Genomic characterization of the human DNA excision repair-controlling geneXPAC

Gene, 136 (1993) 345-348 0 1993 Elsevier Science Publishers

GENE

B.V. All rights reserved.

345

0378-l 119/93/%06.00

07464

Genomic characterization gene XPAC

of the human DNA excision repair-controlling

(Xeroderma pigmentosum; genomic library; phage vector; alternative splicing; promoter activity)

Ichiro Satokata, Kunimitsu Iwai, Toshiro Matsuda, Yoshio Okada and Kiyoji Tanaka Institute for Molecular and Cellular Biology, Osaka University, l-3, Yamada-oka, Suita, Osaka 565, Japan Received by T. Sekiya: 26 April 1993; Revised/Accepted:

24 June/27

June 1993; Received at publishers:

27 July 1993

SUMMARY

We have characterized the human DNA excision repair gene, XPAC (xeroderma pigmentosum group A complementing). This gene of approximately 25 kb consists of six exons. The S-flanking region of the gene has a CAAT box, but no TATA box. The region upstream from the coding sequence of exon 1 is G+ C rich (73%), and has a GC box. Transcriptional mapping analysis suggested that there is one major transcription start point (tsp). The presence of two polyadenylation signals suggests that the two XPAC mRNAs with different 3’ untranslated regions in normal human cells are due to alternative polyadenylations. The promoter activity, measured by transient expression of the cut gene with the 5’ flanking regions, indicated the presence of a functional promoter.

INTRODUCTION

Xeroderma pigmentosum (XP) is an autosomal recessive disorder characterized by extremely high sensitivity of the skin to ultraviolet (UV) light and a high predisposition to skin cancer. Cells from XP patients are hypersensitive to the lethal effect of UV radiation, because they have a defect in repair of UV-induced DNA damage (Cleaver, 1968). To date, eight genetic forms of XP have been identified, but little is yet known about the excision repair system in eukaryotes. XP cells of complementation groups A through G have defects in excision repair, while the XP variant is thought to have an impairment of postCorrespondence to: Dr. K. Tanaka, Biology,

Osaka

University,

Tel. (81-6) 877-5238;

Institute

1-3, Yamada-oka,

for Molecular Suita, Osaka

and Cellular 565, Japan.

Fax (81-6) 877-9136.

Abbreviations: aa, amino acid(s); bp, base pair(s); CAT, Cm acetyltransferase; car, gene encoding CAT, cDNA, DNA complementary to RNA, Cm, chloramphenicol; kb, kilobase or 1000 bp; nt, nucleotide(s); SDS, sodium dodecyl sulfate; tsp, transcription start point(s); UTR, untranslated region(s); UV, ultraviolet; XP, xeroderma pigmentosum; XPAC, XP group A complementing protein; XPAC, gene encoding XPAC, Y, G or T.

replication repair (Friedberg, 1984). Previously, we cloned a mouse DNA-repair gene that complements the defect of group-A XP cells and named it the XP group-A complementing (XPAC) gene (Tanaka et al., 1989). Using mouse genomic fragments containing exons of the XPAC gene, we also cloned human and mouse XPAC complementary DNAs (Tanaka et al., 1990). The aim of present study was the characterization of the human genomic XPAC gene.

EXPERIMENTAL

AND DISCUSSION

(a) Exon and intron structures of the human XPAC gene Using human XPAC cDNA as a probe for plaque hy-

bridization, we isolated four clones from a human genomic DNA library. The precise locations of exon-intron junctions were defined by restriction enzyme mapping and Southern blot hybridization followed by sequencing of appropriate regions of the clones and their alignment with the human XPAC cDNA sequence. The gene has six exons and spans about 25 kb (Fig. 1). All the exon-

346 0 I

5 I

10 I

AEMBL3/ IS-7

4

4

15 I

20 I

b

LEMBL3/lS-31

b

4

AEMBL3IIS-30 4

Exon 1 m

23 I

Exonl

30kb I

25 I

45 a

1

2

3 +

e+-++ f +

s

b F

I.EMBL3/IS-28

6 D-l

I

4 et ---*

5

6

-+

Fig. 1. Structure of the human XPAC gene and sequencing strategy. High-molecular-weight DNA of human leucocytes was partially digested with Sau3A1, size-fractionated through a sucrose gradient, ligated with the BamHI + EcoRI-digested arms of hEMBL3 and packaged using Gigapack Gold (Stratagene). Phage plaques were screened with 32P-labeled human XPAC cDNA probe. Phage DNAs were purified from positive plaques and characterized by restriction enzyme mapping and Southern blot hybridization. The nt sequences of the exons, exon-intron junctions, and the 5’- and 3’-flanking regions were determined by the dideoxy chain termination method. Inserts of four clones covering the XPAC gene are shown at the top. A restriction map of EcoRI (E) and BamHI (B) sites is shown in the middle. The exon/intron structure of the gene is depicted below. Closed and open boxes represent coding and noncoding exons, respectively. The start codon (ATG) and polyadenylation signal (AATAAA) are indicated by vertical arrows. Detailed restriction maps of exons 1-6 and the nt sequencing strategy are shown at the bottom. A, AluI; D, HindIII; E, EcoRI; H, HaeIII; K, KpnI; M, SmaI; P, PstI; S, SacI. The nt sequences determined in this study have been submitted to the GenBank TM/EMBL Data Bank (Accession No. D14533).

intron splice junctions conform to the consensus GT-AG rule. The nucleotide sequences of the exons are identical with those of the corresponding regions of human XPAC cDNA (Fig. 2).

EEXON

INTRON

INTRON

splice acceptor

splice donor 57

5 6

3' poly(A) tail

59 CTTTTACTTTTGTAG

2C AT; 96

2

TTTTCCTTTTCTTAG

B C'C'T" 131 0 .LSP A GAT 186 187 Il.3"al ATT GTG 226

3

TTAATCTGTTTTCAG

GATTTTGTGTTGTAG AAATTTTTTTTTCAG

&

T%

4

In primer extension analysis, two major fragments were detected, corresponding to tsp at nt - 160 and - 161 (Fig. 3). We propose that transcription starts at nt - 161 (Fig. 4A), because the appearance of subfragments may be caused by the bulky 5’ cap structure on the mRNA (Weaver and Weissmann, 1979) and most eukaryotic mRNAs start with the adenine of the cap consensus signal T/GCAGT (Bucher, 1990).

58

ay G GGA G 94 95 Pro c CCA G 129 130 cyB Ar TGC AG 184 185 IS" Ol" TTA CAG 221 225 LYllG AAAG

1

(b) Transcription start point @VP)

GTTTGGGCGTCCGCG

(c) S- and 3’4anking GTAAAGTGAGTTTTT GTACTTATTTTAGAT GTCTCTAATAAGTTG GTAGATGGCCACATT

Fig. 2. Structural details of organization of human XPAC intron-exons. The nt sequence of each intron-exon junction is shown. Vertical lines represent intron-exon borders. The corresponding aa at the beginning and end of each exon are shown by three letters and numbered as reported previously (Tanaka et al., 1990).

regions

The 5’-flanking region contains a CAAT box but no canonical TATA box. The region from nt -200 to - 1 has a high content of G and C residues (73%). There is a GC box, which is thought to be the binding site of transcription factor Spl (Dynan and Tjian, 1985) in the 5’-flanking region (Fig. 4A). The 3’-UTR is in exon 6. There are two polyadenylation signals (AATAAA), suggesting that the two XPAC mRNAs in normal cells are due to alternative polyadenylations. In addition to polyadenylation signals, a pentanucleotide CAYTG, which is suggested to mediate poly(A)

347

GATC

A

12

-672 GCCTTGGAGCAAGTCCTGCATTTGCCT -645 GAGGTACACACGCAAGTTAACTGGGAGTTTcAACCcAGGTCTTCTCATTTTA?!ATCTAAG -585 GCTGTGTCTCTAGGCCGTGTATCCX+cATAA?.ATcAGGACTATcATCTGTTGGGCTCTAT -525 WLCTGGCACTACTACTACTCACTGTTACCATTTTTTGAGG -465 TGTCCTGGT~CTAATCCTCCCAAAGACTCGGTAAATGGTTCCATAATTATCCTCATT -405 TTACCACTGAAGAAACTGAACTCTGGTTAAATAACTTGCCCAAGGTCACACAGACTTTAA -345 CTGTC~GACTTAACCCAGGCCTGACTCCTA sac I, Al" I -285 GCGAGACCTATTTTAAAGGTGACCAGGTCGTGAGATATGATCT~G~C~CCCTTCTC

nt 250-

-225 CCGGATGACAAGAGAGCAGGTAGTTAGGCGGGTACTCCGTGTCCGCGCATACCCAGACTC l

240~

-165 GCCCAGCGQXGBiC AGGCCACCCCGAGCCCCTTAACTGCGCAGGCGCTCTCACTCAGAA SmaI -105 AGGCCGCTGGGTGCGGGAGcGCAGAGGcGTGU@&& CTGGCTCGCTCGGCGTGCAGT +1 GGTCCTCGGAGTGGGCCAGAGATGGCGGCGGCCGAC - 45 GCGCGTGCGTGG~G AlU I Alu I + 16 GGGGCTTTGCCGGA~GGCGGCTTTAGAGCAACCCGCGGAGCTGCCTGCCTCGGTGCGG

230~ 220*

6

210-

G1Y Ly* Hot w808 TAT GAA AAA ATG TGA TTTTTTAGTTCAGTGACCTGTTTTATAGAATTTTAT l

859 BTTTBAAA?.GGAAATTTAGATTGGTCCTTTTCAAAATTCS-----1246 TGTGTTTTTTAGGACGATTTCTGTCTCCACGATGGTGGAAT 1302 GCTGGAAAAAGCCCTAATAGCAGMLATAAA@~?T.GAGTTGTACGAGTCTGATTATG 1358 TTTTCTGTACTCTTGGGCC

Fig. 4. Nucleotide human

sequences

of the 5’- and 3’-flanking

XPAC gene. (A) S-Flanking

region. The sequence

regions

of the

is numbered

from the first nt of the ATG start codon. The upstream sequence is indicated by negative numbers. The tsp is shown by an asterisk, CAAT boxes

are underlined,

pertinent restriction names. The 30-mer

and the GC box is doubly

underlined.

Some

enzyme sites are underlined with their respective synthetic primer, complementary to the antisense

Fig. 3. Primer extension analysis. For mapping of tsp in the human XPAC gene, primer extension analysis was performed as described

sequence used in primer extension Flanking region. Corresponding

(Kingston,

asterisk marks the stop codon. Two polyadenylation signals are doubly underlined, and a CAYTG motif and ATTTA motif are underlined.

1987a). Total RNA was extracted

the standard method by oligo(dT)-cellulose

from WI38VA13

cells by

(Maniatis et al.. 1982). Poly(A)+RNA was selected chromatography. A 30-nt primer complementary

analysis, is shown by an arrow. (B) 3’aa are shown by three letters. The

to nt 30-59 of exon 1 was synthesized, and labelled at the 5’ end using [Y-~‘P]ATP. The end-labeled primer was hybridized to poly(A)+RNA (10 ug) and was extended

in the presence

products gel and

in 6% polyacrylamide/S M urea denaturing Lanes: 1, yeast transfer RNA (10 ug);

were separated autoradiographed.

of reverse transcriptase.

2, human poly(A)+RNA. Primer-extended fragments arrows. G, A, T and C, dideoxy sequencing ladders human

XPAC gene cloned in a M13mp18

S-GTTTTCCCAGTCACGAC.

The

are indicated by of the exon 3 of

using Ml3 universal

primer

which were used as size markers.

formation through interaction with RNA of the U4 small ribonucleoprotein (Berget, 1984) was found after the second poly(A) site. T-rich and G +T-rich sequence motifs were located downstream from the poly(A) sites. These motifs have been shown to function synergistically and to be important for efficient and accurate 3’ end formation (McDevitt et al., 1986; Gil and Proudfoot, 1987). Furthermore, a mRNA stabilizing motif (ATTTA) (Clemens, 1987) was found in the 3’-flanking region (Fig. 4B; Tanaka et al., 1990).

(d) Functional analysis of the XPA C gene promoter

To examine the promoter activity of the 5’ region of the human XPAC gene, we inserted the EcoRI-SmaI fragment (nt -2300 to - 155) or the SacI-SmaI fragment (nt - 334 to - 155) into a site upstream from the promoterless cut gene in plasmid pSVOOcat, and measured CAT enzyme production in WI38VA13 cells (SV40-transformed normal human cells) 48 h after transfection. Weak but significant CAT activities were observed with both the EcoRI-SmaI fragment (pSVOOEScat) and the SacI-SmuI fragment (pSVOOSScat). No CAT activity was detected with pSVOOcat (Fig. 5). These results indicate that the SucI-SmuI region harbors promoter activity.

ACKNOWLEDGEMENTS

This work was supported by grants from the Foundation for Promotion of Cancer Research, The Mitsubishi Foundation, The Senri Life Science

348

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2

3

4

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mRNA Kingston.

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XPAC

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extract

cells; 4, an extract

of pSVOOEScat-transfected

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cells.

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to - 155), respectively,

Hind111 linker.

Samples

cells;

Plasmids

an end-filled fragment (nt

with a Hind111 site of pSVOOcat using

of 1 x lo6 WI38VA13

cells were seeded

into

loo-mm Petri dishes in Dulbecco’s modified minimum essential medium containing 10% fetal bovine serum. The next day, cells were transfected with plasmids (10 ug each) by the ca. phosphate precipitation method. Cells were harvested 48 h after transfection and their CAT activity was measured as described (Kingston, 1987b). The amounts of cell extracts were normalized for protein concentration. Samples were subjected to thin-layer chromatography, analyzed by densitometric

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Freeman,

Gil, A. and Proudfoot, N.J.: Position-dependent sequence elements downstream of AAUAAA are required for efficient rabbit B-globin

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Spots were

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involved in poly-

D.D.,

Seidman,

J.G.,

Smith,

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R.,

J.A.

and

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John

acetyltransfer-

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