford, P., Fichtinger-Schepan, A. M. J., Shellard, S. A.,

Walker, M. C., Masters, J. R. & Hill, B. T. (1988) Cancer Res. 48,3019-3024.

40. Hill, B. T., Shellard, S. A., Hosking, L. K., Fichtinger-Schcpman,A. Mi. J. & Bedford, P. (1990) Int. J. Radiat. Oncol. Biol. Phys. 19,75-83.

41. Page, J. D., Husain, I1., Sancar, A. & Chancy, S. G. (1990) Bio-chemistry 29, 1016-1024.

42. Beilon, S. F., Coleman, J. H. & Lippard, S. J. (1991) Biochemistry30, 8026-8035.

43. Lepre, C. A. & Lippard, S. J. (1990) in Nucleic Acids and Molec-ularBiology, eds. Eckstein, F. & Lilley, D. Mi. J. (Springer, Berlin),Vol. 4, pp. 9-38.

Proc. Nad. Acad. Sci. USA 89 (1992)