Molecular Biology Reports 29: 347–352, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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Potential protein partners for the N-terminal domain of humantopoisomerase I revealed by phage display

Agata M. Trzcinska, Agnieszka Girstun, Agnieszka Piekiełko, Barbara Kowalska-Loth &Krzysztof Staron∗Institute of Biochemistry, Warsaw University, ul. Miecznikowa 1, 02-093 Warsaw, Poland; ∗Author forcorrespondence (Phone: +48 22 5543114; Fax: +48 22 5543116; E-mail: staron@biol.uw.edu.pl)

Accepted 15 July 2002

Key words: partner proteins, phage display, topoisomerase I

Abstract

Phage display procedure was applied to the N-terminal domain of human topoisomerase I. The consensus se-quence identified for clones binding to the N-terminal domain was found in 35 human proteins that are eitherpermanently or temporarily located in the nucleus. They are in majority involved in the DNA repair, transcription,RNA metabolism or cell cycle control. Four of identified proteins: Bub3 protein, Cockayne syndrome protein A,damaged DNA binding protein 2 and GRWD protein belong to WD-repeat proteins and their sequences recognizedby the N-terminal domain are identically localized.

Abbreviations: DHFR – dihydrofoliate reductase; htopo I – human topoisomerase I; htop(1-214) – polypeptidecomprised of the first 214 amino acids of human topoisomerase I.

Introduction

Human topoisomerase I (htopo I) is the main enzymeresponsible for relaxing DNA supercoils [1] and a pro-tein kinase specific for SR proteins [2]. Htopo I mayalso be involved in regulation of initiation of transcrip-tion by RNA polymerase II [3]. Htopo I is a singlepolypeptide composed of 765 amino acids residues.The fragment comprised of the first 214 amino acidsresidues, called the N-terminal domain, is not essentialfor relaxing activity [4] but is considered as a mainregion of htopo I that binds proteins [5]. It includes abinding site for substrate proteins phosphorylated byhtopo I/kinase [6] and binds nucleolin [7], SV40 largeT antigen [8], p53 [9], TATA-binding protein [10] andRING/SR proteins called topors [11]. Two proteins,BTBD1 and BTBD2, have recently been shown tobind outside the N-terminal domain [12]. Several otherproteins: PSF/p54nrb [13], protein kinase CK2 [14],proliferation cell nuclear antigen [15] and poly(ADP-ribose) polymerase I [16] also bind to htopo I althoughthe exact binding region has not yet been established.

Another two proteins, called tof1 and tof2, have beenshown to bind to yeast topo I [17].

Recent findings demonstrate that htopo I is fullymobile in vivo and most likely remains free for tran-sient interactions with other proteins in the nucleus[18]. This suggests that htopo I may potentially in-teract with any protein present in this compartmentif the protein contains an amino acids motif recog-nized by htopo I. Up to now, no motif that bindsto htopo I with high affinity has been identified. Toaddress this problem we employed a phage displayprocedure for identification of amino acids sequencesrecognized by the N-terminal domain of htopo I. Inthis report we present several proteins that could bepotential partners of htopo I.

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Materials and methods

Expression and purification of htop(1-214)

The 5′ region of cDNA, coding for the N-terminal domain of htopo I, was amplified byPCR from the pQE30 based plasmid (Qiagen),containing the fragment of htopo I cDNA cor-responding to the first N-terminal 483 aminoacids of human topo I. Oligo pairs: TTCAT-TAAAGAGGAGAAAACT and GTTCAAGCTTGC-CTTCAGGATAGCGCTC were used. The identity ofthe PCR product was confirmed by DNA sequenc-ing. The product was ligated into BamHI and HindIIIsites of pQE30 expression vector (Qiagen). Htop(1-214) polypeptide coded by this plasmid containedamino acids 1-214 of htopo I and additionally 6 Histag coming from the pQE30 vector at its N-terminalend. Htop(1-214) was overexpressed in E. coli strainM15 (Qiagen) and extracted from bacterial cells un-der native conditions according to Qiagen instruc-tion. Cleared lysate was put on heparine agarosecolumn and eluted with 700 mM NaCl, 7 mM 2-mercaptoethanol, 20 mM Tris-HCl, pH 8. The eluatewas subjected to chromatography on Ni-NTA agaroseaccording to Qiagen instruction. SDS electrophore-sis and Western blotting of protein preparations wereperformed according to [19]. Anti-His (Qiagen) andanti-htopo I (TopoGEN) antibodies were used foridentification of htop(1-214).

Expression and purification of DHFR

To express His-tagged DHFR the pQE-40 plasmid(Qiagen) was used. Expression and purification of theprotein was performed according to Qiagen instruc-tion.

Phage display

Ph.D.-7TM kit (New England Biolabs, Inc.) contain-ing complete linear 7-mer peptide library was usedin all phage display experiments according to theproducer instruction. Before applying phage displaylibrary to htop(1-214) prepanning step was performedwith albumin (100 µg/ml) as a target.

Results and discussion

To be sure of specificity of interactions revealed byphage display procedure we first tested identity and

Table 1. The amino acids sequences present in16 phage clones that interact with the N-terminaldomain of htopo I.

Amino acids sequence Number of clones

KLWVIPQ 3

KLWVLPK 2

KLWQVFP 1

KVWILTP 1

KVWTIPR 1

KVWYITP 1

KCCYIPT 4

KGPPITR 1

KVWDLRS 1

YVTREPR 1

purity of htop(1-214). SDS electrophoresis of htop(1-214) preparation revealed only a single band if stainedwith Coomassie blue (Figure 1). Molecular weight ofthe band corresponded to that calculated for htop(1-214). The band was recognized by anti-His and anti-htopo I antibodies (not shown).

The 3rd round of panning of the phage display pro-cedure applied to htop(1-214) revealed several positiveclones. None of the clones gave a positive reactionwith His-tagged DHFR indicating this way that theclones did not bind to the tag sequence added tohtop(1-214) to facilitate purification of the polypep-tide. The amino acids sequences of 16 randomly se-lected clones are shown in Table 1. Considering thatseveral sequences were repetitions of the same clonewe identified 9 different sequences. The consensussequence established on this basis was as follows:

KBWXB(P, T, R, F)X,

where B and X mean hydrophobic and any residue,respectively.

The consensus sequence revealed by phage dis-play procedure was used for identification of the bestmatches on possible protein partners. A search ofSWISS-PROT database, using FASTA and BLASTP,allowed us to identify 124 human proteins that con-tain an exact match of the consensus. 25 of them areexclusively nuclear proteins and additional 10 maytemporarily be located in this compartment. Lookingfor potential protein partners for htopo I among thetwo latter groups we took into consideration proteinsthat were involved in processes linked with htopo Iactivities, interacted with known htopo I partners and

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Table 2. Nuclear and temporarily nuclear proteins containing the consensus sequence recognisedby the N-terminal domain of htopo I

Protein Location of the sequence Cellular process in which

in the protein the protein is involved

Damaged DNA

Binding Protein 2 (DDB2) 268 KIWDLRQ 274 DNA repair

Cockayne

Syndrome WD-

repeat Protein A 212 KLWDVRR 218 DNA repair and transcription

(CSA)

Serine-protein

kinase ATM 486 KIWCITF 492 DNA repair and cell cycle control

Interferon

Regulatory 72 KAWALFK 78 Transcription and other

Factor 4 (IRF4)

BRCA1-associated

Ring Domain 754 KVWKAPS 760 Transcription and other

Protein 1 (BAR1)

ETS-related

protein NET 43 KLWGLRK 49 Transcription

(ELK-3)

TAR RNA-

binding Protein 2 171 KGWRLPE 177 Transcription

(TRBP)

Trithorax-like

Zinc Finger

Protein 1523 KVWICTK 1529 Transcription

(ALL-1/HRX)

Interferon

Consensus

Sequence Binding 58 KAWAVFK 64 Transcription

Protein (ICSB)

Transcriptional

Regulator ISGF3

Gamma Subunit 60 KAWAIFK 66 Transcription and other

(IRTF)

Ski-interacting

Protein (SKIP) 48 KGWIPRL 54 Transcription

Splicing Factor 3b

Subunit 1 252 KIWDPTP 258 RNA metabolism and other

(S3B1/SAP155)

Polymyositis /

Scleroderma

Autoantigen 1 112 KVWQIRV 118 RNA metabolism

(PM/Scl 1)

U1 Small Nuclear

Ribonucleoprotein 185 KGWRPRR 191 RNA metabolism

70 kDa (Ru17/U1snRNP)

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Table 2. Continued

U2 snRNP

Auxiliary Factor

Large Subunit 90 KYWDVPP 96 RNA metabolism and other

(U2AF65)

U6 snRNA-

associated Sm-like 52 KFWRMPE 58 RNA metabolism

Protein (LSM4)

Gemin 4 (GEM4) 710 KYWPLPK 716 RNA metabolism

Cell Division

Protein Kinase 8 92 KVWLLFD 98 Cell cycle control and transcription

(Cdk8)

Human Mitotic

Checkpoint Protein 121 KLWDPRT 127 Cell cycle control

(hBub3)

107 kDa

Retinoblastoma –

associated Protein 819 KIWTCFE 825 Cell cycle control

(Rbl1)

130 kDa

Retinoblastoma –

associated Protein 867 KIWTCFE 873 Cell cycle control

(Rbl2)

Glutamate-rich

WD-repeat Protein 333 KIWDLRQ 339 Cell proliferation

(GRWD)

Symplekin (SPK) 901 KVWEGFI 907 Differentiation marker

Tyrosine-protein

kinase Jak1 74 KLWYAPN 80 Signal pathway

70 kDa WD-repeat

Tumor-Specific

Antigen Homolog 103 KLWRLPG 109 Tumor antigen homolog

(W70T)

DNA Ligase IV

(DNL4) 457 KYWKPFH 463 DNA ligase

55 kDa Regulatory

Subunit of PP2A 116 KLWKITE 122 Regulatory subunit of protein phosphatase

(2ABG)

72 kDa Tat-

interacting Zinc 471 KPWGITA 477 Biological activity of HIV-1 Tat protein

Finger Protein 492 KLWCFTV 498

(HT2A)

Putative ATP-

dependent RNA

Helicase (Y134/ 59 KFWTFFE 65 RNA helicase

KIAA0134)

Recoverin 153 KIWKYFG 159 Photoresponse

(RECO)

Glycogen

Phosphorylase 364 KAWEITK 370 Carbohydrate metabolism

(PHS3)

351

Table 2. Continued

Apoptotic Protease

Activating 757 KLWDATS 763 Apoptosis

Factor 1 (APAF-1)

Colonic and

Hepatic Tumor

Over-expressed 19 KLWKARL 25 Tumor over-expressed protein

Protein (CTOG)

Delta Tubulin 412 KAWNMFA 418 A specialised microtubule

(TBD) system present during reshaping

of the sperm head

cAMP and cAMP-

inhibited cGMP 3′,5′-cyclic 670 KLWPVTK 676 Signal transduction

Phosphodiesterase

10A (PDE 10A)

(or) had a common structure. List of identified nuclearproteins, their cellular functions and location of theconsensus sequences in polypeptide chains are shownin Table 2. It also includes abbreviations of proteins’names used in the text below.

Three identified proteins are linked with DNA re-pair. DDB2 is a part of the complex involved in therepair of the UV-damaged DNA [20]. Recruitmentof htopo I to DNA repair complex is precedented,because another complex involved in excision repaircomprises poly(ADP-ribose) polymerase [21], that in-teracts with htopo I [16]. CSA is most possibly linkedwith transcription-coupled repair [22]. Protein kinaseATM is engaged in the cellular response to DNA dam-age [23]. ATM directly binds and phosphorylates p53[24] which is also known to interact with htopo I [9,10].

Large group of identified proteins is involved intranscription, similarly as it has been proposed forhtopo I [3]. This group includes: interferon-responsivetranscription factor [25], BRCA1-associated RINGdomain protein [26], protein Elk-3 [27], CSA [28],TAR RNA binding protein of HIV-1 [29] and zinc fin-ger ALL-1 protein [30]. Another large group of iden-tified proteins are participants of RNA metabolismconsidered here because of protein kinase activity ofhtopo I that is directed towards splicing proteins [2].This group includes: spliceosomal protein 155 [31],RNase present in the nuclear exosome complex [32],U1snRNP protein 70K [33], U2AF65 protein [34] andSm-like protein binding to snRNA [35]. The proteincomponent of the exosome and U1 70K protein are,

Figure 1. SDS electrophoresis of htop(1-214) (lane 2). Lane 1:molecular weight markers. The gel was stained with Coomassieblue.

similarly to htopo I, recognized by sera from patientswith scleroderma or lupus erythematosus [36].

Several identified proteins are involved in the cellcycle control: ATM kinase [37], cdk8 kinase [38],hBub3 protein [39] and retinoblastoma-like protein 1[40]. The latter protein forms a complex with SV40large T antigen [39], previously identified as htopo I-interacting protein [8].

It is possible that the recognized sequence presentin some proteins listed above is hidden inside theprotein so that they have appeared in the group ofpotential proteins partners simply by chance. Themore interesting is therefore the observation that fourof identified proteins of different cellular functions

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are built similarly one to the other. DDB2, hBub3,CSA and recently identified GRWD protein (Acc. noQ9BQ67) belong to WD-repeat proteins, known tobe involved in assembling macromolecules [41]. Eachprotein contains 5 WD repeats and the sequence recog-nized by the N-terminal domain of htopo I is alwayspresent in the third but not other repeat. Based onthe spatial model of another WD-repeat protein, β-transducin (PDB database, 1tbg chain c), one may findthe motif homologous to that recognized by the N-terminal domain as accessible from the outside of theprotein.

Phage display procedure did not recognize anyprotein previously identified as interacting with the N-terminal domain of htopo I. One possible reason couldbe that the interaction with peptides containing the re-vealed consensus sequence was very strong and theywere overrepresented among clones remaining afterpanning. In contrast, nucleolin has previously beenshown to bind weakly to the N-terminal domain [7].Another reason could be that interaction through theregion other than binding to identified consensus se-quence needs spatial structures not provided by shortpeptides present in phage display library.

Acknowledgements

This work was supported by the State Committee forScientific Research (KBN, grant 6 P04A 069 15).

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