Mutation Research 362 (1996) 105-I 17

DNA Repair

Isolation of a cDNA encoding a UV-damaged DNA binding factor defective in xeroderma pigmentosum group E cells

Byung Joon Hwang a, Joseph C. Liao a, Gilbert Chu a-b,*

” Department of Medicine. Stanford lJniL,ersig Medical Center, Stanford, CA 94305. USA b Department of Biochemistry, Stanford CJniwrsi& Medical Center, Stanford. CA 94305, USA

Received 24 March 1995: revised 3 August 1995; accepted 7 August 1995

Abstract

XPE binding factor (XPE-BF) is deficient in a subset of patients from xeroderma pigmentosum complementation group

E. Binding activity copurifies with a 125 kDa polypeptide (~125) that binds to DNA damaged by ultraviolet (UV) radiation

and many other agents. We isolated cDNA encoding a polypeptide with a predicted amino acid sequence that matched the sequences of eleven tryptic peptides derived from digestion of XPE-BF purified from human placenta. In vitro transcription and translation of the cDNA yielded a polypeptide of 125 kDa that bound specifically to UV-damaged DNA. Therefore the

cDNA encodes either all or the major component of XPE-BF.

Keywords: DNA repair: Xeroderma pigmentosum; Ultraviolet radiation-induced DNA damage

1. Introduction son, 1992; Scherly et al., 1993) and some of the

Xeroderma pigmentosum (XP) is an autosomal biochemical functions (Hoeijmakers, 1993: Tanaka

recessive disease characterized by extreme skin sen- and Wood, 1994) and physical interactions (Li et al.,

sitivity to ultraviolet radiation, pigmentation abnor- 1994; Park and Sancar, 1994) of these proteins have

malities, and predisposition to skin cancer (Cleaver been characterized.

and Kraemer, 1989). XP cells are defective in nu- Cells from a subset of patients in complementa-

cleotide excision repair. Somatic cell fusion experi- tion group E are deficient in XPE binding factor

ments have defined seven genetic complementation (XPE-BF), a factor that binds to damaged DNA (Chu

groups, A through G, suggesting that at least seven and Chang, 1988). XPE-BF binds to a broad spec-

proteins are involved in the human nucleotide exci- trum of DNA lesions with a range of different

sion repair pathway. XP genes for groups A, B, C, D affinities. It showed high affinity for 6-4 photo-

and G have been cloned (Tanaka et al., 1990; Weber products (Treiber et al., 1992) and relatively lower

et al., 1990; Weeda et al., 1990; Legerski and Peter- affinities for a subset of cyclobutane pyrimidine dimers (Hwang and Chu, 1993; Keeney et al., 1993; Reardon et al., 1993). single-stranded DNA, apurinic

* Corresponding author. Tel.: (415) 725-6442: Fax: (415) 725- sites, and DNA damaged by the anticancer drugs

1420. cis-DDP (cis-diamminedichloroplatinum(I1)) and ni-

0921-8777/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved

SSDI 09’1.8777(95)00040-2

trogen mustard (Payne and Chu, 1994). However,

XPE-BF does not bind to T[cis,syn]T cyclobutane

pyrimidine dimers (Hwang and Chu, 1993; Keeney

et al., 1993). trans-DDP, 4-nitroquinoline-N-oxide,

8-methoxypsoralen. or enzymatically methylated cy-

tosine and adenine (Payne and Chu. 1994). Thus

XPE-BF is a versatile damage recognition protein,

but other proteins must exist for the recognition of

the lesions not bound by XPE-BF.

Several lines of evidence support a role for XPE-

BF in the nucleotide excision repair pathway. First,

human cells selected for resistance to cisplatin ex-

press higher levels of XPE-BF. cross-resistance to

UV, and enhanced DNA repair (Chu and Chang. 1990). Second, when purified XPE-BF protein is

microinjected into XP-E cells that lack binding activ-

ity, nucleotide excision repair is restored to normal

levels (Keeney et al., 1994).

XP group E cells with undetectable XPE-BF bind-

ing activity are only mildly sensitive to UV radiation

(Cleaver and Kraemer, 1989). Nucleotide excision

repair of a UV-damaged DNA substrate can be reconstituted to 50% of maximum levels by combin-

ing purified protein fractions while omitting XPE-BF

(Aboussekhra et al.. 1995). Thus, XPE-BF does not

appear to be essential for the repair of UV-damaged DNA. The importance of its role in the repair of

other forms of DNA damage remains to be deter-

mined. We previously reported the purification of XPE-

BF to near homogeneity (Hwang and Chu, 1993).

About 90% of the purified protein was a 125 kDa

polypeptide (p 125). The remaining 10% of the puri-

fied protein was a 93 kDa polypeptide (~93). which was quantitatively insufficient to account for the

binding activity observed. We have now obtained amino acid sequences from tryptic peptides of both

polypeptides, demonstrating that p93 is proteolyti- tally derived from ~125. Furthermore, a cDNA en- coding an open reading frame containing the se- quenced amino acids was isolated. Transcription and translation of pl25 cDNA in vitro produced a 125 kDa polypeptide capable of binding specifically to

UV-damaged DNA. A UV-damaged DNA binding (UV-DDB) protein

has been purified from monkey CV-I cells and found to consist of a single polypeptide with molecu- lar mass 127 kDa, very similar to that of XPE-BF

(Hirschfeld et al., 1990; Abramic et al.. 1991). How- ever. a number of properties were thought to be

different. For example, UV-DDB protein was re-

ported to have a much more modest affinity for

UV-damaged DNA (Hirschfeld et al.. 1990) than

found for XPE-BF (Hwang and Chu. 1993). Here,

we show that the two proteins are highly homolo-

gous and that the human and monkey proteins both

have a high affinity for UV-damaged DNA.

2. Materials and methods

The following buffers were used: buffer A (I2

mM HEPES, pH 7.9, 60 mM KCI, 5 mM MgCl,, 4 mM Tris-HCI. 0.6 mM EDTA, I mM DTT. 12%

(v/v) glycerol): buffer B (50 mM Tris-HCI, pH 8.5,

380 mM glycine. 2 mM EDTA): buffer C (IO mM

HEPES, pH 7.9, 2 mM EDTA. 2 mM DTT, 10%

(v/v) glycerol, 0.01 o/r (v/v) NP-40).

2.2. Protein sequerlciilg

The elution-digestion-sequencing method (Hwang

et al., 1995) was used to obtain internal amino acid sequences from purified proteins. Briefly, highly pu-

rified protein fractions with XPE-BF binding activity

were resolved by SDS-PAGE. The gel was stained with Coomassie blue. and bands copurifying with the

binding activity were cut out of the gel. The gel slices were treated with Lys-C (which cleaves at Lys

residues) to facilitate the elution of large poly- peptides and each polypeptide was eluted from the gel by passive diffusion in buffer containing 20 mM

Tris-HCI. pH 8.0, and 0.04% SDS. The SDS was

removed from the eluate by extraction with heptane and isoamyl alcohol. The polypeptide was then di- gested to completion with trypsin (which cleaves at Lys and Arg residues), and the tryptic peptides were fractionated by HPLC and sequenced by Edman

degradation.

The cDNA encoding pl25 was obtained by screening a Lambda zap I1 human cDNA library

B. J. Hwnng er al. /Mutation Research 362 (1996) 105- 1 I7 107

from the Jurkat T-cell leukemia cell line. Screening

was performed by plating approximately IO6 phage from the library onto E. coli strain XL-1 Blue on 150 mm diameter dishes and incubating at 37°C.

After the plaques reached 1 mm in size, phage DNA

was transferred onto a solid support by overlaying the plaques with duplicate Hybond-N filters

(Amersham, Arlington Heights, IL). Hybridization

and washing steps were performed under stringent

conditions (Sambrook et al., 1989). Twenty plaques were randomly picked out of about 150 positive

plaques from the first screening, and purified to

homogeneity. The pBluescript (SK-) plasmids were excised from the Lambda zap II DNA according to

the manufacturer’s protocol (Stratagene. La Jolla,

CA).

2.4. III l,itro trunscription und tmrkslation

Labeled protein was synthesized by transcribing

the pl25 cDNA from a T7 promoter with T7 RNA

polymerase and then translating the mRNA in a

rabbit reticulocyte lysate in the presence of

[ ‘5S]methionine according to the manufacturer’s in- structions (Promega. Madison, WI).

2.5. Electrophoretic mobili sh# assay of labeled protein

Protein-DNA complexes were allowed to form in

IO ~1 of buffer A containing 1 ng of DNA probe and 0.4 pg of pRSVcat plasmid DNA to mask the effects

of nonspecific DNA binding proteins. The in vitro

translated 35S-labeled protein was added last. The reaction mixture was incubated at room temperature

for 30 min and then resolved by non-denaturing gel

electrophoresis through 5% polyacrylamide in buffer B. The gel was fixed in 50% methanol and 10%

acetic acid for 2 h. soaked in Amplify (Amersham, Arlington Heights, IL) for 30 min, and dried onto

Whatman 3MM paper. The sticky surface of the dried gel was treated with baby powder, which al- lowed direct contact of the gel with X-ray film, which was then exposed at - 80°C. The cassette was completely dried before developing the X-ray film by putting the cassette in 37°C incubator with the door open.

2.6. Electrophoretic mobility shift assay with labeled DNA probe

The UV-irradiated DNA probe f148 and crude

extracts from HeLa and CV-1 cells were prepared as

described previously (Hwang and Chu, 1993). Bind- ing activity to the UV-irradiated f148 probe was

measured by an electrophoretic mobility shift assay

as described previously (Hwang and Chu. 1993).

2.7. UV-damaged DNA cellulose of/K@ chromatog- raphy

UV-damaged DNA cellulose was prepared as de-

scribed previously (Hwang and Chu, 1993). A mini-

column was made by packing 10 yl of UV-damaged

DNA cellulose in a 200 ~1 Pipetman tip and equili-

brated with buffer C. In vitro translated j5S-labeled ~125 polypeptide was loaded onto the column as 50

~1 of the reticulocyte extract. The column was

washed extensively with 600 ~1 of buffer C contain- ing 0.2 M NaCl, and eluted with 50 ~1 of buffer C

containing I .O M NaCl. Fractions were stored at

- 80°C.

2.8. SDS-PAGE

Denatured proteins were resolved by SDS-PAGE

gel with 7.5% polyacrylamide by the method of

Laemmli (Laemmli, 1970). After the electrophoresis, proteins were stained with Coomassie blue. To visu-

alize 35S-labeled protein, the gel was exposed to X-ray film as described above.

3. Results

3.1. Cloning of p125 cDNA of XPE-BF

Human XPE-BF was purified as described previ-

ously (Hwang and Chu, 1993). The most highly

purified fractions contained a predominant 125 kDa polypeptide (p 125) and a minor 93 kDa polypeptide (~93). Table 1 lists the amino acid sequences ob- tained from tryptic peptides generated from 20 pmol of p 125 and 10 pmol of ~93. Eight peptide sequences from pi25 were identical to the amino acid sequence

108 B.J. Hwang rt nl./Mututior~ Research 362 f1996) 105-117

predicted by a cDNA cloned from a monkey protein that binds to UV-damaged DNA, UV-DDB protein

(Takao et al., 1993). A ninth peptide sequence from

~125 was different at a single amino acid from both

a different p 125 peptide and the corresponding pep-

tide beginning at position 823 in UV-DDB. This

ninth peptide sequence could be due to either an

error in amino acid sequencing or to a polymorphism

in the purified protein, which was originally obtained

from ten pooled placentas.

Two peptide sequences from p93 were also identi-

cal to the amino acid sequence predicted by the UV-DDB cDNA. Furthermore, these two peptide

sequences and one of the peptide sequences from

p 125 (corresponding to positions 5 14, 847 and 108 1)

were identical to sequences obtained directly from

Table I Homology between human XPE-BF and monkey UV-DDB proteins

purified UV-DDB protein. The 10 identical peptide

sequences span 96 (or 8.4%) of the 1140 amino acids

in the ORF predicted by the UV-DDB cDNA. These results together with the precise concordance in

molecular weight indicate that ~125 is the human

homolog of the monkey UV-DDB protein.

The predicted amino acid sequence for UV-DDB

cDNA was nearly identical to a partial cDNA ob-

tained by random cDNA cloning from human brain mRNA (Takao et al., 1993). To isolate the full

length human cDNA, a 425 bp DNA fragment corre-

sponding to the 3’ untranslated region of the human cDNA was amplified by PCR from genomic DNA

obtained from HeLa cells. The 425 bp DNA frag-

ment was used as a probe to screen a human cDNA library from Jurkat cells. Approximately IO6 phage

Position Amino Acid Sequence Species

191 TYEVSLR Human ~125 [K] _______ Monkey UV-DDB

219 ? ?MLLLE Human ~125 [R]LF----- Monkey UV-DDB

514 ALYYLQIHPQELR Human ~125 [K]-----__-_____ Monkey UV-DDB*

823 ??NTYFIVGPAMVYPE Human ~125 [K]DP_--__-_T---_--EAEPK Monkey UV-DDB

823 DPNTYFIVGTAMVYPEEAEP Human ~125 [K]-----__-_----_---_-_K Monkey UV-DDB

847 IVVFQYS Human p93 [R] -------DGK Monkey UV-DDB*

867 ?AVYSMVEF Human ~125 [K]G--------NGK Monkey UV-DDB

1082 ?EPATGFIDG Human p93 [KIT---------DLIESFLDISR Monkey UV-DDB*

1104 MQEWANLQYDDG Human ~125 [K]-_____--_____SGm Monkey UV-DDB

1121 REATADD Human ~125 [K] -------LIK Monkey UV-DDB

1132 ?VEELTRI Human ~125 [K]V----___H Monkey UV-DDB

* . indicates sequences also obtained by direct amino acid sequencing.

[ ] indicates that in the deduced monkey sequence, the amino acid just before the first amino acid in the human ~125 peptide was always a

lysine [K] or arginine [R], as expected from trypsin digestion. Internal arginines were found in the peptides starting at amino acid positions

I 121 and 1132. showing that in those cases the trypsin digestion was incomplete. The human peptide starting at position 823 was found in

two different HPLC peaks and contained either threonine or proline at position 832, perhaps representing a polymorphism in human ~125. since the protein was purified from 10 pooled placentas, For every remaining peptide there was exact conservation between the human and

monkey sequences, as indicated by the dashes.

B.J. Hwang et al./Mutation Resrurch 362 (19961 105-117 109

were screened, and a cDNA of 4.2 kbp (~125 cDNA)

was obtained. All of the 11 peptide sequences ob-

tained from the ~125 and p93 proteins were con- tained in the predicted amino acid sequence from the ~12.5 cDNA. This shows that the cDNA encodes

~125 and that p93 is a protein degradation product of

p125.

3.2. Sequence analysis

The p 125 cDNA clone was 422 1 nucleotides long.

The first ATG in the cDNA was at nucleotide 110

and conferred an open reading frame encoding a

polypeptide of 1140 residues (Fig. 1). Using the

BLAST database search program (Altschul et al.,

19901, a near complete homology with the monkey UV-DDB (GenBank accession number L202 16)

(Takao et al., 1993) was found. The two sequences

are identical in length, with 98.4% identity in the ORF starting from the presumed initiation codon at

nucleotide 110 of the cDNA clone (54 differences

out of 3420 bp) and a 99.6% identity in amino acid

sequence (5 differences out of 1140 residues). The

five amino acid differences were scattered and re-

1 MSYNYWTAQ KPTAVNGCVT GHFTSAEDLN LLIAKNTRLE IYVVTAEGLR

51 PVKEVGMYGK IAVMELFRPK GESKDLLFIL TAKYNACILE YKQSGESIDI

101 ITRAHGNVQD RIGRPSETGI IGIIDPECRM IGLRLYDGLF KVIPLDRDNK

151 ELKAFNIRLE ELHVIDVKFL YGCQAPTICF VYQDPQGRHV KTYEVSLREK

201 EFNKGPWKQE NVEAEASMVI AVPEPFGGAI IIGQESITYH NGDKYLAIAP

251 PIIKQSTIVC HNRVDPNGSR YLLGDMEGRL FMLLLEKEEQ MDGTVTLKDL

301 RVELLGETSI AECLTYLDNG VVFVGSRLGD SQLVKLNVDS NEQGSYWAM

351 ETFTNLGPIV DMCWDLERQ GQGQLVTCSG AFKEGSLRII RNGIGIHEHA

401 SIDLPGIKGL WPLRSDPNRE TYDTLVLSFV GQTRVLMLNG EEVEETELMG

451 FVDWQTFFC GNVAHQQLIQ ITSASVRLVS QEPKALVSEW KEPQAKNISV

501 ASCNSSQVVV AVGRALYYLO IHPOET,RQIS HTEMEHEVAC LDITPLGDSN

551 GLSPLCAIGL WTDISARILK LPSFELLHKE MLGGEIIPRS ILMTTFESSH

601 YLLCALGDGA LFYFGLNIET GLLSDRKKVT LGTQPTVLRT FRSLSTTNVF

651 ACSDRPTVIY SSNHKLVFSN VNLKEVNYMC PLNSDGYPDS LALANNSTLT

701 IGTIDEIQKL HIRTVPLYES PRKICYQEVS QCFGVLSSRI EVQDTSGGTT

751 ALRPSASTQA LSSSVSSSKL FSSSTAPHET SFGEEVEVHN LLIIDQHTFE

801 VLHAHQFLQN EYALSLVSCK LGKDPNTYFI VGTAMVYPEE AEPKQGRm

851 WDGKLQT VAEKEVKGAV YSMVEFNGKL LASINSTVRL YEWTTEKDVR

901 TECNHYNNIM ALYLKTKGDF ILVGDLMRSV LLLAYKPMEG NFEEIARDFN

951 PNWMSAVEIL DDDNFLGAEN AFNLFVCQKD SAATTDEERQ HLQEVGLFHL

1001 GEFVNVFCHG SLVMQNLGET STPTQGSVLF GTVNGMIGLV TSLSESWYNL

1051 LLDMQNRLNK VIKSVGKIEH SFWRSFHTER KTEPATGFID GDLIESFLDI

1101 SRPKMQEWA NLQYDDGSGM KREATADDLI KWEELTRIH

Fig. 1. Primary structure of ~125. Translation of the longest open reading frame of the ~125 cDNA starting at the first AUG codon is shown.

Sequences identified by direct peptide sequencing of ~125 and p93 proteins are underlined. The nucleotide sequence has been deposited with GenBanks accession number U32986.

I IO B.J. Hwatl!: et al./Mutation Research 362 II9961 105-117

sulted from single base pair changes at the DNA level.

The BLAST search also identified other proteins with significant homology to the C-terminal region

of ~125, as previously reported for UV-DDB (Takao

et al., 1993), including an uncharacterized protein from the slime mold Dic~ostelium discoideum (Sydow et al., 19921, an uncharacterized protein

from rice Oryza satica (Sasaki and Minobe, 1993),

and a POU domain protein from the zebra fish Brachyodanio rerio (Johansen et al., 1993). The

region of homology in the POU domain protein was

just upstream and did not overlap the POU domain

motif. A search for conserved structural motifs using

the Prosite search program (IntelliGenetics, Moun-

tain View, CA) was unrevealing. XPE-BF binds to a broad spectrum of DNA le-

sions. including those induced by UV radiation, de-

naturation, depurination, cisplatin and nitrogen mus- tard (Chu and Chang, 1988; Payne and Chu, 1994).

We previously reported that the photoreactivating

enzyme. photolyase from the yeast Saccharomyces cereuisiae, also binds to the same broad spectrum of

DNA lesions with similar relative affinities (Patter-

son and Chu, 1989; Fox et al., 1994). To see if this remarkable concordance in binding activities might

be reflected in protein structure, we compared the amino acid sequences of XPE-BF and S. cerer+siae photolyase. No regions of significant homology were

found. Recently, goldfish (Yasuhira and Yasui, 1992)

and marsupial (Kate et al., 1994) photolyases were cloned and found to be significantly homologous to

each other but not to the photolyases from S. cere- Lisiae. E. coli, or other microorganisms. We found no significant homology between XPE-BF and the

goldfish or marsupial photolyases. The BLITZ program (Sturrock and Collins, 1993)

uses the Smith and Waterman best local similarity algorithm to search for local sequence homologies

(Smith and Waterman, 1981). BLITZ found a signif-

icant region of homology between the N-terminal

regions of XPE-BF (amino acid 209-249) and an-

other DNA binding protein, the galactose operon repressor GalR from E. co/i (amino acids 56-95).

Comparison between these two regions revealed 35% identity and 70% similarity (Fig. 2). The number of

results from a search of the data base that by chance

would have this degree of homology had an expected value of 7.79 X 10ph. (Expected values below 0.05

are considered to be significant.)

3.3. 111 l+tro transcription arid translution of ~12.5

cDNA yields a 125 kDa polypeptide w!hich biuds specifically to W-damaged DNA

The p 125 cDNA was transcribed in vitro from the

T7 promoter and then translated with rabbit reticu-

locyte lysate in the presence of [“5S]methionine. A 125 kDa polypeptide was specifically synthesized

from ~125 cDNA but not from the control vector

(Fig. 3, lanes I and 2). Polypeptides smaller than

125 kDa were also present, representing either degra- dation of the ~125 kDa polypeptide or mis-initiated

translation of the ~125 message. Of note, the first ATG was contained in the sequence TAGACATGT,

which represents a relatively poor match to the con-

sensus Kozak sequence CCACCATGG (Kozak,

1986). To see if the translated ~125 was capable of

binding to damaged DNA, the ‘5S-labeled ~125 pro-

tein (L) was loaded onto a UV-damaged DNA cellu-

lose column (Fig. 3, lanes 3-8). Most of the labeled protein eluted from the column with the flow-through

(F) and the first wash (W,). The second and third washes (Wz and W,) yielded no detectable protein, demonstrating that all the unbound protein had been

successfully washed from the column. A small por- tion of the in vitro translated ~125 protein was eluted

(E) from the column with 1M NaCl (Fig. 3, lane 8).

GalR 56 QQTTETVGLWGDVSDPFFGAMVKAVEQVAYHTGN-FLLIG 95 I: I:::1 I::11 II::: 1 ::I[ (::I I:

XPE-BF 209 QENVEAEASMVIAVPEPFGGAIIIGQESITYHNGDKYLAIA 249

Fig. 2. Sequence homology between XPE-BF and the galactose operon repressor. Using BLITZ (Sturrock and Collins, 1993) to search for

local similarity, a region of homology between XPE-BF (amino acid 209-249) and the galactose operon repressor (GalR) from E. cdi

(amino acid 56-95) was identified. Notations: identity (1); conserved changes (:); gap c-j.

When the “S-labeled ~125 protein was tested for

its ability to bind to damaged DNA by the elec- trophoretic mobility shift assay, no specific complex with a UV-damaged DNA fragment was observed in

the loaded extract CL), the flow-through (F), or the wash (W, ) (Fig. 4A, lanes l-3 and 5-7). However.

after incubation of the high salt eluate (E) with the

DNA fragment, the electrophoretic mobility of ~125

protein was shifted to a new position Bl on the gel

(Fig. 4A, lane 4). To see if the DNA probe was

shifted to the same position, parallel incubations

were done of the loaded extract CL) and each of the column fractions (F, W, and E) with the same UV-

damaged DNA fragment, now labeled with “P (Fig. 4A. lanes 5-S). There was a large and specific

increase of radioactivity in band Bl when the DNA

probe was labeled with “P (compare lanes 4 and 8). By contrast, the levels of radioactivity from the

unbound p 125 protein (band P) were independent of

whether the DNA probe was labeled (compare lanes

l-4 to lanes S-8). Furthermore. band B 1 is located

at a position very near, if not identical, to the

mobility shift of the DNA fragment when the labeled fragment was incubated with a human cellular ex- tract (data not shown). Taken together. these data

demonstrate the formation of a protein-DNA com-

plex between the labeled ~125 protein and UV-

damaged DNA.

The binding specificity of the eluted ~125 protein (El was tested by comparing the effects of incubat-

ing intact or UV-damaged probe DNA with the

j5S-labeled protein. The intact DNA failed to pro-

duce a shift in electrophoretic mobility of the labeled

protein (Fig. 4B. lanes 1 and 2), while UV-damaged

probe DNA shifted the mobility to Bl (Fig. 4B, lane

3). Some of the labeled ~125 protein in fraction E migrated at the top of the gel (Fig. 4B, lanes 1 and 2;

Fig. 4A. lanes 4 and 8) perhaps because small amounts of UV-damaged high molecular weight

DNA was eluted from the column along with the

protein. The specificity for damaged DNA was tested

further by investigating the effect of competition for

Expression of ~125 cDNA in vitro yields a 125 kDa polypeptide, some of which binds to UV-DNA cellulose

5 Z. $2 5%

UV-DNA cellulose fractions

BB L F W, W, W, E M (ma)

1 2 345678

Fig. 3. Expression of ~125 cDNA in vitro yields a 125 kDa polypeptide. some of which binds to UV-DNA cellulose. Protein was

synthesized in vitro by transcribing I pg of pcDNA3(pl25) (lanes I. 3-S) or pcDNA3 (lane 2) with the T7 promoter and translating the

message in the presence of [35S]methionine in a rabbit reticulocyte lysate as described in Materials and methods. The in vitro labeled

products were resolved by SDS-PAGE in a 7.5% gel, Coomassie stained. and then fluorographed. The ‘SS-labeled proteins from the

translation of pcDNA3(p125) were then loaded onto a UV-DNA cellulose column. The loaded proteins (L). flow-through (F), first wash

(W,). second wash (Wz), third wash (W,), and I M NaCl elution (E) were collected and 10% of each fraction was then resolved by

SDS-PAGE (lanes 3-8). Coomassie stained. and fluorographed.

B.J. Hwang et al./Mutation Research 362 (10961 105-117

The ~125 fraction eluted from UV-DNA cellulose binds specifically to UV damaged DNA

A B Probe UV-f148 32P-UV-f148 PmtleuV o-+++

Fraction mm CompetitoruV 0 0 0 - +

rb Ir ’ &&A#‘&

Bl- P- -W y” . . J’! B;-, y w -“.rr -

F-

123456789 12345

Fig. 4. The ~125 fraction eluted from UV-DNA cellulose binds specifically to UV damaged DNA. (A) ~125 fractions after UV-DNA

cellulose chromatography assayed for binding to a UV-damaged f148 probe. Protein was translated from pcDNAXp125) and the loaded

protein CL), flow-through (F), first wash (W, ), and eluate (E) (see Fig. 3) were incubated with either unlabeled (lanes I-4) or “P-labeled

DNA probe (lanes 5-8) and resolved by electrophoresis in a nondenaturing 5% polyacrylamide gel. The fraction marked 0 (lane 9)

represents the result of a binding reaction omitting any protein. The electrophoretic migration is marked for free f148 DNA probe (F).

unbound “S-labeled ~125 protein (P), and a complex between the DNA probe and ~125 protein (Bl). (B) Binding specificity of ~125

fraction E. The fraction of ~125 protein eluted from UV-DNA cellulose (E) was assayed in a binding reaction with 1 ng of unlabeled f148

probe. Probe DNA was omitted (lane l), added as undamaged DNA (lane 2), or added as UV-damaged DNA (lanes 3-5). The binding

reactions contained 400 ng of undamaged competitor plasmid DNA. An additional 50 ng of unlabeled competitor plasmid DNA was either

omitted (lanes I-3). added as undamaged DNA (lane 4). or added as UV-damaged DNA (lane 5).

the labeled protein between the UV-damaged probe

DNA (a 148 bp DNA fragment) and a 50-fold excess

of either intact or UV-damaged plasmid DNA (a 5027 bp supercoiled DNA circle). The protein-DNA

complex in Bl was unaffected by undamaged plas- mid DNA but was competed away by the UV-

damaged plasmid DNA (Fig. 4B, lanes 4 and 5).

Thus in vitro translated ~125 protein bound specifi- cally to UV-damaged DNA.

3.4. Human XPE-BF and monkey UV-DDB proteins

haue the same specificity for W-damaged DNA

The amino acid sequences obtained from purified human XPE-BF demonstrate that XPE-BF is the human homolog of the monkey UV-DDB protein.

The predicted amino acid sequences from the human and monkey cDNAs were 99.6% identical and both

purified proteins migrated with the same apparent molecular weight on SDS-PAGE (Abramic et al., 1991; Hwang and Chu, 1993). However, other pub- lished properties of UV-DDB differ significantly

from those reported for XPE-BF. First, the reported

native molecular weight of UV-DDB suggested that

it exists as a homodimer in solution (Abramic et al., 199 1 ), while XPE-BF exists primarily as a monomer

(Hwang and Chu, 1993). Second, UV-DDB was reported to bind to 6-4 pyrimidine dimers but not to

cyclobutane pyrimidine dimers (Hirschfeld et al., 19901, while XPE-BF binds to both 6-4 (Treiber et

al., 1992) and some cyclobutane pyrimidine dimers (Hwang and Chu, 1993; Keeney et al., 1993; Rear-

don et al., 1993). Third, the reported specificity of UV-DDB for UV-damaged DNA was lOO-fold less

than observed for XPE-BF.

To address these differences, we compared the UV-damaged DNA binding activities in monkey and human extracts. First, the reported difference in na- tive molecular weights was not supported by the data. The native molecular weight of UV-DDB was based on gel filtration data and the assumption of a globular shape for UV-DDB. XPE-BF migrated in the same position on gel filtration as UV-DDB rela- tive to the same molecular mass standards. However,

B.J. Hwnng et al./Mutation Research 362 (1996) 105-117 II3

the estimate of its native molecular mass of 134 kDa

was based on data from both gel filtration and

sedimentation, and was therefore independent of any a priori assumption of the shape of the protein

(Hwang and Chu, 1993).

Second, the reported failure of UV-DDB to bind

to cyclobutane dimers was based on the absence of

any change in binding activity when the UV-irradia-

ted DNA probe was treated with photoreactivating enzyme to remove the cyclobutane pyrimidine dimers. These experiments used unpurified or only

partially purified protein from CVI monkey cell

extracts and irradiated the DNA probe with a single,

relatively high UV dose (Hirschfeld et al., 1990;

Human XPE-BF and monkey W-DDB proteins have the same specificity for W-damaged DNA

Competitor ds W-d.5 w-ss I In*-

88 08 08 08 ng ooo~- -cI~-~cIcIcI-- Probeuv + -+++++++++++++

Protein - + + + + + + + + +-+ + + + +

HeLa

BZ-

Bl- YUUY 111

cv-1

B2- u

Bl- ruwr urr

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Fig. 5. Specificity of XPE-BF and UV-DDB proteins for damaged DNA. Crude extracts (0.3 pg) from HeLa and CV-I cells (upper and

lower panels, respectively) were incubated with labeled f148 probe DNA (0.2 ng) in the presence of different amounts of unlabeled

competitor DNA, pUC18 linearized by digestion at a single site by Hind III. The probe DNA was UV treated (lanes I and 3-15) or

untreated (lane 2). Protein extract was omitted (lane 1) or added (lanes 2-14). Control binding reactions omitted pUC18 competitor DNA

(lanes I-3). Competition was carried out with intact double-stranded DNA (ds) (lanes 4-6), UV-irradiated double-stranded DNA (UV-ds)

(lanes 7-9). single-stranded DNA (ss) (lanes IO- 12), or UV-irradiated single-stranded DNA (UV-ss) (lanes 13- 15). Single-stranded DNA

was prepared by heating at 95°C for 5 min followed by rapid cooling.

Abramic et al., 199 1). By contrast, the experiments

with XPE-BF were done with highly purified protein

and a range of UV doses, which demonstrated that a

change in binding activity after photoreactivation of

the DNA probe was best seen at intermediate UV doses and less well seen when the dose was either

too high or too low (Hwang and Chu. 1993). We

made crude extracts from human and monkey cells

(HeLa and CVI 1 and assayed both extracts for changes in binding activity after photoreactivation of

the DNA probe. In both cases, a small change in binding activity was seen at intermediate UV doses

(data not shown).

Finally, the specificities of UV-DDB and XPE-BF for UV-damaged DNA were compared to each other

in an electrophoretic mobility shift assay. Extracts

from monkey CVI and human HeLa cells showed the same high specificity for UV-damaged single or

double-stranded DNA: 1 ng of UV-damaged com-

petitor DNA inhibited more than 80% of the binding but 1000 ng of intact DNA did not inhibit the

binding at all (Fig. 5). Thus, UV-DDB had the same

relative specificity for UV-damaged DNA as XPE- BF.

4. Discussion

4.1. cDNA encoding XPE-BF

Several pieces of evidence indicate that we have

isolated a cDNA that encodes the 125 kDa polypep-

tide (~125) of the UV-damaged DNA binding pro- tein, xeroderma pigmentosum group E binding factor

(XPE-BF). First, the open reading frame of the cDNA encodes a polypeptide of an appropriate molecular

weight. Second, the predicted amino acid sequence is matched by the sequence of IO different tryptic

peptides from purified XPE-BF. Third, expression of the cDNA in vitro produced a polypeptide that was capable of binding specifically to UV-damaged DNA.

Does the binding of in vitro synthesized ~125 to UV-damaged DNA imply that ~125 is the sole com- ponent of XPE-BF? The native molecular mass of XPE-BF estimated from its Stokes radius and sedi- mentation coefficient is 134 kDa + 20 kDa (Hwang and Chu. 1993). The uncertainty in this determina-

tion is large enough to be consistent with the possi-

bilities that XPE-BF is either a monomer of ~125 or

a heterodimer of ~125 and a second low molecular

weight protein. Since the rabbit reticulocyte extract

in which p 125 was expressed contained endogenous binding activity for XPE-BF, any cofactor necessary

for conferring binding activity to ~125 could have

been supplied by the rabbit reticulocyte extract.

Therefore, we cannot rule out the possibility that XPE-BF contains factors in addition to ~125. How-

ever, the data do demonstrate that we have isolated the cDNA encoding ~125. which is at least the major

component of XPE-BF.

Keeney et al. ( 1993) reported the copurification of

XPE-BF from HeLa extracts with two polypeptides of 124 kDa and 41 kDa. However, fractions contain-

ing only the 124 kDa polypeptide contained binding activity. Thus, the 41 kDa polypeptide may associate

with the larger subunit, but is not needed for binding

activity. Assendelft et al. also reported the purifica- tion of a UV-damaged DNA binding protein of 125

kDa from HeLa extracts that appears to be identical

to XPE-BF (Assendelft et al., 1993). When partially purified protein was resolved by SDS-PAGE, trans- ferred to nitrocellulose. renatured, and probed with a

UV-damaged oligonucleotide, binding activity was seen only in a band at 125 kDa. This evidence

supports the likelihood that XPE-BF binds to DNA

as a monomer of ~125. Only a small portion of ~125 expressed in vitro

was active for binding activity. This may have been

due to a number of possible deficiencies in the reticulocyte extract. including improper folding, lim-

iting amounts of a cofactor necessary for binding, limiting amounts of enzymes for post-translational modification, or improper initiation of translation

from the DNA substrate. Additional studies will be

required to investigate these possibilities. Even though p 125 alone may be sufficient for

binding activity, additional proteins appear to inter- act with it. presumably as part of the nucleotide excision repair pathway. For example, the 41 kDa protein that copurified with ~125 altered the DNA footprint on a substrate containing a single 6-4 TT dimer (Reardon et al., 1993). Indeed, the finding that some XP group E cells contain intact binding activ- ity (Kataoka and Fujiwara, 1991; Keeney et al., 1992) might be explained if those cells contain ~125

protein with normal binding activity but with mis-

sense mutations in a region of ~125 that interacts

with other nucleotide excision repair proteins.

4.2. Human XPE-BF and monkey (IV-DDB

Amino acid analysis of peptides generated from purified human ~125 revealed that it is the human

homolog of a monkey protein of the same molecular

weight, UV-DDB (Takao et al., 1993). The predicted

amino acid sequences of the human and monkey

proteins were 99.6% identical. This result appeared to be inconsistent with differences in the properties

of XPE-BF and UV-DDB previously reported. In

particular, there were reported differences in the native molecular weight, binding activity for cy-

clobutane pyrimidine dimers, and relative affinity for UV-damaged over undamaged DNA. However, it

now appears that these differences were due to dif- ferences in the assays or in the interpretation of

similar experimental results, rather than to real dif- ferences in the proteins. Most significantly, when

extracts were tested in parallel in the same assay

system with the same DNA probe, we found that the

monkey and human proteins exhibited the same high

specificity for UV-damaged DNA.

4.3. Homology between XPE-BF and galactose re-

pressor

A search for proteins homologous to XPE-BF

revealed a previously unreported but highly signifi-

cant homology to a region near the N-terminus (amino acids 56-95) of the galactose repressor GalR, which belongs to the lactose repressor family of

transcription regulators (Weickert and Adhya, 1992).

Recently, the crystal structure of one of the family

members, the purine repressor, PurR, bound to DNA was solved (Schumacher et al., 1994). The N-termi- nal helix-turn-helix motif (residues 4 to 23) binds to

the major groove of the DNA operator sequence. The helix-turn-helix (helices 1 and 2) together with helix 3 (residues 30 to 43) form a globular DNA binding subdomain that is connected to a corepressor binding domain by a hinge region consisting of helix 4 (residues 48 to 56) followed by four extended

residues. Significantly. the hinge region binds deeply

in the minor groove producing a kink in the DNA of

45 degrees. Since the numbering of amino acids in

PurR and GalR are identical in this region, XPE-BF

is highly homologous to a part of GalR (56 to 95) that includes 5 residues from the hinge region and a part of the corepressor binding domain consisting of

p strand A (61 to 661, helix I (72 to 881, and b

strand p (91 to 96) that forms a structure located

directly above the kink in the DNA.

The binding of XPE-BF to damaged DNA may occur with the DNA in a kinked conformation, since

several XPE-BF substrates, including 6-4 pyrimidine dimers. cyclobutane pyrimidine dimers. and cisplatin

adducts. are known to introduce severe kinks in the

DNA (Payne and Chu, 1994). Furthermore, the bind- ing of XPE-BF to the 6-4 pyrimidine dimer produces

additional kinking in the DNA, indicated by a new

DNAse hypersensitive site in the footprint (Reardon

et al., 1993). Thus, part of the DNA recognition domain of XPE-BF may bind to the minor groove in

damaged DNA at a site in the DNA that is either

already bent or easily deformable by the formation

of the protein-DNA complex.

4.4. Genetic d.efect in XP group E?

Is the gene for XPE-BF defective in XP group E individuals? Takao et al. (Takao et al., 1993) ana-

lyzed three different XP group E cell lines. two of

which lacked XPE-BF binding activity. by probing Northern blots with UV-DDB cDNA. Because of the

high homology between UV-DDB and XPE-BF. the

hybridization signal corresponded to XPE-BF mRNA. which was found to be normal in abundance and size in each of the XP group E individuals.

Thus, elucidation of the molecular defect in XP

group E awaits a more detailed analysis of the

XPE-BF gene.

Acknowledgements

This work was supported by NIH grant CA 44949 and Graham and Jane Nissen. The authors thank Douglas Brutlag, Tod Klingler, Paul Mitsis, Kim Rathmell, Vaughn Smider, and Sara Cheng for valu- able discussions and critically reading the manuscript.

116 B. J. Hwang et nl. /Mutation Research 362 (1996/ 105-I 17

References

Aboussekhra, A., M. Biggerstaff, M.K.K. Shivji, J.A. Vilpo, V.

Moncollin, V.N. Podust, M. Protic, U. Hubscher. J. Egly and

R.D. Wood (199.5) Mammalian DNA nucleotide excision re-

pair reconstituted with purified protein components, Cell. 80,

859-868.

Abramic, M., A. Levine and M. Protic (1991) Purification of an

ultraviolet-inducible, damage-specific DNA-binding protein

from primate cells, J. Biol. Chem., 266, 22493-22500.

Altschul, S.F., W. Gish, W. Miller, E.W. Myers and D.J. Lipman

(1990) Basic local alignment search tool, J. Mol. Biol.. 215.

403-410.

Assendelft, G.B.V., E.M. Rigney and I.D. Hickson (1993) Purifi-

cation of a HeLa nuclear protein that binds selectively to DNA

irradiated with ultra-violet light, Nucleic Acids Res.. 21,

3399-3404.

Chu, G. and E. Chang (19881 Xeroderma pigmentosum group E

cells lack a nuclear factor that binds to damaged DNA,

Science, 242. 564-567.

Chu, G. and E. Chang (1990) Cisplatin-resistant cells express

increased levels of a factor that recognizes damaged DNA,

Proc. Nat]. Acad. Sci. USA, 87, 3324-3327.

Cleaver, J.E. and K.H. Kraemer (1989) Xeroderma pigmentosum,

in: C.R. Striver. A.L. Beaudet, W.S. Sly and D. Valle (Eds.),

The Metabolic Basis of Inherited Disease. McGraw-Hill. New

York, pp. 2949-297 1.

Fox, M., B. Feldman and G. Chu (19941 A novel role for DNA

photolyase: binding to drug-induced DNA damage is associ-

ated with enhanced cytotoxicity in yeast, Mol. Cell. Biol.. 14.

8071-8077.

Hirschfeld, S., A. Levine, K. Ozato and M. Protic (1990) A

constitutive damage-specific DNA-binding protein is synthe-

sized at higher levels in UV-irradiated primate cells, Mol.

Cell. Biol.. 10. 2041-2048.

Hoeijmakers, J. (1993) Nucleotide excision repair II: from yeast to

mammals. Trends Genet., 9. 21 I-217.

Hwang, B.J. and G. Chu (19931 Purification and characterization

of a protein that binds to damaged DNA, Biochemistry, 32,

1657-1666.

Hwang, B.J., A. Smith and G. Chu (19951 Internal sequence

analysis of proteins eluted from polyacrylamide gels, Submit-

ted.

Johansen. T., U. Moens, T. Holm, A. Fjose and S. Krauss (19931

Zebrafish pou[c]: a divergent POU family gene ubiquitously

expressed during embryogenesis, Nucleic Acids Res.. 21,

475-483.

Kataoka, H. and Y. Fujiwara (1991) UV damage-specific DNA-

binding protein in xeroderma pigmentosum complementation

group E, Biochem. Biophys. Res. Commun., 175. 1139-l 143.

Kato. T., T. Todo, H. Ayaki, K. Ishizaki, T. Morita, S. Mitra and

M. Ikenaga (19941 Cloning of a marsupial DNA photolyase

gene and the lack of related nucleotide sequences in placental

mammals, Nucleic Acids Res., 22, 4119-4124.

Keeney, S., G.J. Chang and S. Linn (19931 Characterization of

human DNA damage binding protein implicated in xeroderma

pigmentosum E. J. Biol. Chem., 268, 21293-21300.

Keeney, S., A. Eker. T. Brody, W. Vermuelen. D. Bootsma, J.

Hoeijmakers and S. Linn (19941 Correction of the DNA repair

defect in xeroderma pigmentosum group E by injection of a

DNA damage-binding protein, Proc. Natl. Acad. Sci. USA, 9 1,

4053-4056.

Keeney, S., H. Wein and S. Linn (19921 Biochemical heterogene-

ity in xeroderma pigmentosum complementation group E,

Mutation Res., 273. 49-56.

Kozak, M. (19861 Point mutations define a sequence flanking the

AUG initiator codon that modulates translation by eukaryotic

ribosomes, Cell. 44. 283-292.

Laemmli. U. (19701 Cleavage of structural proteins during the

assembly of the head of bacteriophage T4. Nature. 227. 680-

685.

Legerski. R. and C. Peterson (19921 Expression cloning of a

human cDNA repair gene involved in xeroderma pigmento-

sum group C. Nature, 359, 70-73.

Li. L., S. Elledge, C. Peterson, E. Bales and R. Legerski (19941

Specific association between the human DNA repair proteins

XPA and ERCCI. Proc. Nat]. Acad. Sci. USA, 91,5012-5016.

Park, C. and A. Sancar (19941 Formation of a ternary complex by

human XPA. ERCCl and ERCC4(XPFl excision repair pro-

teins, Proc. Natl. Acad. Sci. USA, 91, 5017-5021.

Patterson, M. and G. Chu (19891 Evidence that xeroderma pig-

mentosum cells from complementation group E are deficient

in a homolog of yeast photolyase. Mol. Cell. Biol.. 9. 5105-

5112.

Payne, A. and G. Chu (1994) Xeroderma pigmentosum group E

binding factor recognizes a broad spectrum of DNA damage,

Mutation Rea.. 3 10. 89-102.

Reardon, J.T.. A.F. Nichols, S. Keeney, C.A. Smith. J.S. Taylor,

S. Linn and A. Sancar (I 9931 Comparative analysis of binding

of human damaged DNA-binding protein (XPE) and ,%-

cherichin co/i damage recognition protein (uvrA1 to the major

ultraviolet photoproducts: T[c,s]T, T[t,s]T. T[6-4]T and T[De-

war]T. J. Biol. Chem.. 268, 21301-21308.

Sambrook. J.. E. Fritsch and T. Maniatis (19891 Molecular

Cloning: a Laboratory Manual Cold Spring Harbor Laboratory

Press, Cold Spring Harbor, New York.

Sasaki, T. and Y. Minobe (19931 GenBank accession number

D15449,

Scherly, D.. T. Nouspikel, J. Corlet. C. Ucla. A. Bairoch and S.

Clarkson (1993) Complementation of the DNA repair defect in

xeroderma pigmentosum group G cells by a human cDNA

related to yeast RAD2, Nature, 363, 182-185.

Schumacher. M., K. Choi, H. Zalkin and R. Brennan (19941

Crystal structure of LacI member. PurR. bound to DNA:

minor groove binding by (Y helices, Science, 266, 763-770.

Smith, T.F. and M.S. Waterman (198 1) Identification of common

molecular subsequences, J. Mol. Biol., 147, 195-197.

Sturrock, S.S. and J.F. Collins (19931 MPsrch version 1.3. Bio-

computing research unit, University of Edinburgh. Edinburgh.

Sydow, L., H. Alexander and S.J. Alexander (19921 GenBank

accession number X65937,

Takao. M., M. Abramic, M. Moos, V. Otrin. J. Wootton, M.

McLenigan. A. Levine and M. Protic (19931 A 127 kDa

component of a UV-damaged DNA-binding complex, which is

B.J. Hwang et al./Muration Research 362 (1996) 105-I 17 117

defective in some xeroderma pigmentosum group E patients. is

homologous to a slime mold protein, Nucleic Acids Res., 2 I,

4111-4118.

Tanaka. K.. N. Miura, I. Satokato, I. Miyamoto. M. Yoshida, Y.

Satoh, S. Kondo, A. Yasui, H. Okayama and Y. Okada (1990)

cDNA sequence of the xeroderma pigmentosum group A gene

encoding a protein with a zinc finger structure. Nature, 348,

73-76.

Tanaka, K. and R. Wood (1994) Xeroderma pigmentosum and

nucleotide excision repair of DNA, Trends Biochem. Sci.. 19.

83-86.

Treiber. D., Z. Chen and J. Essigmann (1992) An ultraviolet

light-damaged DNA recognition protein absent in xeroderma

pigmentosum group E cells binds selectively to pyrimidine

(6-4) pyrimidone photoproducts. Nucleic Acids Res.. 20.

5805-5810.

Weber, C.. E. Salazar, S. Stewart and L. Thompson (1990)

ERCC2: cDNA cloning and molecular characterization of a

human nucleotide excision repair gene with high homology to

yeast RAD3, EMBO J., 9, 1437-1447.

Weeda. G.. R. Van Ham. W. Vermeulen, D. Bootsma, A. van der

Eb and J. Hoeijmakers (1990) A presumed DNA helicase

encoded by ERCC-3 is involved in the human repair disorders

xeroderma pigmentosum and Co&dyne’s syndrome, Cell. 62.

777-79 I,

Weickert, M. and S. Adhya (1992) A family of bacterial regula-

tors homologous to Gal and Lac repressors. J. Biol. Chem..

267. I5869- 15874.

Yasuhira, S. and A. Yasui (1992) Visible light-inducible pho-

tolyase gene from the goldfish Carassius aumfus, J. Biol.

Chem., 367. 25644-25647.