Cisplatin- and UV-damaged DNA lure the basal transcription factor

The EMBO Journal Vol.16 No.24 pp.7444–7456, 1997

Cisplatin- and UV-damaged DNA lure the basal

transcription factor TFIID/TBP

Paul Vichi

, Fre

ric Coin,

Jean-Paul Renaud, Wim Vermeulen

J.H.J.Hoeijmakers

, Dino Moras

and Jean-Marc Egly

Institut de Ge

tique et de Biologie Mole

culaire et Cellulaire, BP 163,

F-67404, Illkirch Cedex, Universite

Louis Pasteur, Strasbourg, France

and

Department of Cell Biology and Genetics, Medical Genetics

Center, Erasmus University Rotterdam, P.O. Box 1738,

3000 DR Rotterdam, The Netherlands

Present address: University of Vermont, Department of Molecular

Physiology and Biophysics, Burlington, VT 05405, USA

Corresponding author

P.Vichi and F.Coin contributed equally to this work

A connection between transcription and DNA repair

was demonstrated previously through the characteriz-

ation of TFIIH. Using ﬁlter binding as well as in vitro

transcription challenge competition assays, we now

show that the promoter recognition factor TATA box-

binding protein (TBP)/TFIID binds selectively to and

is sequestered by cisplatin- or UV-damaged DNA,

either alone or in the context of a larger protein

complex including TFIIH. Computer-assisted 3D struc-

tural analysis reveals a remarkable similarity between

the structure of the TATA box as found in its TBP

complex and that of either platinated or UV-damaged

oligonucleotides. Thus, cisplatin-treated or UV-irradi-

ated DNA could be used as a competing binding site

which may lure TBP/TFIID away from its normal

promoter sequence, partially explaining the phenome-

non of DNA damage-induced inhibition of RNA syn-

thesis. Consistent with an involvement of damaged

DNA-speciﬁc binding of TBP in inhibiting transcrip-

tion, we ﬁnd that microinjection of additional TBP in

living human ﬁbroblasts alleviates the reduction in

RNA synthesis after UV irradiation. Future anticancer

drugs could be designed with the consideration of

lesion recognition by TBP and their ability to reduce

transcription.

Keywords: cisplatin/DNA repair/TBP/TFIID/UV

irradiation

Introduction

Nucleotide excision repair (NER) is essential for the

genomic repair of UV-induced pyrimidine dimers or bulky,

helix-distorting chemical adducts caused by numerous

compounds such as acetylaminoﬂuorene (AAF) or the

anticancer drug, cisplatin (Zamble and Lippard, 1995).

Early investigations aimed at elucidating how cells respond

to damage induced by such diverse agents demonstrated

a transcriptionally linked subpathway of NER in which

7444

lesions in transcribed genes were repaired preferentially

(Bohr et al., 1985). A further bias was shown by the

increased removal of damage in the coding versus the

non-coding strand and was suggested to result from

participation of the arrested RNA polymerase II (RNA

pol II) elongation complex which might serve to recruit

repair machinery (Mellon et al., 1987; Leadon and

Lawrence, 1991; Sweder and Hanawalt, 1992).

More recently, it was established that the basal transcrip-

tion factor TFIIH, critical for transcription, was also an

intricate component of NER (for reviews, see Hoeijmakers

et al., 1996; Svejstrup et al., 1996). This dual function

suggests that TFIIH, as part of the transcription initiation

complex, is well positioned to assist in the rapid removal

of lesions in transcribed genes. What remains unclear is

how the TFIIH complex, an essential transcription factor

as well as part of the core NER machinery required for

both global and transcription-coupled repair, is shared

between these two distinct processes. Difﬁculties in under-

standing the regulation/function of TFIIH in repair versus

transcription reﬂect the involvement of numerous proteins

and their relative interactions in each process. This is

complicated further by differences in in vitro conditions

required to support each process. As a result, studies

concerned with the role of TFIIH during in vitro NER or

transcription are usually performed in the absence of one

process. However, the notion that tight connections exist

between NER and transcription, and the dual involvement

of components in both processes, prompted us to investi-

gate whether damaged DNA produces a high afﬁnity site

which is able to sequester factors supporting NER and/or

transcription (Iyer et al., 1996). We test this hypothesis

by developing a challenge in vitro transcription assay

using an undamaged transcription unit as a template in

the presence of either UV-irradiated or cisplatin-damaged

DNA as a competitor. This assay still examines a selective

function of TFIIH (transcription) but, through pre-incub-

ation of damaged DNA with whole cell extracts or puriﬁed

factors, allows processes of NER to be initiatied and allows

the observation of the relative inﬂuence of interactions

between such factors on the ability of these extracts to

support transcription. Our results indicate that the presence

of damaged DNA leads to an inhibition of transcription

from an independent and transcriptionally viable template.

The most likely interpretation of this trans-effect on

transcription is the sequestration of TFIIH in repair events,

rendering it unavailable for transcription initiation. How-

ever, administration of extra TFIIH had only a relatively

minor effect on recovery of RNA synthesis. Unexpectedly,

addition of the recombinant TATA box-binding protein

TBP or the whole TFIID also appeared to largely abolish

the inhibition of transcription. TBP was found sub-

sequently to bind strongly to different types of damaged

DNA. Computer-assisted 3D structural analysis reveals a

Damaged DNA lures transcription factor TBP/TFIID

Fig. 1. (A) Design of transcription competition experiment.

(B) Description of DNA fragments used for the in vitro transcription

competition assay. AdMLP and F562 transcription templates are

generated by EcoRI–SalI and EcoRI–SphI restriction digestion of

plasmid pUC309 and give rise to 309 and 220 nt RNA transcripts

respectively. Both contain the AdMLP promoter (hatched bars).

Fragment F879 was created by restriction digestion of pUC309 with

BamHI and SspI. This fragment does not contain any promoter

sequence.

remarkable similarity between the structure of the TATA

box as found in its TBP complex and that of platinated

oligonucleotides. Consistent with an involvement of

damaged DNA-speciﬁc binding of TBP resulting in the

inhibition of transcription, we also ﬁnd that microinjection

of additional TBP into living human ﬁbroblasts alleviates

the reduction in RNA synthesis after UV irradiation.

The sequestration of this crucial transcription factor by

DNA lesions could partially explain the overall reduction

in RNA synthesis observed in vivo after genotoxic treat-

ment, and thus represents part of the cellular response to

DNA damage.

Results

Addition of damaged DNA inhibits in vitro

transcription

The presence of DNA lesions recognized by TFIIH, alone

or in concert with other NER proteins, may reduce the

availability of TFIIH or other transcription factors, thereby

inhibiting the formation of a functional transcription

initiation complex. To investigate this possibility, we

developed a crude in vitro transcription competition assay

(Figure 1A).

HeLa whole cell extract (WCE) was pre-incubated with

various amounts of cisplatin- or UV-damaged DNA under

conditions which only allow the formation of the pre-

incision complex, one of the ﬁrst steps of NER. The

following incision/excision and resynthesis steps are

inhibited by the low ATP concentration and the absence

of dNTP respectively (Calsou and Salles, 1994; Moggs

et al., 1996). After the ﬁrst 15 min, an AdMLP reporter

template was introduced and the reactions were continued

for an additional 15 min to allow the formation of pre-

initiation transcription complexes and for any redistribu-

tion of factors including TFIIH between damaged DNA

7445

and transcription template. RNA synthesis was then initi-

ated by addition of NTPs and quantiﬁed by the production

of a 309 nt transcript. Pre-incubation of WCE with a

UV-irradiated 879 bp fragment (F879 UV1) (containing

~3–4 lesions per DNA molecule, Jones and Wood, 1993)

lacking promoter sequences (Figure 1B) inhibited tran-

scription from the AdMLP template (Figure 2A, compare

lanes 2–5 with lanes 6–9). Similarly, pre-incubation of the

3 kb pSK plasmid containing ~30 cisplatin-induced DNA

crosslinks (Hansson and Wood, 1989) also inhibited tran-

scription compared wirh undamaged DNA (Figure 2B,

compare lanes 2–8 with lanes 9–15).

Quantiﬁcation of the synthesis of RNA transcript

(309 nt) from several experiments repeatedly shows that

both UV- and cisplatin-damaged DNA inhibit transcription

from an AdMLP template 3- to 4-fold more than un-

damaged DNA (see Figure 2A and B, lower panels). Note

that 50% transcription inhibition is observed with a 2-fold

excess of cisplatinated sites compared with TATA promoter

sites, in the conditions described in Figure 2B.

To ensure that inhibition of the 309 nt transcript reﬂected

a titration of factors on damaged DNA necessary to

support transcription, we included a ﬁxed concentration

of a competitor DNA in the reaction but varied the

ratio of damaged to undamaged fragment. Under these

conditions, inhibition of transcription was observed as a

function of the increase in damaged fragment present

(Figure 2A, lanes 10 and 11). To demonstrate further that

inhibition resulted from the loss of critical transcription

factors on damaged DNA rather than non-speciﬁc protein–

DNA or DNA–DNA interactions, we performed transcrip-

tion competition experiments using two transcribable tem-

plates: the AdMLP reporter template (product 5 309 nt)

and a second AdMLP-containing template, F562, which

produces a 220 nt transcript (Davison et al., 1983). We

reasoned that if a decrease in the production of the 309 nt

transcript resulted solely from DNA–DNA or non-speciﬁc

protein interactions, then transcription from a transcribable

competitor may also be inhibited when presented in

an undamaged form. However, we observed that pre-

incubation of increasing amounts of UV-irradiated F562

resulted in a more effective inhibition of transcription from

the AdMLP template than pre-incubation with undamaged

F562 DNA (Figure 2C, compare the 309 nt transcript

from lanes 8–13 with lanes 2–7). The presence of un-

damaged F562 decreased transcription somewhat from the

AdMLP reporter template (309 nt), as might be expected

since transcription factors would now be shared between

two active promoters; however, the F562 template was

still capable of being transcribed (220 nt), suggesting that

the presence of two different DNAs in our reaction was

not leading to interactions which rendered the templates

unavailable for transcription. Only in the presence of UV-

irradiated F562 did we also observe the absence of the

220 nt transcript (Figure 2C, lanes 8–13). The lack of the

220 nt product demonstrates that transcription factor(s)

which are associated with damaged DNA (F562 UV1)

are unable to support transcription from that template but,

more importantly, are also unavailable to participate in

transcription from an independent, undamaged template

(AdMLP). Together with data presented in Figure 2A,

these experiments demonstrate that the reaction conditions

support transcription from two templates and that DNA

P.Vichi et al.

Fig. 2. Inhibition of in vitro transcription from the AdMLP by the

presence of damaged DNA. (A) Transcription of AdMLP (50 ng) is

performed with 30 µg of WCE in the presence of increasing amounts

of UV-irradiated (3 kJ/m

) (lanes 2–5) or non-irradiated (lanes 6–9)

F879 DNA fragment. Upper panel: autoradiogram; lower panel:

densitometric quantiﬁcation of autoradiogram (309 nt band), presented

as the percentage of transcription from the AdMLP as a function of

the amount of F879 competitor DNA. Transcription in the absence of

competitor equals 100%. (B) Transcription of AdMLP (50 ng) is

performed with 20 µg of WCE in the presence of increasing amounts

of plasmid pSK CP(–) (lanes 2–8) or CP(1) (lanes 9–15). Top panel:

autoradiogram showing transcription from the AdMLP (309 nt);

bottom panel: graph representing quantiﬁcation by PhosphoImage

analysis of the autoradiogram. (C) Inhibition of transcription by

UV-irradiated F562 DNA. Transcription of AdMLP is performed with

20 µg of WCE in the presence of increasing amounts of non-irradiated

(lanes 2–7) or UV-irradiated (1.5 kJ/m

) (lanes 8–13) F562 DNA

fragment. The size of each transcript is indicated.

containing UV- or cisplatin-induced lesions inhibits tran-

scription because it interacts with components absolutely

required for transcription from a TATA promoter.

7446

Fig. 3. Transcription factors are associated with damaged DNA.

(A) UV-irradiated (1.5 kJ/m

) (25 ng) or non-irradiated (25 ng) F562

DNA fragment was incubated as described in Figure 2C with 30µgof

WCE. Recovery was obtained with the addition of 10 µgofWCE

(lanes 3 and 5) to reactions inhibited by non-irradiated and

UV-irradiated F562 DNA respectively. (B) Puriﬁed recombinant TBP

and TFIIB as well as puriﬁed TFIIH from HeLa cells (hydroxyapatite

fraction, Humbert et al., 1994) were analysed by SDS–PAGE.

puriﬁed reconstituted in vitro transcription system (RTS). Cisplatin-

damaged pSK DNA is pre-incubated with all components of the RTS

before addition of AdMLP and either of the various transcription

factors indicated at the top of the ﬁgure. The added amount of factors

being tested for their ability to restore transcription was the same as

that used to assemble the reconstituted assay, effectively doubling the

concentration during the recovery. (D) The scanned transcription

activities of (C) are reported together with values of two other

experiments. In the shaded and chequered columns, 40 ng of damaged

DNA is added; the chequered columns contain TBP. The means 6 SE

are indicated.

TBP is sequestered by DNA damage

In order to determine the speciﬁc transcription factors

contributing to the loss of activity in the presence of

damaged DNA, we carried out transcription competition

experiments in which WCE or puriﬁed transcription factors

were added to the transcription reaction together with the

AdMLP fragment. Addition of WCE increases transcrip-

tion from either AdMLP (309 nt) or F562 (220 nt) (Figure

3A, compare lanes 2 and 3 with lanes 4 and 5). When

added to a reaction inhibited by UV-damaged DNA, WCE

signiﬁcantly restored the level of transcription (compare

Damaged DNA lures transcription factor TBP/TFIID

Fig. 4. Preferential binding of TBP to damaged DNA. (A)

P-Labelled F879 DNA was UV irradiated at different doses as indicated in the right part

of the panel, incubated with various amounts of TBP and tested for its retention on nitrocellulose ﬁlters. Graphs represent the percentage of DNA

retained on the ﬁlters as a function of the amount of TBP for four different doses of UV and for an undamaged F879 DNA fragment. Quantiﬁcation

was performed using a PhosphoImage analyser; 100% represents the total counts obtained when 1 µl of each DNA probe (input) was spotted onto

Whatman paper and simultaneously exposed with the nitrocellulose ﬁlter. (B) Inhibition of AdMLP-dependent transcription in a highly puriﬁed

reconstituted in vitro transcription system is dependent on the UV dose. The reconstituted transcription assay was carried out in the presence of

increasing amounts of non-irradiated (lanes 2–5), 500 J/m

(lanes 6–9) and 1000 J/m

(lanes 10–13) UV-irradiated F879 DNA. (C) The transcription

reaction was carried out in an RTS with F879 UV (1) (1000 J/m

) or (–) DNA probe as competitor. Recovery of transcription after inhibition with

competitor DNA was obtained by adding TBP. (D) The ﬁlter binding assay was performed as described in (A), except that a PvuI restriction

fragment (1084 bp) of pSK cisplatin-treated CP(1) or untreated CP(–) was used as the DNA probe. (E) The transcription reaction was carried out in

an RTS with an increasing amount of cold CP(–) or CP(1) pSK PvuI DNA probe (see D) as competitor. Recovery of transcription after inhibition

with CP(1) DNA was achieved by adding various amounts of TBP. (F) The transcription reaction was carried out as in (E) except that puriﬁed

TFIID was used in place of TBP. Recovery of transcription was obtained by adding various amounts of TFIID roughly corresponding to 6–15 ng of

TBP according to Western blot analysis.

lanes 1, 4 and 5). The weak 220 nt product from the F562

UV1 template (lane 5) is either non-speciﬁc or results

from residual undamaged template. Together, these results

demonstrate that our assay is responsive to new factors

and that we are below saturating conditions with respect

to total protein (compare lanes 2 and 3 with lanes 4 and

5). Furthermore, these results also demonstrate that UV

lesions in the damaged F562 template remain a signiﬁcant

block to transcription even in the presence of additional

proteins (compare lanes 3 and 5).

Besides TFIIH, WCE contains many other factors which

may contribute to the observed restoration of transcription.

In addition, controlling the relative amounts of individual

factors within a WCE is virtually impossible. We therefore

used a reconstituted transcription system (RTS) in order

to determine effectively the role of each transcription

factor. Thus in vitro transcription competition experiments

were performed with an RTS containing highly puriﬁed

TBP, TFIIB, TFIIE, TFIIF, TFIIH, TFIIA and RNA pol

II (Humbert et al., 1994; see also Figure 3B). The RTS

was pre-incubated with cisplatin-damaged DNA, and the

ability of each puriﬁed transcription factor to restore

activity was determined. Addition of TBP, and to a lesser

extent addition of TFIIB and TFIIH, restored transcription

(compare Figure 3C and D, lane 3, with lanes 6–8),

7447

whereas addition of TFIIA, TFIIE or RNA pol II had no

effect (lanes 9–11).

However, the addition of TFIIH and TFIIB, either alone

or in combination with TBP, did not lead to a signiﬁcantly

greater degree of restoration than that provided by TBP

itself, suggesting that this was the limiting primary factor

after incubation of the RTS with damaged DNA. In fact

addition of increasing amounts of either TBP or TFIID,

in an RTS already inhibited by cisplatin-treated DNA

(Figure 4E and F), was able to restore activity to the

initial level of transcription without inducing similar

increases in the reaction performed in the absence of

competitors (Figure 4E, compare lane 1 with lanes 8 and

9). Together these results suggest that TBP interacts

directly with the DNA lesion, whereas TFIIH and TFIIB

may either be associated with DNA lesions through the

TBP, or drive transcription through stabilization of the

TBP–promoter complex (see Discussion and Ge

rard et al.,

1991). Furthermore, TBP and TFIIB as well as puriﬁed

TFIIH were shown to be free of repair proteins (Figure

3B; see also Aboussekra et al., 1995), indicating that

binding to damaged DNA as well as recovery of transcrip-

tion is not the result of a contaminating repair factor.

Although TFIIH has been reported to be recruited to the

DNA lesion site in conjunction with other factors (Park

P.Vichi et al.

et al., 1995), the added presence in our reconstituted

transcription system of recombinant xeroderma pig-

mentosum group A (XPA) protein did not signiﬁcantly

affect transcription (data not shown).

TBP interacts directly with damaged DNA

The ability of TBP, TFIIB and TFIIH to restore transcrip-

tion inhibited by the presence of damaged DNA suggested

that these factors may interact with DNA lesions. We

investigate this possibility using a standard nitrocellulose

ﬁlter binding assay. As illustrated in Figure 4A, treatment

of F879 DNA, which does not contain a TATA element

(Figure 1B), with increasing doses of UV irradiation

(Figure 4B; 100–1500 J/m

) resulted in a corresponding

increase in the amount of DNA retained by a ﬁxed

concentration of TBP. The functional signiﬁcance of

this interaction is demonstrated further in the following

experiment. The RTS, containing all of the basal transcrip-

tion factors, was pre-incubated with F879 DNA fragment

damaged by irradiation with either 500 or 1000 J/m

, and

assayed for its ability to support transcription from the

AdMLP reporter template. In agreement with observations

from the crude transcription assay and nitrocellulose

ﬁlter binding assays, the presence of damaged DNA

preferentially inhibited the production of the 309 nt

transcript (Figure 4B, compare lanes 2–5 with 6–9 and

10–13). The inhibition can be reversed by back addition

of TBP (Figure 4C).

TBP also recognizes cisplatin-damaged DNA, as judged

by the nitrocellulose ﬁlter binding assay (Figure 4D). Pre-

incubation of the RTS containing recombinant TBP with

cisplatin-damaged DNA resulted in a speciﬁc inhibition

of transcription from the AdMLP template (Figure 4E,

compare lanes 2 and 3 with lanes 4 and 5). Transcription

can be restored completely after readdition of TBP (lanes

6 and 7). It is worthwhile noting that our assay is saturated

with respect to all individual components for a given

concentration of AdMLP template. For example, we

noticed that addition of TBP did not result in a general

increase in the level of transcription (Figure 4E, compare

lane 1 with lanes 8 and 9). When the same experiment

was performed with an RTS containing TFIID (the native

transcription factor that contains TBP) instead of TBP,

cisplatinated DNA also inhibited the transcription reaction

(Figure 4F, lanes 1–5); here too, transcription is restored

upon addition of an excess of TFIID (lanes 6–7), thus

demonstrating that in the context of TFIID, TBP can still

bind to damaged DNA.

TFIIB, which was also able to restore transcription, was

unable to retain a damaged or undamaged fragment

(data not shown), suggesting that its contribution to the

inhibition/restoration of transcription is indirect, most

probably through stabilization of TBP on the promoter.

Attempts to use ﬁlter binding assays to demonstrate a

speciﬁc association of highly puriﬁed TFIIH alone with

damaged DNA were also unsuccessful. Highly puriﬁed

TFIIH retained, although weakly, similar levels of both

damaged and undamaged DNA, suggesting that binding

to damaged DNA probably occurs through interactions

with other repair factors such as XPA (Park et al.,

1995) or the transcription factor TBP, as suggested by

transcription recovery experiments.

The ability of puriﬁed TBP to bind to damaged DNA

7448

Fig. 5. The TATA box-containing fragment competes with damaged

DNA. (A) Gel shift analysis of rTBP binding to cisplatin-damaged

DNA. Binding was performed with the indicated cisplatin-damaged

CP(1) or undamaged CP(–) 36mer DNA probe (0.5 ng; 10 000

c.p.m.), rTBP (20 ng) and/or rTFIIB (20 ng). Then 10 and 50 ng of a

64mer AdMLP fragment containing the TATA box (TATA) or 10 and

50 ng of PvuI pSK CP(1) and CP(–) DNA fragment were added as

competitors as indicated. Note that the radioactive material in the

wells is due to recombinant TBP–DNA probe aggregates.

(B) Inhibition of TBP binding to UV-irradiated (1.5 kJ/m

) F879 DNA

was performed by addition of increasing amounts of the TATA DNA

fragment (64mer) also used in the EMSA. When heat inactivated

(47°C), TBP interacts weakly with DNA.

was also observed in standard electrophoresis mobility

shift assays (EMSAs). A

P-labelled DNA probe, either

undamaged or containing a single 1,3-GpTpG cisplatin

crosslink, was incubated with rTBP and non-speciﬁc DNA

(see Materials and methods), and complex formation was

detected by a change in the migration pattern of DNA on

acrylamide gels. EMSA revealed the formation of a TBP–

DNA nucleoprotein complex that was speciﬁc for damaged

DNA (Figure 5A, lanes 2 and 12). Moreover, the formation

of a TBP–damaged DNA complex was reduced speciﬁcally

with increasing concentrations of an unlabelled cisplatin-

damaged PvuI pSK DNA fragment as compared with a

non-damaged fragment (compare lanes 7 and 8 with

lanes 9 and 10). The addition of an unlabelled fragment

containing a TATA box (TATA) alsoresulted in competition

with TBP binding to the cisplatin-damaged DNA probe

(compare lane 2 with lanes 5 and 6), supporting conclu-

Damaged DNA lures transcription factor TBP/TFIID

Table I. Effect of TBPr injection on transcription in living human ﬁbroblasts

Exp. Microinjected UV irradiation Incubation RNA synthesis

DNA repair

sample

(J/m

) time (h)

(grains/nucleus) (grains/nucleus)

1 400 ng/µl rTBP 16 1 20.0 6 1.0

Non-injected 16 1 21.0 6 1.0

2 400 ng/µl rTBP 0 1 12.0 6 1.02

40 ng/µl rTBP 0 1 14.0 6 1.0

4 ng/µl rTBP 0 1 21.0 6 1.0

Non-injected 0 1 22.0 6 1.0

3 16 ng/µl rTBP 0 1 74.0 6 2.0

Non-injected 0 1 73.0 6 2.0

16 ng/µl rTBP 16 1 54.0 6 3.0

Non-injected 16 1 34.0 6 1.0

4 16 ng/µl rTBP 16 3 43.5 6 3.5

50 ng/µl TFIIB 16 3 27.8 6 1.0

Non-injected 16 3 27.0 6 2.5

5* 16 ng/µl rTBP 16 3 42.0 6 3.0

Non-injected 16 3 25.0 6 1.0

6 16 ng/µl rTBP 30 3 34.0 6 1.0

Non-injected 30 3 19.0 6 1.0

Injections were performed in control ﬁbroblasts (C5RO), except for Exp 5 where ﬁbroblasts of a CS-B patient (CS1AN) are injected.

Time of incubation after UV irradiation and before pulse labelling with radioactive nucleotides.

RNA synthesis (with and without UV challenge), autoradiographically measured by [

H]uridine incorporation (Vermeulen et al., 1994) expressed as

grains/nucleus 6 SEM (at least 50 nuclei for each sample are counted).

UV-induced DNA repair synthesis (UDS), measured autoradiographically as the [

H]thymidine incorporation, expressed as grains/nucleus 6 SEM

(.50 nuclei/sample).

*CS-B cells also presented in Figure 6B.

sions that the shifted probe corresponded to a TBP–DNA

complex. TFIIB alone was unable to shift the damaged

DNA probe, and no supershift was observed when TFIIB

was added to the TBP–damaged DNA complex (see lanes

3 and 4 respectively). These conclusions were supported

by additional ﬁlter binding assays assessing the relative

afﬁnity of TBP for a DNA lesion in the presence of the

normal TATA sequence. UV-irradiated F879 DNA (Figure

1B) was incubated with TBP in the presence of the TATA

element of the AdMLP (TATA). The results illustrated in

Figure 5B conﬁrm a preferential association of TBP with

damaged DNA (column sets for TBP concentration 0.7–

10 ng) and indicate that signiﬁcantly greater amounts of

the TATA fragment were required to compete the binding

of TBP on UV-damaged DNA to similar levels to that

observed with undamaged DNA (column sets for TATA

competitor, concentration 260–5200 fmol). A rough

estimation indicates that ~650 fmol of TATA box-con-

taining fragment (~200-fold) is necessary to achieve a

50% competition inhibition of the association of highly

puriﬁed TBP with the 3–5 fmol of damaged sites induced

by UV irradiation (according to Jones and Wood, 1993)

(see also Figure 3C). As a control, inactivated TBP,

previously incubated for 15 min at 47°C (Nakajima

et al., 1988), shows no interaction with UV-damaged or

undamaged DNA. Together, these results indicate for the

ﬁrst time that two types of DNA lesions, induced by either

UV irradiation or cisplatin treatment, serve as binding

targets for TBP.

TBP microinjection protects cells from UV-induced

inhibition of RNA synthesis

UV-induced DNA damage in the genome causes a transient

inhibition of overall transcription. One possibility is that

this suppression is at least in part due to sequestration of

TBP by DNA lesions. Therefore, we reasoned that micro-

7449

injection of extra TBP might partly relieve this UV-induced

inhibitory effect. To test this possibility, rTBP was micro-

injectedintonormalprimary ﬁbroblasts.TheeffectonNER,

transcription and UV-induced inhibition of transcription

was assessed by incubating the cells after rTBP injection,

in the presence of

H-labelled thymine (for repair synthesis)

H-labelled uridine (to measure transcription). NER and

transcription were quantiﬁed by counting the number of

autoradiographic grains above the nuclei of injected cells

and compared with the non-injected cells on the same slide.

Initial experiments (Table I, Exp 1 and 2; using rTBP at a

concentration of 400 ng/µl)indicated that the injected rTBP

by itself did not affect DNA repair (Exp 1) but caused a

strong inhibition of transcription (Exp 2). Apparently, the

excess TBP (we calculate that we injected ~40–60% of the

total cellular TBP content) squelches factors interfering

with normal transcription, a fact that was also observed

after transient overexpression of TBP (S.Buratowsky and

P.Chambon, personal communications). To avoid a domin-

ant-negative effect on regular transcription, we titrated TBP

to a concentration at which no inhibition was observed (4

and 16 ng/µl, see Exp 2 and Exp 3 respectively). As shown

in Table I (Exp 3, 4 and 6), injection of 16 ng/µl rTBP

induced a clear protective effect against the inhibition of

transcription caused by different doses of UV irradiation

(see also Figure 6A). The partial relief of transcription was

observed 1 and 3 h after UV exposure and was independent

of transcription-coupled repair, as injection in Cockayne

syndrome (CS)-B ﬁbroblasts (which exhibit a selective

defect in transcription-coupled repair) also stimulated UV-

suppressed RNA synthesis (Exp 5 and Figure 6B). Micro-

injection of TFIIB (50 ng/µl, Exp 4) and cdk7–cyclinH–

MAT1 complex (50 ng/µl) failed to reverse the UV-induced

transcription inhibition, indicating that the observed protec-

tion was TBP speciﬁc. These ﬁndings showthat at least part

of the suppression of transcription by UV can be overcome

P.Vichi et al.

Fig. 6. Effect of TBP on transcription in vivo. Micrograph of C5RO normal (A) and CS-B patient (B) ﬁbroblasts injected (indicated by an arrow)

1 h before UV irradiation (16 J/m

) showing an increase in the overall RNA synthesis. In each panel, one has to compare the autoradiographic

grains above the nuclei of injected cells (indicated by an arrow) with the surrounding nuclei.

by exogeneous TBP. This is consistent with the idea that

after UV irradiation, sequestration of endogeneous TBP

to DNA lesions takes place, contributing to the damage-

induced inhibition of transcription.

Similarities between the TATA box and

cisplatin-damaged DNA

We investigated the structural implications of these observ-

ations by comparing the structure of a platinated DNA

with its TATA counterpart from the human TBP–TATA

complex. The only available crystal structure of a 1,2-

cisplatin adduct on an oligonucleotide is that of a double-

stranded DNA dodecamer containing a central G∧G site

(cis-[Pt(NH

)

-{d(GpG)-N7(G

), N7(G

)}] intrastrand

crosslink) (Takahara et al., 1995), hereafter referred to as

GGPG. Superposition of GGPG onto the TATA box

DNA (TATA) revealed a strikingly similar overall shape,

especially in the central region, although the detailed

conformations of the base pairs differ in the two structures

(Figures 7A and 8A and B). The following analysis is

based on the optimal superposition of the original structural

data without any energy minimization. An r.m.s.d. of 2.1

Å was found between the backbone atoms of TATA and

GGPG (compared with an r.m.s.d. of 8.8 Å between the

backbone atoms of TATA and BDNA, the canonical

B-DNA dodecamer, and an r.m.s.d. of 5.7 Å between the

7450

backbone atoms of GGPG and BDNA, compare Figure

7B and C). To calculate a realistic r.m.s.d. value, we have

taken into account the different orientation of the base

pairs in the two structures which leads to a shift of one

nucleotide on one strand (Figure 7A). This explains how

the two overall structures can ﬁt so well in spite of

important differences in base pair conformations. Both

TATA and GGPG are bent towards the major groove and

partially unwound without disrupting the Watson–Crick

hydrogen bonding pattern. In the TBP–TATA complex,

the saddle-shaped TBP core wraps around the DNA in

the minor groove (Chasman et al., 1993; J.L.Kim et al.,

1993; Y.Kim et al., 1993; Juo et al., 1996). The pronounced

bend is induced by the insertion of phenylalanine side

chains into the ﬁrst and last steps of the TATA element,

and is favoured by the intrinsic bendability of the TA

steps. In the GGPG structure, a similar bend is produced

by the coordination of the platinum ion to the N7 nitrogen

atoms of two adjacent guanines on the same strand, which

forces the destacking of the complementary bases. As a

result, the cisplatin adduct mimicks the distorted conform-

ation of TATA in its complex with TBP. In both molecules,

the structure exhibits an abrupt B- to A-form transition

near the bent portion, with a pronounced opening and

ﬂattening of the minor groove, as illustrated in the two by

two superpositions of TATA, BDNA and GGPG (Figure 7).

Damaged DNA lures transcription factor TBP/TFIID

Fig. 7. Stereoviews of the two by two superpositions of the crystal structures of the TATA box from the human TBP–TATA complex (TATA, PDB

code 1TGH; Juo et al., 1996), of the DNA dodecamer containing a central cis-[Pt(NH

)

-{d(GpG)-N7(G

), N7(G

)} intrastrand crosslink (GGPG,

PDB code 1GPG; Takahara et al., 1995), and of a canonical B-DNA dodecamer (BDNA, PDB code 1BNA; Drew et al., 1981). The superpositions

were optimized using the LSQ options of O (Jones et al., 1991) and displayed with SETOR (Evans, 1993). TATA is shown in red, GGPG in cyan,

with the platinum ion and the nitrogen atoms of its amine ligands in green, and BDNA in yellow. (A) GGPG–TATA; (B) BDNA–TATA;

Indeed, TBP appears to dock exceedingly well on

GGPG (Figure 8A and B), the complex showing very

few stereochemical clashes at the level of the inserted

phenylalanines. An optimal ﬁt would require a small

adaptation of DNA with a minimal energy cost. The

buried surface in the TBP–GGPG complex calculated

from the docked structures is 2707 Å

compared with

3090 Å

in the TBP–TATA complex, the slightly lower

value arising from the more pronounced bend in GGPG,

as seen in Figure 8A. Note that the complex with GGPG

has not been energy-minimized. In both cases, the contacts

with TBP are essentially hydrophobic, with only a few

exceptions such as the hydrogen bond bridges formed by

Asn163 and Asn253 with the two central T

A base pairs

in the TBP–TATA complex. When BDNA is docked with

TBP, the interface drops down to 2241 Å

, and the

7451

minor groove forms a cavity which suppresses the contacts

between the protein side chains and the bases. Instead,

phosphate groups point towards the protein surface, making

the binding of TBP to DNA in a B conformation very

unfavourable (Figure 8C). The importance of the inter-

actions with the bases is supported by the observation that

two TBP mutants at the 253 position are defective in DNA

binding (Arndt et al., 1995; Lee and Struhl, 1995). As a

consequence, the helical twist is nearly zero at the central

step, the two base pairs being stacked directly on top of one

another with a 25° roll angle (Juo et al., 1996). This value

can be compared with the 26° roll angle observed between

the two central G*C base pairs in GGPG (Takahara et al.,

1995). Accordingtoourdockingexperiment,thetwoaspar-

agine residues are accommodated at the TBP–GGPG inter-

face, providing different hydrogen bonding patterns,

P.Vichi et al.

Fig. 8. Stereoviews of the TBP–DNA interfaces displayed with SETOR. (A) View along the long axis of TBP of the human TBP–TATA complex

showing GGPG superposed to TATA as in Figure 6A. TATA is shown in red, GGPG in cyan, with the platinum ion and the nitrogen atoms of its

ammine ligands shown in green, and TBP is displayed in yellow in a ribbon representation. (B) Close view in the same orientation as in Figure 6

showing TBP (in white) sitting on the minor groove. The Asn163 and Asn253 side chains are coloured in magenta. (C) View of BDNA docked with

TBP in the same orientation as in Figure 7B, showing the cavity at the interface and the unfavourable orientation of the central phosphate groups.

Asn163 and Asn253 contacting N3 of G

and O2 of C

respectively. Thus these interactions do not discriminate

between different sequences, but rather are involved in the

recognition of a particular type of DNA structure.

7452

Discussion

We present data which suggest that repair and transcription

may be linked at a more fundamental level than previously

Damaged DNA lures transcription factor TBP/TFIID

thought. Earlier work suggested a role for an arrested

RNA pol II complex at a DNA lesion (Donahue et al.,

1994), followed by participation of TFIIH in the recogni-

tion and incision/excision of the DNA damage, leading to

preferential repair (Moggs et al., 1996). Our work pre-

sented here suggests that TBP/TFIID, an essential com-

ponent which nucleates the formation of an active

transcription complex, recognizes and binds directly to

DNA lesions induced by UV irradiation or cisplatin

treatment.

TFIID/TBP directly binds damaged DNA

Using in vitro transcription challenge competition assays,

we show that TBP/TFIID, either in the context of a crude

cellular extract (WCE) and therefore in the presence of

all repair proteins, or in the presence of all the general

transcription factors and RNA pol II, associates with

damaged DNA. This association appears to be relatively

rapid and persistent, since changing either the time of pre-

incubation of WCE with damaged DNA or increasing the

pre-initiation time in the presence of both the competitor

and AdMLP DNA had little effect on the overall level of

inhibition induced by damaged DNA (data not shown).

The so-called TATA-binding protein, TBP, either free or

associated with the TBP-associated factors, TAFs (named

TFIID), directly interacts with damaged DNA as demon-

strated by nitrocellulose ﬁlter binding, gel shift and tran-

scription competition experiments. This was also observed

in reactions containing a TATA box, indicating that a

lesion can bind TBP efﬁciently even in the presence of

its speciﬁc (and natural) binding site. Moreover, the

strength of interaction of TBP with either UV-, cisplatin-

(the present study) or AAF- (unpublished results) damaged

DNA will be a function of the nature of the damage and

the surrounding sequences, which will each contribute to

the overall distortion in the DNA helix.

Transcription challenge competition assays show that

not only TBP but also TFIIB and TFIIH, although to a

lower extent, are required to restore AdMLP transcription

activity previously inhibited by the presence of a damaged

DNA fragment (Figure 3B). This is not particularly

surprising in light of the fact that TFIIH contains subunits

with zinc ﬁnger motifs (Humbert et al., 1994; data not

shown) and has been shown to interact with TBP (Ge

rard

et al., 1991). However, attempts to demonstrate a speciﬁc

association, using gel shift experiments, between TFIIH

or TFIIB alone or in combination with TBP and damaged

DNA were unsuccessful (data not shown). TFIIH recruit-

ment to the excision complex was shown to occur through

other factors present in the crude cellular extract (Nocentini

et al., 1997). It is possible that the association of TFIIB

and TFIIH with a DNA lesion or with a TBP-damaged

DNA complex could be rather weak compared with the

interactions which are required during the formation of

an active transcription complex. Partial restoration by

TFIIB and TFIIH may therefore reﬂect some interactions

with DNA lesions or the ability of these factors to disrupt

binding of TBP to damaged DNA. If TBP alone exhibits

a stronger afﬁnity for damaged DNA, rather than for its

natural TATA target, addition of TFIIB or TFIIH may

stabilize existing binary TBP–TATA box complexes, dis-

placing the thermodynamic equilibrium away from the

damaged DNA complex towards the formation of a

7453

functional transcription initiation complex. The inability

of TFIIA, previously shown to stabilize TBP–TATA box

interactions (Buratowski et al., 1989), to recover transcrip-

tion inhibited by damaged DNA competitor may reﬂect

this being exclusively a promoter function. It also must

be borne in mind that the damaged cisplatin structure is

not completely identical to the TATA box structure and

thus may not be targeted equally by all the basal transcrip-

tion factors, e.g. TFIIA, TFIIB and TFIIH.

In vivo evidence for TBP/TFIID binding to damaged

DNA

To investigate a possible in vivo role of TBP/TFIID in

the cellular response to DNA damage, we performed

microinjection into living cells. The absence of an effect

on UV-induced DNA repair synthesis, under conditions

in which transcription was strongly inhibited, indicates

that both processes, although requiring the participation

of common factors such as TFIIH, are largely independent.

Furthermore, the lack of squelching by excess TBP on

NER suggests that TBP does not interact with essential

NER factor(s), and is therefore not implicated directly in

the NER process. However, microinjection of a well-

deﬁned concentration of TBP was found to protect cells

from the reduction in transcription (overall RNA synthesis)

caused by UV exposure, whereas microinjection of either

TFIIB or the three components of the CAK complex

(Rossignol et al., 1997) had no signiﬁcant effect. One

possible explanation for the relief provided by TBP is that

when exogeneously added, this protein binds directly to

lesions that would otherwise have trapped endogenous

transcription-competent TFIID or other SL1 or TFIIIB

complexes (two transcription factors which include TBP

and are associated with RNA pol I and RNA pol III

transcription respectively). The result of microinjection

then is to increase the pool of TBP-containing complexes

such as TFIID (Colgan and Manley, 1992) which would

be available for damage and/or promoter recognition. This

conclusion is supported by our ﬁlter binding studies. The

fact that TFIIB microinjection did not relieve transcription

inhibition may reﬂect the weak afﬁnity of TFIIB for the

damage and/or its preference for the transcription reaction

in the context of an in vivo situation in which TFIIB plays

a crucial role in the activation process. Although the

microinjection experiments are not simple to interpret,

they ﬁt well with our model in which TBP/TFIID binding

to lesions is at least partly responsible for the general

drop in transcription exerted by UV irradiation and other

DNA-damaging treatments.

TBP recognizes a typical 3D structure

The almost perfect match between the TBP core, as found

in its complex with the TATA box, and GGPG strongly

supports the present results of a speciﬁc binding of TBP

to cisplatin 1,2-adducts. In the former case, the TBP–

TATA interaction is an induced ﬁt, while in the latter,

TBP seems to bind to a pre-formed, bent DNA in a lock-

and-key fashion. As noted by Juo et al. (1996), TBP

recognizes the intrinsic bendability of the TATA box more

than the base pair sequence per se. It is the opening of

the minor groove induced by the protein which allows a

snug ﬁt of the concave surface of TBP against DNA. In

the 1,2-cisplatin G∧G adduct, the minor groove is already

P.Vichi et al.

exposed, inviting TBP. The limited number of polar

interactions with the bases, as discussed above, discards

any strong sequence speciﬁcity and favours a structural

recognition. As for the relationship with the UV-damaged

DNA, it is of interest to note that the crystal structure of

an oligonucleotide containing a cyclobutane-type thymine

dimer in complex with T4 endonuclease V also exhibits

a sharp kink around the thymine dimer portion, splitting

the duplex into two halves of B-DNA with a 60° inclination

between the two helical axes (Vassylyev et al., 1995), i.e.

two 30° bends. In this case too, the DNA shape is very

reminiscent of that of TATA bound to TBP, although the

thymine dimer undergoes different constraints from its

interaction with the endonuclease, including the ﬂipping-

out of the adenine base complementary to the 59-thymine

of the dimer, which allows the excision process to take

place. The structural similarity of the deformation indicates

that this type of UV-damaged DNA could also bend

towards the major groove through interaction with TBP,

due to the instability of the TT step introduced by the

crosslinking of the two adjacent thymines.

It thus appears that various genotoxic and antitumour

agents could turn GC-rich sequences into potential sites for

TBP. This structural correlation supports the experimental

observation that several damaging factors have similar

effects, suggesting that in all cases the DNA bendability,

with a marked tendency to adopt locally an A-form

conformation with a ﬂat, widened minor groove, is the

common property.

Implications of TFIID/TBP binding to damaged

DNA

The structural similarities between the TATA box and

DNA lesions implies an important basal role for TBP.

Indeed, TBP, either directly or within the context of one

of the multiprotein complexes SL1, TFIID and TFIIIB,

allows the initiation of transcription from the three classes

of promoters. The damage caused by agents such as UV

irradiation and cisplatin treatment results in an altered 3D

structure of the DNA similar to the one adopted by the

TATA sequence. These different lesions, forming a kind

of TATA-like 3D structure, may then be recognized by

TBP, with functional implications; they may serve as a

lure for TFIID/TBP, diverting it from its natural promoter

target, explaining the loss of transcription observed in

cells after DNA damage. In addition, binding of TBP to

damaged DNA could also serve to alter the equilibrium

of TFIIH associated with transcription or repair complexes.

Less TBP bound to promoter sequences would result in a

decrease in the number of pre-initiation complexes to

which TFIIH may be recruited and would lead to an

increase in the availability and association of TFIIH with

repair proteins. In this manner, TBP may stimulate the

repair function of TFIIH indirectly. However, such a

hypothesis does not exclude a possible role, if any, for TBP

in the ﬁrst step of NER, simply through the recognition of

the lesion in conjunction with XPA and RPA, and the

recruitment of TFIIH. It remains to be determined how

TBP damage recognition leads to a decrease in overall or

selective transcription, resulting in apoptosis. Binding of

TBP could shield the lesion from repair proteins unless it

can be translocated efﬁciently. Furthermore, the persistent

presence of bound TBP may be responsible for the

7454

increased cytotoxicity of DNA-damaging agents in CS

cells (Mayne and Lehmann, 1982).

Although this kind of molecular decoy has been pro-

posed previously [hUBF, a transcription factor involved

in rRNA synthesis, was shown to be hijacked by cisplatin

adducts (Treiber et al., 1994)], this is the ﬁrst time that a

functional consequence of this type of interaction

(hijacking) has been demonstrated. Depending on the

outcome, future anticancer drugs could be designed with

the consideration of lesion recognition by TBP, taking

into account the speciﬁc type of lesion, its afﬁnity for

TBP and its tendency to compete transcription.

Materials and methods

Materials

HeLa WCEs, as well as all the components of the in vitro reconstituted

transcription system, were as described in Humbert et al. (1994).

Substrates used for in vitro transcription or ﬁlter binding analysis

were generated by restriction digestion of either pUC309 or pSK plasmid

DNA. pUC309 was created by ligation of an EcoRI–BamHI fragment,

corresponding to sequences –677 to 133 of the AdMLP (∆ –372/–34),

to the BamHI–SalI fragment from pBR322. The resultant fragment was

cloned into the EcoRI–SalI sites of pUC19, generating the pUC309

plasmid. Competitor DNA fragments of 879 (F879) and 562 bp (F562)

were generated by restriction digestion of pUC309 with BamHI–SspI

and EcoRI–SphI, respectively (Figure 1B). The ﬁnal competitor used

was the 3 kb Bluescript, pSK1 plasmid (Stratagene). F562 and F879

were damaged by UV irradiation, at 0.1 mW/cm

with a germicidal

UV-C lamp. pSK was treated with cisplatin for 15 h in the dark at 37°C

and at a drug:nucleotide ratio of 0.005 (Hansson and Wood,1989).

The CP(–) or CP(1) fragments used in the ﬁlter binding assays were

generated by restriction digestion with PvuI of pSK undamaged or

damaged by cisplatin. The AdMLP DNA probe (64mer) was created by

annealing synthesized, complementary oligonucleotides corresponding

to regions –40 to 124 of the AdMLP.

The 32mer 59-TCTTCTTCTTCTTCTGTGCACTCTTCTTCTCT-39

containing a single GpTpG (highlighted sequence) was allowed to react

with cisplatin (Moggs et al., 1996). After ethanol precipitation, the

presence of a 1,3-intrastrand cisplatin d(GpTpG) DNA crosslink was

conﬁrmed by analysis of the oligonucleotide on a 12% acrylamide gel.

The 36 bp DNA probe used in EMSA was created by annealing the

damaged CP(1) or undamaged CP(–) DNA with its complementary

oligonucleotide, leaving a 59 overhang at each extremity. The DNA was

ﬁlled and radiolabelled with [

P]dATP (3000 Ci/mmol) in the presence

of Klenow and puriﬁed on G50 Sephadex columns.

Crude transcription assay

Approximately 15–30 µg of HeLa WCE were incubated with varying

amounts of competitor DNA in a 50 mM Tris–HCl pH 7.9 buffer

containing 10% glycerol, 1 mM EDTA, 0.5 mM dithiothreitol (DTT)

and 5 mM MgCl

. Reactions (ﬁnal volume 20 µl) were incubated for

15 min at 28°C, at which point 50 ng of the AdMLP template (EcoRI–

SalI) were added and pre-initiation of transcription allowed to continue

for 15 min. Transcription was then initiated by addition of NTPs

including [α-

P]CTP (400 Ci/mmol). The ﬁnal volume of the reaction

was 25 µl, and transcription was carried out for 45 min at 28°C. The

RNA transcripts were then analysed by autoradiography and quantiﬁed

directly by counting on a PhosphoImage analyser.

The reconstituted transcription assay containing puriﬁed transcription

factors TBP, TFIIA, TFIIB, TFIIE, TFIIH, TFIIF and RNA pol II was

modiﬁed to include an initial incubation step to allow the potential

binding of transcription factors to a damaged or undamaged fragment

and carried out as described above. TFIID was derived from a subfraction

of the TFIIH puriﬁcation procedure (Ge

rard et al., 1991).

Filter binding assay

Puriﬁed recombinant human TBP was combined with various DNA

probes: UV-damaged or undamaged 879 bp fragment was labelled

with [

P]dATP (3000 Ci/mmol) using the Klenow fragment of DNA

polymerase; the damaged or undamaged PvuI fragment from plasmid

pSK was labelled by exploiting the exonuclease activity of Klenow in

the presence of [

P]dCTP (3000 Ci/mmol) and subsequent ﬁlling by

Damaged DNA lures transcription factor TBP/TFIID

addition of cold nucleotides before puriﬁcation. Approximately 1 ng of

probe corresponding to 5000 c.p.m. was combined with varying amounts

of TBP, in 20 µl of the transcription buffer containing 60 µg/ml bovine

serum albumin (BSA), 500 ng of poly(dGdC) and 5 mM MgCl

, for

30 min at 30°C. Reactions were applied to a 0.45 mm nitrocellulose

membrane (Millipore) using the 96-well Hybri-dot Manifold (BRL), pre-

soaked in 0.4 mM KOH, washed with distilled water and pre-equilibrated

in the reaction buffer without BSA. Filters were air dried and directly

exposed to a PhosphoImage screen, for quantiﬁcation, or a Biomax ﬁlm

(Kodak). One µl of input DNA corresponding to the same volume used

in each reaction was spotted on Whatman ﬁlter paper as a control for

determination of the percentage of DNA retained on nitrocellulose ﬁlters.

The amount of radioactivity retained in the presence of TBP was

measured, background counts (radioactivity retained in the absence of

protein) subtracted, and the amount was divided by the level of

radioactivity present in the input.

Miroinjection of rTBP into human ﬁbroblasts

Microneedle injection into homopolykaryons of ﬁbroblasts derived from

a repair-competent individual (C5RO) and of a CS-B patient (CS1AN)

was performed as described earlier (Vermeulen et al., 1994). RNA

synthesis was determined by pulse labelling the cells for 1 h with

[

H]uridine (10 µCi/µl), whereas NER was determined by [

H]thymidine

(10 µCi/µl) incorporation after UV irradiation (16 J/m

) and autoradio-

graphy. TPB and TFIIB were diluted into phosphate-buffered saline

(PBS) containing BSA.

Electrophoretic mobility shift assays

EMSA reaction mixtures (20 µl) contained 0.2 ng of

P-labelled 36 bp

DNA probe (10 000 c.p.m.), 500 ng of poly(dGdC) in a 50 mM Tris–

HCl pH 7.9 buffer containing 80 mM KCl, 5 mM MgCl

, 0.1 mM

EDTA, 500 ng of BSA, 10% glycerol, 0.5 mM DTT, 0.01% NP-40, and

rTBP and/or rTFIIB, when indicated. After 30 min of incubation at

30°C, glycerol was added to a ﬁnal concentration of 20% and applied

to a 4% native polyacrylamide gel. Protein–DNA complexes were

electrophoresed in 25 mM Tris–19 mM glycine buffer at room temper-

ature. Gels were dried and exposed to Biomax ﬁlm (Kodak).

Structural analysis

The crystallographic coordinates of the human TBP–TATA box complex

(Juo et al., 1996) and of a double-stranded DNA dodecamer containing

a central G∧G site (cis-[Pt(NH

)

-{d(GpG)-N7(G

), N7(G

)}] intrastrand

crosslink) (Takahara et al., 1995) were extracted from the PDB (Bernstein

et al., 1977; access codes 1TGH and 1GPG respectively). In the platinated

DNA crystal structure, two duplexes are found in the asymmetric unit

but appear to be almost identical (r.m.s.d. value of 0.3 Å between the

two molecules). The structure superpositions were done using the LSQ

options of the program O (Jones et al., 1991). The TBP–DNA interfaces

were analysed and displayed with the program GRASP (Nicholls et al.,

1993). For comparison, the Dickerson’s dodecamer (Drew et al., 1981)

was used as canonical B-DNA (PBD code 1BNA), and noted BDNA.

Acknowledgements

We thank P.Hanawalt, P.Chambon and T.Seroz for fruitful discussion.

We thank M.Chipoulet and A.Fery for their excellent technical assistance

and R.Ripp for help with SETOR. P.V. was supported by NSF and ARC

fellowships. This work was supported by grants from the INSERM, the

CNRS, the Ministe

re de la Recherche et de l’Enseignement Supe

rieur,

the Association pour la Recherche sur le Cancer and the Direction des

Recherches Etudes et Techniques.

References

Aboussekhra,A.M. et al. (1995) Mammalian DNA nucleotide excision

repair reconstituted with puriﬁed protein components. Cell, 80, 859–

868.

Arndt,K.M., Ricupero-Hovasse,S. and Winston,F. (1995) TBP mutants

defective in activated transcription in vivo. EMBO J., 14, 1490–1497.

Bernstein,F.C., Koetzle,T.F., Williams,G.J.B., Meyer,E.F., Brice,M.D.,

Rodgers,J.R., Kennard,O., Shimanouchi,T. and Tasumi,M. (1977) The

Protein Data Bank: a computer-based archival ﬁle for macromolecular

structures. J. Mol. Biol., 112, 535–542.

Bohr,V.A., Smith,C.A., Okumoto,D.S. and Hanawalt,P.C. (1985) DNA

repair in an active gene: removal of pyrimidine dimers from the

7455

DHFR gene of CHO cells is much more efﬁcient than in the genome

overall. Cell, 40, 359–369.

Buratowski,S., Hahn,S., Guarente,L. and Sharp,P.A. (1989) Five

intermediate complexes in transcription initiation by RNA polymerase

II. Cell, 56, 549–561.

Calsou,P. and Salles,B. (1994) Properties of damage-dependent DNA

incision by nucleotide excision repair in human cell-free extracts.

Nucleic Acids Res., 22, 4937–4942.

Chasman,D.I., Flaherty,K.M., Sharp,P.A. and Kornberg,R.D. (1993)

Crystal structure of yeast TATA-binding protein and model for

interaction with DNA. Proc. Natl Acad. Sci. USA, 90, 8174–8178.

Colgan,J. and Manley,L. (1992) TFIID can be rate limiting in vivo

for TATA-containing, but not TATA-lacking, RNA polymerase II

promoters. Genes Dev., 6(2), 304–315.

Davison,B.L., Egly,J.-M., Mulvihill,E.R. and Chambon,P. (1983)

Formation of stable preinitiation complexes between eukaryotic class

B transcription factors and promoter sequences. Nature, 301, 680–686.

Donahue,B.A., Yin,S., Taylor,J.S., Reines,D. and Hanawalt,P.C. (1994)

Transcript cleavage by RNA polymerase II arrested by a cyclobutane

pyrimidine dimer in the DNA template. Proc. Natl Acad. Sci. USA,

91, 8502–8506.

Drew,H.R., Wing,R.M., Takano,T., Broka,C., Tanaka,S., Ikatura,K. and

Dickerson,R.E. (1981) Structure of a B-DNA dodecamer: conformation

and dynamics. Proc. Natl Acad. Sci. USA, 78, 2179–2183.

Evans,S.V. (1993) SETOR: hardware lighted three-dimensional solid

model representations of macromolecules. J. Mol. Graphics, 11,

134–138.

rard,M., Fischer,L., Moncollin,V., Chipoulet,J.-M., Chambon,P. and

Egly,J.-M. (1991) Puriﬁcation and interaction properties of the human

RNA polymerase B(II) general transcription factor BTF2. J. Biol.

Chem., 266, 20940–20945.

Hansson,J., and Wood,R.D. (1989) Repair synthesis by human cell

extracts in DNA damaged by cis- and trans-diammine dichloro

platinum(II). Nucleic Acids Res., 17, 8073–8091.

Hoeijmakers,J.H.J., Egly,J.-M. and Vermeulen,W. (1996) TFIIH: a key

component in multiple DNA transactions. Curr. Opin. Genet. Dev., 6,

26–33.

Humbert,S., van Vuuren,H., Lutz,Y., Hoeijmakers,J.H.J., Egly,J.-M. and

Moncollin,V. (1994) p44 and p34 subunits of the BTF2/TFIIH

transcription factor have homologies with SSL1, a yeast protein

involved in DNA repair. EMBO J., 13, 2393–2398.

Iyer,N., Reagan,M.S., Wu.,K.-J., Canagarajah,B. and Friedberg,E.C.

(1996) Interactions involving the human RNA polymerase II

transcription:nucleotide excision repair complex TFIIH, the nucleotide

excision repair protein XPG, and Cockayne syndrome group B (CSB)

protein. Biochemistry, 35, 2157–2167.

Jones,C.J. and Wood,R.D. (1993) Preferential binding of the xeroderma

pigmentosum group A complementing protein to damaged DNA.

Biochemistry, 32, 12096–12104.

Jones,T.A., Zou,J.Y., Cowan,S.W. and Kjeldgaard,M. (1991) Improved

methods for building protein models in electron density maps and the

location of errors in these models. Acta Crystallogr., A47, 110–119.

Juo,Z.S., Chiu,T.K., Leiberman,P.M., Baikalov,I., Berk,A.J. and

Dickerson,R.E. (1996) How proteins recognize the TATA box. J. Mol.

Biol., 261, 239–254.

Kim,J.L., Nikolov,D.B. and Burley,S.K. (1993) Co-crystal structure of

TBP recognizing the minor groove of a TATA element. Nature, 365,

520–527.

Kim,Y., Geiger,J.H., Hahn,S. and Sigler,P.B. (1993) Crystal structure of

a yeast TBP/TATA-box complex. Nature, 365, 512–520.

Leadon,S.A. and Lawrence,D.A. (1991) Strand-selective repair of DNA

damage in the yeast GAL7 gene requires RNA polymerase II. J. Biol.

Chem., 267, 23175–23182.

Lee,M. and Struhl,K. (1995) Mutations on the DNA-binding surface of

TATA-binding protein can speciﬁcally impair the response to acidic

activators in vivo. Mol. Cell. Biol., 15, 5461–5469.

Mayne,L.V. and Lehmann,A.R. (1982) Failure of RNA synthesis to

recover after UV irradiation: an early defect in cells from individuals

with Cockayne’s syndrome and xeroderma pigmentosum. Cancer Res.,

42, 1473–1478.

Mellon,I., Spivak,G. and Hanawalt,P.C. (1987) Selective removal of

transcription-blocking DNA damage from the transcribed strand of

the mammalian DHFR gene. Cell, 51, 241–249.

Moggs,J.G., Yarema,K.J., Essigmann,J.M. and Wood,R.D. (1996)

Analysis of incision sites produced by human cell extracts and

puriﬁed proteins during nucleotide excision repair of a 1,3-intrastrand

d(GpTpG)-cisplatin adduct. J. Biol. Chem., 271, 7177–7186.

P.Vichi et al.

Nakajima,N., Horikoshi,M. and Roeder,R.G. (1988) Factors involved in

speciﬁc transcription by mammalian RNA polymerase II: puriﬁcation,

genetic speciﬁcity, and TATA box-promoter interactions of TFIID.

Mol. Cell. Biol., 8, 4028–4040.

Nicholls,A. (1993) GRASP: Graphical Representation and Analysis of

Surface Properties. Columbia University, New York.

Nocentini,S., Coin,F., Saijo,M., Tanaka,Y. and Egly,J.-M. (1997) DNA

damage recognition by XPA protein promotes efﬁcient recruitment of

TFIIH. J. Biol. Chem., 272, 22991–22992.

Park,C.H., Mu,D., Reardon,J.T. and Sancar,A. (1995) The general

transcription-repair factor TFIIH is recruited to the excision repair

complex by the XPA protein independent of the TFIIE transcription

factor. J. Biol. Chem., 270, 4896–4902.

Rossignol,M., Kolb-Cheynel,I. and Egly,J.-M. (1997) Substrate

speciﬁcity of the cdk-activating kinase (CAK) is altered upon

association with TFIIH. EMBO J., 16, 1628–1637.

Svejstrup,J.C., Vichi,P. and Egly,J.-M. (1996) The multiple roles of

transcription/repair factor TFIIH. Trends Biochem. Sci., 21, 346–350.

Sweder,K.S. and Hanawalt,P.C. (1992) Preferential repair of cyclobutane

pyrimidine dimer in the transcribed strand of a gene in yeast

chromosomes and plasmids is dependent on transcription. Proc. Natl

Acad. Sci. USA, 89, 10696–10700.

Takahara,P.M., Rosenzweig,A.C., Frederick,C.A. and Lippard,S.J. (1995)

Crystal structure of double-stranded DNA containing the major adduct

of the anticancer drug cisplatin. Nature, 377, 649–652.

Treiber,D.K., Zhai,X., Jantzen,H.-M. and Essigman,J.M. (1994)

Cisplatin–DNA adducts are molecular decoys for the ribosomal RNA

transcription factor hUBF (human upstream binding factor). Proc.

Natl Acad. Sci. USA, 91, 5672–5676.

Vassylyev,D.G., Kashiwagi,T., Mikami,Y., Ariyoshi,M., Iwai,S.,

Ohtsuka,E. and Morikawa,K. (1995) Atomic model of a pyrimidine

dimer excision repair enzyme complexed with a DNA substrate:

structural basis for damaged DNA recognition. Cell, 83, 773–782.

Vermeulen,W. et al. (1994) Three unusual repair deﬁciencies associated

with transcription factor BTF2 (TFIIH). Evidence for the existence of

a transcription syndrome. Cold Spring Harbor Symp. Quant. Biol.,

59, 317–329.

Zamble,D.B. and Lippard,S.J. (1995) Cisplatin and DNA repair in cancer

chemotherapy. Trends Biochem. Sci., 20, 435–439.

Received on July 23, 1997; revised on September 19, 1997

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