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A Human Poly(ADP-ribose) Polymerase Gene Family (ADPRTL):cDNA Cloning of Two Novel Poly(ADP-ribose)

Polymerase Homologues

Magnus Johansson1

Karolinska Institute, Division of Clinical Virology F68, Huddinge University Hospital, S-141 86 Stockholm, Sweden

Received November 30, 1998; accepted February 19, 1999

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Posttranscriptional modification of nuclear proteinsy poly(ADP-ribosyl)ation in response to DNA strandreaks plays an important role in DNA repair, regula-ion of apoptosis, and maintenance of genomic stabil-ty. A 113-kDa human poly(ADP-ribose) polymerasePARP) has previously been identified and cloned.owever, there is evidence that additional enzymesith PARP activity exist in mammalian cells. I have

dentified and cloned the cDNAs of two novel '60-kDauman proteins that are 40 and 31% identical to theatalytic C-terminal domain of PARP. These proteins,amed PARP-2 and PARP-3, lack the DNA-binding andutomodification domains. PARP-2 and PARP-3RNAs were detected in 16 different human tissues asajor bands of 2.0 and 2.2 kb, respectively. Radiationybrid analysis assigned the PARP-2 gene (HGMW-pproved symbol ADPRTL2) to chromosome 14q11.2–12 and the PARP-3 gene (HGMW-approved symbolDPRTL3) to 3p21.1–p22.2. This report shows the ex-

stence of a human PARP gene family with at leasthree closely related members. © 1999 Academic Press

Poly(ADP-ribosyl)ation of several nuclear proteins isn early cellular response to DNA strand breaks (4). A13-kDa chromatin-associated human poly(ADP-ri-ose) polymerase (PARP)2 has been cloned and therotein carefully characterized (3). The N-terminal do-ain of PARP contains two zinc fingers that bind toNA strand breaks. Binding of PARP to damaged DNAllosterically activates the catalytic C-terminal domaino synthesize complex ADP-ribose polymers usingAD1 as substrate. The central PARP domain con-

ains several acceptor sites for poly(ADP-ribose) poly-ers, and this region is automodified when the enzyme

Sequence data from this article have been deposited with theMBL/GenBank Data Libraries under Accession Nos. AF085734 andF083068.

1 Telephone: 146-8-58581306. Fax: 146-8-58587933. E-mail:agnus.johansson@mbb.ki.se.2 Abbreviation used: PARP, poly(ADP-ribose) polymerase.

enomics 57, 442–445 (1999)rticle ID geno.1999.5799

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888-7543/99 $30.00opyright © 1999 by Academic Pressll rights of reproduction in any form reserved.

s activated. The physiological function of PARP andoly(ADP-ribosyl)ation has been addressed in studiesn transgenic PARP 2/2 mice. These mice are viablend develop normally, but they are highly sensitive tolkylating agents and g-irradiation (10, 14). Cells de-ived from the mice also exhibit signs of genomic in-tability and show delayed repair of DNA strandreaks (14, 15). The PARP 2/2 mice were believed toe completely deficient in poly(ADP-ribosyl)ation, but aecent report shows that cells derived from PARP 2/2ice retain the ability to synthesize poly(ADP-ribose)

olymers (11). Although the amount and length of poly-ADP-ribose) polymers were decreased in the PARP/2 cells, the cells exhibited induction of poly(ADP-

ibosyl)ation in response to DNA damage (11). Theseata suggest that additional enzymes with PARP ac-ivity exist in mammalian cells. Recently, tankyrase, aelomere-associated enzyme, was cloned and shown toatalyze poly(ADP-ribosyl)ation (13).I searched the expressed sequence tag library of theenBank database with the sequence of human PARP

3) to identify cDNA clones that encoded novel se-uence-related human proteins. cDNA clones that en-oded two proteins similar, but not identical, to humanARP were identified (Integrated Molecular Analysisf Genomes and Their Expression Clone ID: 51469,81891, 295907, 505009, 505390, and 1028271) (7).he full-length cDNAs of the novel proteins wereloned from a human fetal brain cDNA library by rapidmplification of cDNA 59 ends (Marathon-ReadyDNA; Clontech). The translated products of the 1910-nd 1880-bp cDNAs obtained (GenBank Accession Nos.F085734 and AF083068)3 were 40 and 31% identical

o human PARP at best alignment (Fig. 1A). I namedhe encoded proteins PARP-2 and PARP-3, based onheir high level of similarity to PARP (referred to asARP-1). The PARP-2 cDNA encoded a 534-amino-acidrotein with a calculated molecular mass of 60.8 kDa,

3 The HGMW-approved symbols for the genes described in thisaper are ADPRTL2 and ADPRTL3.

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nd the PARP-3 cDNA encoded a 533-amino-acid pro-ein with a mass of 60.1 kDa. Both cDNAs containedtop codons upstream of the predicted translationtart, which indicates that the cDNA clones containedhe complete open reading frames. PARP-2 andARP-3 were accordingly smaller than PARP-1, andoth novel proteins lacked the N-terminal DNA-bind-

FIG. 1. Alignment of human PARP-1 with the two novel human hcid sequences. Black boxes indicate conserved residues. (B) SchematLS, nuclear localization signal.

ng domain as well as the central automodificationomain (Fig. 1B). Attempts to produce recombinantARP-2 and PARP-3 in bacteria to verify their enzy-atic activity failed, because the enzymes were toxic to

acteria in the expression system (data not shown).owever, it is likely that PARP-2 and PARP-3 are

atalytically active, since the amino acid residues

ologues, PARP-2 and PARP-3. (A) Alignment of the predicted aminolignment of the three enzymes. Zn-1 and Zn-2, zinc finger structures;

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nown to be important for synthesis of poly(ADP-ri-ose) polymers are highly conserved in these enzymes.urthermore, a truncated mutant of PARP-1 lackinghe DNA-binding and automodification domains re-ains catalytic activity, which shows that the 53-kDa-terminal domain of PARP is sufficient for poly(ADP-

ibose) polymerization (12). However, the truncatedutant is not activated by damaged DNA, whereas the

oly(ADP-ribosyl)ation in PARP-1 2/2 mice is acti-ated by DNA-damaging agents (11). A recent reporthows that plants have an enzymatically active PARPomologue of size similar to those of PARP-2 andARP-3 (1). The plant PARP homologue lacks the clas-ical DNA-binding domain of PARP, but it is activatedy damaged DNA in vitro. It is possible that PARP-2nd PARP-3 are regulated by a similar mechanism.Northern blot analysis showed, as expected, the

ARP-1 mRNA transcript as an '4-kb band that wasresent at similar levels in all human tissues investi-ated (Fig. 2). PARP-2 and PARP-3 mRNAs were alsobiquitously expressed in all tissues as major bands of.0 and 2.2 kb, respectively. An additional PARP-2and of 4 kb was detected in muscle tissues, and weakands of 2.0–2.5 kb were seen for both PARP-2 andARP-3 in several tissues. The origin of the additionalands is unknown, but they may represent differentranslated or nontranslated splice variants. Theorthern blots confirmed that the major transcripts ofARP-2 and PARP-3 were shorter than the PARP-1ranscript, and the mRNA sizes corresponded well tohe sizes of the cloned cDNAs.

FIG. 2. Northern blot analysis of PARP-1, PARP-2, and PARP-3RNA expression. Human multiple tissue Northern blots (Clontech)ere probed with [a-32P]dCTP-labeled cDNA fragments of PARP-1

bp 94–898), PARP-2 (bp 1370–1854), or PARP-3 (bp 1081–1787). Anctin cDNA probe was used to verify the abundance of mRNA in eachane.

The gene encoding human PARP-1 is located at chro-osome 1q42 (2, 3). A sequence-tagged site derived

rom the 39 region of a PARP-3 expressed sequence tagas identified in GenBank (SGC30878). This markeras located at chromosome 3p21.1–p22.2 (5). The chro-osome location of the PARP-2 gene was determined

sing the GeneBridge 4 Radiation Hybrid Mappinganel (Research Genetics, Huntsville, AL). The cellybrid clones were screened for the presence or ab-ence of a PCR-amplifiable marker for the PARP-2 loci59-GAAGCTCCCATCACAGGTTAC and 59-AGGTAC-GCATACG-GACCTGGTTGGGGTTA). The radiationybrid analysis indicated that the PARP-2 gene was

ocated 8.45 cR distal to marker D14S264. This markers located at chromosome 14q11.2–q12 (5). Two puta-ive PARP pseudogenes have been located to 13q34 and4q24 by in situ hybridization with a PARP-1 generobe (2). The gene at 13p34 has been shown to be arocessed pseudogene closely related to PARP-1 (8, 9).he sequence of the 14q24 locus is not yet analyzed,ut studies on somatic cell hybrids suggest that it isocated distal to 14q13 and proximal to 14q32 (3). TheARP-2 gene marker at 14q11.2–q12 is located proxi-al to this region, and there may accordingly be two

eparate PARP gene loci on chromosome 14. The 3p21egion, where PARP-3 is located, is frequently alteredn several types of solid malignant tumors (6). Consid-ring the role of poly(ADP-ribosyl)ation in mainte-ance of genomic stability, PARP-3 should be consid-red a candidate tumor suppressor gene in the 3p21egion.The identification and cloning of the two PARP ho-ologues will be the basis for future studies on the role

nd physiological function of the different members ofhe PARP gene family. A key experiment to under-tand the importance of the enzymes will be to produceells and animal models that are deficient in expres-ion of PARP-2 and PARP-3 as well as multiple genenockouts that completely lack poly(ADP-ribosyl)-tion.

ACKNOWLEDGMENT

This work was supported by a grant from the medical faculty of thearolinska Institute.

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