Vol. March

167, 16,

No.

2, 1990

BIOCHEMICAL

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Pages

1990

701-710

CHARACTERIZATION OF A PUTATIVE PROMOTER REGION OF THE HUMAN POLY(ADP-RIBOSE) POLYMERASE GENE: STRUCTURAL SIMILARITY TO THAT OF THE DNA POLYMERASE p GENE Tsutomu Ogura, Hiroshi Nyunoya*, Mitsuko Takahashi-Masutani, Masanao Miwa#, Takashi Sugimura and Hiroyasu Esumi Biochemistry Cancer Center #Institute

Division and *Virology Division, National Research Institute, Tsukiji, Tokyo 104, Japan of Medical Science, Ibaraki 305,

University Japan

of Tsukuba,

Received January 30, 1990 SUMMARY: The 5'-flanking region of the human poly(ADPribose) polymerase gene was isolated and characterized. The nucleotide sequence of a part of the poly(ADP-ribose) polymerase gene completely matched that of the cDNA. The transcriptional initiation sites (cap sites) of this gene, located about 166-bp upstream from the translational initiation site, were identified by Sl mapping analysis. Neither CAAT box nor TATA box was found within 500-bp upstream from the cap sites of poly(ADP-ribose) polymerase gene. The 200-bp immediately upstream of the cap site had a high G+C content (76.5%) and contained double repeats of the sequence CCGCCC, putative Spl binding sites, and a palindromic structure. The 5'-flanking region of poly(ADPribose) polymerase gene also showed promoter activity in chloramphenicol acetyltransferase assay and structural similarity to that of DNA polymerase p qene. @1990Academic Press.

Inc.

Poly(ADP-ribose) is

a nuclear

polymerase

enzyme that

poly(ADP-ribosyl)ation

(PADPRP) (EC 2.

catalyzes

4. 2. 30)

a DNA-dependent

of various

nuclear

proteins.

This

enzyme reaction is suggested to play roles in the regulation of various important cellular phenomena, DNA repair,

sister

differentiation, Full-length and sequenced of expression conditions function

chromatid exchanges, cell and transformation (l-3).

such as

proliferation,

cDNA for human PADPRP has been isolated Recently, we observed that the level (4-6). of PADPRP mRNA varied

of the

cells

of poly(ADP-ribose)

(7,8).

in different

However,

biological

the biological

and the mechanism of 0006-291x/90$1.50 701

Copyright 0 1990 by Academic Press. Inc. All rights of reproduction in any form reserved.

Vol.

167,

No.

regulation

of

understood. In the of

BIOCHEMICAL

2, 1990

regulation

expression, region of

PADPRP gene

present of

work,

AND

BIOPHYSICAL

expression to

we isolated PADPRP gene

are

elucidate

poly(ADP-ribose)

RESEARCH

not the

polymerase

and characterized from human leukocyte

MATERIALS

yet

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well

molecular

basis

gene the promoter genomic DNA.

AND METHODS

on and CmcterWon of Human Genomlc Clones. A cosmid Lorist B (9) genomic library constructed with a Sau3AI partial digest of human peripheral blood leukocyte genomic DNA (10) was provided by Dr. M. Terada of Approximately 200,000 clones were screened this Institute. with a 155-bp fragment of a cDNA clone of the region from the translational initiation site to the KpnI site as a Cosmid DNAs of positive clones were subcloned probe (4). into pTZ19R or pTZ19U vectors and the nucleotide sequences of both strands were determined by the exonuclease III/mung bean nuclease 5'- or 3'-end deletion procedure (11) and the and dideoxy7-deaza-deoxyguanosine 5 '-triphosphate ribonucleotide chain termination procedure using bacteriophage T7 DNA polymerase (12). alysis of the 5'-End of Human PADPRP mRNA. Total RNA was prepared from human T cells (Jurkat), osteosarcoma cells (HOS), and neusoblastoma cells (IMR-32) by guanidine Poly(A)+ RNA was prepared by thiocyanate extraction (13). A 21-mer single oligo(dT)-cellulose chromatography (14). strand oligonucleotide, 5'-GAGCGCACCGAACACGCCGCA-3', corresponding to the nucleotide +33 to +53 of the 5'noncoding sequence was synthesized by the solid-phase phosphoramidite method on an Applied Biosystem DNA synthesizer, model 380A. For Sl nuclease mapping analysis, a uniformly [32P]dCTP-labeled 369 base, single strand DNA probe corresponding to the nucleotide -316 to +53 of the 5'-flanking sequence was prepared by the primer extension method using the above mentioned oligonucleotide as a primer (15). The probe was mixed with 10 pg of total RNAs in Sl hybridization buffer (75% deionized formamide, 20 mM Tris-HCl, pH7.0, 400 mM NaCl, 1 mM EDTA, 10 mM DTT, 0.1 mM SDS) (15) . The mixture was heated for 15 min at 80°C, and then incubated for 12 hr at 42'C. Sl nuclease digestion was carried out for 60 min at 37OC in Sl buffer (300 mM NaCl, 3.3 mM ZnS04, 60 mM sodium acetate, pH4.8, 5 pg/ml salmon sperm DNA, 1,000 units/ml of Sl nuclease). Sl digestion products were electrophoresed on a 6% denaturing sequencing gel together with a dideoxy-sequenceing ladder priming from the same primer site. Construction of Plasmids for Chloramohenicol Acetvltransferase Assay. A 2.0 kilobase Bali-Bali fragment of the PADPRP gene was subcloned into a multicloning site of pTZ19U to construct PADPRP Bal/pTZlgU. PADPRP Bal/pTZlgU was subjected to KpnI/XbaI double digestion, and the DNA fragment was digested with exonuclease III and mung bean nuclease for preparation of the 3'-oriented deletion plasmid. The digested 1,846-bp fragment from nucleotide -1776 to +70 was purified, and then ligated with chloramphenicol acetyltransferase (CAT) gene from pSV2-CAT I

I

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plasmid (16), and the pEHT22 vector constructed with Epstein-Barr virus shuttle vector ~201 (17) and the 1acZ gene from pucll9. As positive control DNA for CAT assay, CMV-CAT/pEHT22, which contained an enhancer and a promoter of the immediate early genes of human cytomegalovirus (la), was also constructed from the pCMVti plasmid (19) and pEHT22 vector. Assay. Human T cells (Jurkat) and human B cells (BJAB) were cultured in RPMI-1640 containing 10% fetal calf serum. For assay of CAT activity, 20 pg of plasmid DNA were transfected to the cells by DEAE-dextran procedure (20). CAT activity was assayed 48 hr after transfection by the method described in our previous paper (21).

RESULTS AND DISCUSSION Isolation ribose)

of the Polymerase

Twelve 155-bp probe

independent containing

ribose) genomic

polymerase library.

ribose)

polymerase

designated blotting (5,

5'-Fm Gene

isolated

from

with the poly(ADP-

a human

cosmid

Previous studies showed that poly(ADPgenes were classified into two types, using

In the

present

the

work,

polymerase

on the basis

gene

5 '-portion

of

isolated

genomic clones

genes were also

of their

by Southern

cDNA as a probe

restriction

of

classified

into

maps (data

not

shown). The sequenced region of the noncoding 5'-exon the open reading frame of the PADPRP gene completely matched

those

On the other PADPRP-related

-

Human Poly(ADP

hybridized of human

as PADPRP and PADPRP-related

poly(ADP-ribose) two types

of the

genomic clones the 5 '-sequence

cDNA were

analysis

22).

Region

of cDNA over hand,

the

a length

nucleotide

gene hybridized

was shown to have high

of 280 bases

(Fig.

sequence of a part with

homology

but

the

5'-portion

was not

and 1). of the

of cDNA

identical

to

the nucleotide sequence of the cDNA, and the region sequenced of this gene of up to 550 bases had no intron (data not shown). These sequencing data suggest PADPRP gene is an active gene, and PADPRP-related processed

that gene is a

pseudogene. ion of the

Tr-lation

I

I

II

.

Site

by Sl

ase Mn Sl nuclease mapping analysis was carried identification of the transcriptional initiation the PADPRP gene. After Sl nuclease digestion 703

out

for site of of hybrids

Vol.

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No.

BIOCHEMICAL

2, 1990

AND

BIOPHYSICAL

RESEARCH

-1716

TTCTCTAAGTAAAACTlTTATTTGTTCCATCATATT!lTCCACTTATTCTGTTTACC!l?KA

-1656

AAATATCTTTTTTTTTTMTTTT

-1596

TGGCACTATCATGGCTCACCACAGCCTCAACCTTCAGGGCC

-1536

AGCCTCCCGAGTAGATGGGACTACAGGCACCTGCC!XCACCCCCAGCTAATTTTTGT?LGA

-1476

GACAAGGTTTTGCCATGTTGTCCAGGCTGGTCTTGAACTCCTGGGCTCAAGGGATCCGGC

-1356

CAACGTATCTAACTGGTTAACTTlTAGGATTCGGCCTATGTCTCACAACCTTCTl!GC

-1296

TTAC TCAACATCCTTGTCTCTTAAGCCACTAGCTTCTTCTCTATGG~~CAClTTlTAT

-1236

GAGTTTTATTCATCTGCTTATTTTTC!~TATCCTCTATACCAGPAATTGAATATTTTCAAAT

-1176

AAAGCACACTC&TGTTACAATCTTTGttAATG

-1116

AAACCAATTTTAATAAACTATATTTTGAAGTATAGTTCTATATT

-1056

GCCAGGTGCAGTGGCTCATGCCTGTAATCCCAGCAFil?lYTGGGAAGTCGAGGTGGGAGGAT

GAGACAGGGTCACACTGTCACCCAGGCTAGAGTCCAG

GAAAAAAAAAATGCATAGGATTAGAAAAG AAACAACAAGATCTAG

-996

TGCTTGAGGCCAGGGG!PK!AAGACCAGCCTGGGCAACATGGAGA~~CCCCATCTCTTT

-936

CTTTACACACACACACACACACACACACAAAA

-676

CCACAATTTCGAGTAGTGATGAGCTTXATAATATTTCGAGTTATCACCAACAACTGTAAA --___-------__-_-

-816

CTAACA

-756

CTGGTATl'TGTAGTAAATTCATAATAIUGATAATAAAGGAAATGCTAGGTTTCAGlTGGTATTTTGTCC

-696

CGACGG'J?2TGTGGACGGCAGGTTAGAACGCCCGTCCAAGCCAGGAGGGTGGACCTAGCAC

-636

TGCAGGGTCCACCTCGGGCCAATCAACTATATTCCCGAGGCCCGG

-576

ACCCAGCTGCCCTCAGGG

-516

CATGCCCCTGATCCCAGCACTTCGGGAGGCTGJiGGCG.TGAAGATCACTTGTAGCAGGAGT

-456

TTGAGACCAGTCTAGCCAACTTGGCGAGACCCTGTCCCT

-396

AGCCAGTl'GTGGTGAGCGCCTGTAGTCCCCAGCTACTCGGGAGGCTGAGGTGGGAGGATCG

-336

CTGGGCTC!AGGAGTTCCJ~ZACTGCAG~GCCATGATGGCGGCACTGCACTCCAGCGCGG

-276

TGAGACTCAGTCT

-216

AAGTAAGACTTCCTGGGKAGAACAA~GGGGTGGCGCCGGGTCCTCCAAAGAGCTA

-156

CTAGCTCAGCCCAAGCCCCGCCTCGGCCCCCAGGGCAGCGCCGGC

TATCTGATAGCAACAGGTGCAGTCATTA

TGAAUCGTCTGTGATGACTATTGCCCACAAAGTCACAGGTACTGCTAATACTC

GAGAGAGGACACACTTAAGAGTTTGGGGCCGGCGTGGTAGCT

VTTTTTTTAATT

CAAAAATAAA?LGGGGGAGGGGTTGGGGGTAAAATTAGTTGC

-96

AGGCGCCCGGGAAAClf??CCCGGCCGGCAGGGCQZG~GCCCGCCGGCCj???i??i$G

-36

TGGACGCGGGTTCCGTGGCGTTCCCGCGGCCAGGdCAGCAATCTATCAGGGAACGGCG .a.* II** a..,

25

GTGGCCGGTGCGGCGTGTTCGGTGCGCTCTGGCCGCTGGCCGC~GCCGTGGCGGCTG~T~CG

85

CACGCGAGGCGGCGAGGCGGCAAGCGTGTGTTTCTAGGTCGTGGCGTCGGGCTTCCGGAG

145

CTTTGGCGGCAGCTAGGGGAG-iiiGGCGGAGTCTTCTTCGGATA

205

CGCCAAGAGCGGGCGCGCCTCTTGCAAGAAA

265

COMMUNICATIONS

TGCAGCGAGAGCATCCCCAAGGACTCGCT 1st intron

CCGGATGGCCATCATGGTGCAGGTGCGGGCCGCTGTGCGGCGGGG

704

Vol.

167,

No.

2, 1990

BIOCHEMICAL

between

the

probe

several

fragments

nucleotides IMR-32 cells Furthermore,

nuclease showed sites

isolated

previously

translational

Evidence

of

was

located

cDNA

was

To demonstrate

(positive

the

plasmid, introduced

5 and 6),

also

indicating

whereas

strongly

These PADPRP cDNA from the

that

activity

of

to the nucleotide of PADPRP gene,

and this construct, into Jurkat or

with forming

of these promoter)

out

our

full-length.

almost

the

this cells (Fig.

indicates

that

named BalBJAB cells by gene was of Jurkat

showed

was

transfected 3, lanes

(Fig not

3,

lanes

detected

in

2,

PADPRP gene

is

an active

gene.

1. Nucleotide sequence of the 5'-flanking region of the PADPRP gene. A region of 2,085-bp of the 5'-flanking Nucleotide +1 region of the PADPRP gene is presented. denotes the 5'-most proximal transcriptional site which was determined by Sl nuclease

initiation mapping analysis,

the gene are indicated

by negative

numerals. The untranslated region of the mRNA is indicated by underline. The translated region starting with the first ATG codon is indicated by double underline. The broken lines show typical CAAT and TATA sequences. Complete matching of the putative Spl binding sequences are presented by open boxes. Arrows under the PADPRP sequence The mark the position of a putative palindromic structure. zigzag line indicates the nucleotide sequence of a 21-mer single strand oligonucleotide used as a primer in Sl mapping analysis. The star symbols under the PADPRP sequence indicate the transcriptional initiation sites.

705

3,

with CAT/pEHT22 1 and 4). This finding

Fiq.

and residues preceding

a

CMV-CAT/pEHT22

activity

activity

PADPRP gene, -1776 to +70 was subcloned

of this Extracts

and Bal-CAT/pEHT22

acetylchloramphenicol extracts (without

HOS or

The length same result

The 5'-end of 166-bp upstream

(4),

promoter

transfected

control)

RNA from

not shown). transcriptional

The transient expression by measuring CAT activity.

and BJAB cells

46 and 42

Activity

1846-bp, which corresponds of the 5'-flanking sequence

transfection. monitored

Total

(data several

site

Promoter

into CAT/pEHT22 CAT/pEHT22, was

53,

cells,

the 5'-end of PADPRP mRNA. DNA product also gave the

initiation

for

2).

PADPRP gene.

isolated

about

Jurkat

COMMUNICATIONS

results (data not shown). analysis was also carried

mapping analysis that there are

initiation

previously

of

(Fig.

gave identical primer extension

RESEARCH

RNA from

lengths

detected

for determination of of the major extended of Sl results

BIOPHYSICAL

DNA and total with

were

AND

Vol.

167,

No.

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2, 1990

GATC

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12

Jurkat

BJAB

AcCM CM -+

02

03

1

2

3

4

5

6

Determination of the transcriptional initiation Fiq. 2. site of the PADPRP gene by Sl nuclease mapping analysis. The uniformly labeled, single strand DNA probe for Sl mapping was synthesized as described in Materials and Methods. Samples of 10 pg of yeast tRNA (lane 1) or total RNA isolated from Jurkat cells (lane 2) were hybridized to the probe. After Sl nuclease digestion, the products were Arrowheads separated on a 6% denaturing sequencing gel. indicate the positions of protected bands. Lanes G, A, T and C show a dideoxy-sequencing ladder priming from the same site as the size markers. Fiq. 3. Expression of CAT activities in cells transfected with recombinant plasmids containing 5'-flanking fragments of the PADPRP gene. A 1,846-bp fragment, which corresponds to the nucleotide -1776 to +70 of the 5'-flanking sequence was subcloned into a CAT/pEHT22 plasmid. Jurkat or BJAB cells were transfected with 20 pg of Bal-CAT/pEHT22, (lanes (without promoter) (lanes 1 and 4), or 3 and 61, CAT/pEHT22 CMV-CAT/pEHT22 (positive control) (lanes 2 and 5). After transfection, 60 ~1 of cell extracts were used for assay of CAT activity. The autoradiogram shows the conversion of chloramphenicol (CM) to its acetylated products (AcCM).

Analvsis

of

the

Nucleotide

Seauence

of

the

5’-Flankinq

Reaion The group expressed promoter

of

PADPRP cellular in

gene

is

considered

housekeeping most,

functions

to genes

if

not

of

several

be that

tissues.

all,

housekeeping 706

a member are

of

the

continuously

Recently, genes

the have been

Vol.

167,

No.

BIOCHEMICAL

2, 1990

well characterized upstream of the G+C content

(23-28). transcriptional

(23-28),

TATA box regions

PADPRP gene within 200-bp high

completely

and

contain

either

has

that

the

palindromic

the

transcriptional Comoarison Ribose) was

regulation structure

PADPRP gene

may also

between Polvmerase

It has induced

the

of

the a

expression

between the which catalyze damaged

are

six

Spl binding found in 1).

the

PADPRP

promoter to play

of

upstream

region of an important

those

genes

(23-26,

just

upstream

be important

of

the

for

PADPRP gene.

5'-Flankins

there

-78

of

composed

Resions

of

PADPRP gene, a gap-filling

DNA (32),

and the reaction

we compared

Polv(ADP-

biosynthesis (3). To

mechanism(s)

same

the

8 Gene

been known that poly(ADP-ribose) in DNA damaged mammalian cells whether

of

has

-45, just palindromic

Gene and DNA Polvmerase

investigate P gene, synthesis

of

to

of

palindromic regulation

five

structure

nucleotide -71 1). Previously,

transcriptional of the

which

region

role

Thus,

of the

or TATA box

of the putative (30), were also -150 to -39 (Fig.

5'-flanking

an interesting

site

genes,

in

have been observed in the genes and have been suggested

cap

a

the 5'that of the

site. Furthermore, of the cap site

structures several 31) .

Like CAAT box

sequences

13 G-C pairs from the of the cap site (Fig.

in

is

and double repeats of a 5 '-CCGCCCsequence at positions

14 similar

We found

which

(76.5%),

nucleotide residues match those site (5'-GGGCGGor 5'-CCGCCC-) the region from the nucleotide

gene

island,

upstream of the cap immediately upstream

matching

and -42,

COMMUNICATIONS

site (29). Other common features of these genes are the absence of

not

G+C content

RESEARCH

Their sequences immediately initiation site have a high

and CAAT box (23-28). of other housekeeping

does

SOO-bp region

BIOPHYSICAL

and a CpG-rich

potential methylation the promoter region typical flanking

AND

of

gene

DNA polymerase and repair the

5'-flanking

region of both genes. between the 5'-flanking and mouse DNA polymerase

There is sequences p genes

we found the following 5'-flanking regions of DNA polymerase p genes

structural similalities between the the PADPRP gene, and human and mouse (Fig. 4). First, there are several 707

no significant homology of PADPRP gene, and human (27, 28). Interestingly,

Vol.

400 I

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-12w I

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-1000 I

I

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I

-600 I

-600 I

I

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-400 I

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I

-200 I

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I

ll I

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200 1

I

) CAAT

CAAT

TATA

TATA

I

’

I

I

Poly(ADP-Ribose) Polymerase

Human

DNA Polymerase

p

Mouse

DNA Polymerase

p

CCGCCC

CAAT

I

Human

CCGCCC

I

I

I

TATA I

CCGCCC

I

I

I

I

I

I

I

I

Fig. 4. Diagram of the 5'-flanking regions of human poly(ADP-ribose) polymerase gene, and human and mouse DNA polymerase p genes. The numbering of the nucleotides is relative to the 5'-most proximal transcriptional initiation site. Major sites for transcriptional initiation are indicated by vertical lines and arrows. The putative Spl binding sequences are indicated by closed circles. The CAAT and TATA boxes are shown by closed and open squares, respectively.

transcriptional region flanking

and

(about

sites.

upstream

regions

sequences rich

initiation

immediately of

both

of genes

palindromic 75%).

Third,

Second, the have

cap

putative

CAAT

200-bp

the of

the

Spl

5'binding

and are extremely

structures, the

site

box

and

TATA

box

of

genes are also located in a region more than SOO-bp upstream of the cap site. In addition, we found that

G+C both

in

the mouse tissues examined, the levels of mRNA of the PADPRP gene were high in the testis, spleen, thymus, and brain,

whereas expression of the PADPRP gene was low in the heart, kidney, and liver (unpublished data). The

lung, mouse DNA polymerae expression

p gene shows an identical

in mouse tissues

(33).

pattern

These findings

suggest

that the mechanism(s) of regulation of PADPRP gene expression may be similar to that of DNA polymerase during

recovery

of mammalian cells

of

p gene

from DNA damage.

ACKNOWLEDGMENTS: The authors thank Drs. Terada and K. Shimotohno for supplying the human cosmid genomic library and ~201 plasmid, respectively, and for valuable suggestions. They are also grateful to Drs. M. Nagao, H. Shima, N. Kato and T. Akagi for valuable discussion. This work was supported in part by a Grant-in-Aid from the Ministry of Health and Welfare for the Comprehensive loyear Strategy for Cancer Control, Japan, Grants in Aid for Cancer Research from the Ministry of Education, Science and Culture, and Haraguchi Memorial Cancer Research Fund. M. Takahashi-Masutani is a recipient of a Research Resident 708

Vol.

167,

No.

Fellowship Research.

BIOCHEMICAL

2, 1990

from

the

AND

Foundation

BIOPHYSICAL

for

Promotion

RESEARCH

of

COMMUNICATIONS

Cancer

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Herzog, H., Zabel, B. U., Schneider, R., Auer, B., Hirsch-Kauffmann, M., and Schweiger, M. (1989) Proc. Natl. Acad. Sci. USA, & 3514-3518. Araki, E., Shimada, F., Uzawa, H., Mori, M., and Ebina, Y. (1987) J. Biol. Chem., 2h2, 16186-16191. Ishii, S., Xu, Y. -H., Stratton, R. H., Roe, B. A., Merlino, G. T., and Pastan, I. (1985) Proc. Natl. Acad. Sci. USA, & 4920-4924. Valerio, D., Duyvesteyn, M. G. C., Dekker, B. M. M., Weeda, G., Berkvens, Th. M., van der Voorn, L., van Ormondt, H., and van der Eb., A. J. (1985) EMBO J., L 437-443. Singer-Sam, J., Keith, D. H., Tani, K., Simmer, R. L., Shively, L., Lindsay, S., Yoshida, A., and Riggs, A. D. (1984) Gene, 32. 409-417. Widen, S. G., Kedar, P., and Wilson, S. H. (1988) J. Biol. Chem., J?L& 16992-16998. Yamaguchi, M., Hirose, F., Hayashi, Y., Nishimoto, Y., and Matsukage, A. (1987) Mol. Cell. Biol., 1, 20122018. Bird, A. P. (1986) Nature (London), a 209-213. Dynan, W. S., and Tjian, R. (1983) Cell, L 79-87. Koiwai, O., and Morita, A. (1988) Biochem. Biophys. Res. Commun., a 91-100. Wang, T. S. -F., and Korn, D. (1980) Biochemistry, & 1782-1790. Hirose, F., Hotta, Y., Yamaguchi, M., and Matsukage, A. (1989) Exp. Cell Res., % 169-180.

23. 24. 25.

26. 27. 28. 29.

30. 31. 32. 33.

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