Reveals Homology with Bacterial DNA Ligases (DNA Replication/Accessory Proteins/DNA-Binding Protein) PETER D

Home , Replication factor C

Proc. Natl. Acad. Sci. USA Vol. 90, pp. 11543-11547, December 1993 Genetics Cloning of the large subunit of activator 1 (replication factor C) reveals homology with bacterial DNA ligases (DNA replication/accessory proteins/DNA-binding protein) PETER D. BURBELO*, ATSUSHI UTANI*, ZHEN-QIANG PANt, AND YOSHIHIKO YAMADA* *Laboratory of Developmental Biology, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892; and tProgram in Molecular Biology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Research Center, 1275 York Avenue, Box 97, New York, NY 10021 Communicated by Jerard Hurwitz, August 12, 1993 (receivedfor review July 23, 1993)

ABSTRACT We have cloned a gene encoding a DNA- merase (12). In eukaryotes, the five-protein complex called binding protein by Southwestern screening of a murine cDNA Al (RF-C), consisting in humans of 145-, 40-, 38-, 37-, and library with a double-stranded oligonucleotide containing the 36.5-kDa subunits, is required to assemble PCNA and poly- sequence from the bidirectional promoter of the al and a2 merase 8 on the DNA template (2-4, 13). These five protein collagen IV genes. The middle portion of this 1131-amino acid subunits bind to DNA at the primer termini to form a complex protein has a region homologous to bacterial DNA ligases, and that in the presence of ATP binds PCNA (2, 14, 15). This the more carboxyl portion contains several domains homolo- complex of primed DNA-Al(ATP)-PCNA can then bind gous to p40, p38, p37, and p36.5 subunits of activator 1 (Al, polymerase 6, resulting in effi'cient chain elongation. Addi- also called replication factor C), a human replication protein tionally, the A1-PCNA complex in the presence of single- complex. Western blotting revealed that antiserum generated stranded-DNA-binding protein (HSSB; also called RP-A) is against part of the recombinant protein reacted specifically also able to activate the polymerase e activity at higher salt with the 145-kDa component of the purified human Al com- concentrations (3, 15). plex, indicating that it is the murine counterpart of the Al In this report, we describe the cloning of the murine large p145. Characterization of the DNA-binding activity of the subunit ofAl and find that it has similarity to the four smaller recombinant fusion protein by gel mobility-shift assay revealed subunits ofAl and has a DNA-binding domain similar to that that it had a preference for a run ofpyrimidines on one strand. of bacterial DNA ligases.t Deletion analysis using recombinant proteins revealed that the DNA ligase-like domain was required for DNA-binding activity. The finding that the region required for the binding of MATERIALS AND METHODS murine Al p145 to DNA has similarity to a domain found in Materials. Reagent-grade materials were used throughout. DNA ligases suggests that this region may be utilized by both The following double-stranded oligonucleotides were synthe- proteins in recognizing DNA. sized by the standard phosphoramidite method with an Ap- plied Biosystems model 391 DNA synthesizer: AP-1, AP-2, DNA replication is a highly controlled process regulated by a AP-3, CRE, CIV-1 (5'-TTCCTCCCCTTGGAGGAG- large number of proteins and enzymes which form multiple CGCCGCCCG-3'), and CIV-2 (5'-TTCCTCCCCTTGGAG- protein-DNA and protein-protein complexes (1). The DNA GAGCG-3'). Two variant double-stranded oligonucleotides of polymerases involved in replication have the ability to rapidly the CIV-2 site were also synthesized (the underline denotes duplicate very long stretches ofDNA without dissociation (1). the substitutions): CIV-M1 (5'-TTCCGGTGGTTGGA- Reconstitution experiments using purified human and yeast GGAGCG-3') and CIV-M2 (5'-TTCCTCCCCTTAAIAAA- proteins have revealed that the ability of DNA polymerases 8 GCG-3'). Oligonucleotides were labeled using [y32P]ATP and and e to replicate large stretches ofDNA is not inherent in the T4 polynucleotide kinase. The PQE-10 expression vector and DNA polymerases but rather is due to a number ofassociated nickel chelate Sepharose were obtained from Qiagen accessory proteins dedicated to maintaining the processive (Chatsworth, CA). action of these DNA polymerases (2-4). Southwestern Screening and cDNA Cloning. Poly(A)+ RNA The use of multisubunit proteins to clamp the DNA poly- from a whole 14-day mouse embryo was isolated. Both a merase onto primed DNA is not restricted to eukaryotes but random-primed and an oligo(dT)-primed library were con- is a universal process, also occurring in bacteriophage T4 and structed with AZAPII as described by the manufacturer Escherichia coli (reviewed in refs. 5-7). In each system a (Stratagene), using 4 ,ug of poly(A)+ RNA for each. Approx- multisubunit polymerase accessory protein complex [eukary- imately 3 x 106 recombinants were obtained for each library. otic activator 1 (Al, also called replication factor C, RF-C), The murine 14-day random primed library was screened by T4 gene products 44/62 (g44/62), E. coli ycomplex] functions the Southwestern technique using a double-stranded CIV to recognize the primed template and load a second poly- oligonucleotide according to Vinson et al. (16). Positive merase accessory protein [eukaryotic proliferating-cell nu- clones were plaque-purified following four rounds of screen- clear antigen (PCNA), T4 g45, E. coli /3] on the DNA in the ing. Eight additional clones were obtained using various 5'- presence of ATP. In E. coli, the y complex (y66'x subunits) and 3'-end fragments as probes with both the random and places a dimer of the clamp protein (,8 subunit) on the DNA, oligo(dT) libraries. Plasmids were banded in cesium chloride which then recruits the DNA polymerase III core (8-10). The and sequenced by the dideoxynucleotide method with Se- (3 dimer has been shown to form a ring-shaped clamp around quenase (United States Biochemical). Sequencing used a the DNA (11). In T4, ATP is required for the association of combination of controlled unidirectional deletions and se- the g44/62 (4:1) complex and the g45 protein on primed DNA, quence-deduced oligonucleotide primers. and ATP hydrolysis is required to add the g43 DNA poly- Abbreviations: Al, activator 1; PCNA, proliferating-cell nuclear The publication costs of this article were defrayed in part by page charge antigen. payment. This article must therefore be hereby marked "advertisement" iThe sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. U01222). 11543 Downloaded by guest on October 1, 2021 11544 Genetics: Burbelo et al. Proc. Nati. Acad. Sci. USA 90 (1993) Recombinant Protein and Antiserum Production. Recom- teins (19). A Southwestern screening protocol was used to binant proteins were produced from various restriction frag- clone proteins that interact with the CIV element. This ments or PCR fragments of the Al p145 (MSW) cDNAs. protocol is based on phage recombinant protein recognition These fragments were subcloned into the BamHI-HindIII or of the radioactive CIV DNA binding site. Southwestern BamHI-Sal I site of the PQE-9 bacterial expression vector screening of 8 x 10-'phages with a 27-bp CIV probe contain- (Qiagen) containing sequence encoding a tag of six histidine ing the large stem-loop structure of the bidirectional pro- residues at the N terminus. All the constructs discussed in moter gave 8 positive plaques after the first screening. Two this report were confwrmed by DNA sequence analysis. clones remained positive in later screenings and were plaque Fusion proteins were extracted with 6 M guanidine hydro- purified. Sequencing of both clones revealed that they were chloride and purified on a nickel-affinity column as described identical and contained a 1.4-kb open reading frame. These by the manufacturer. Affinity-purified fusion protein (amino clones were designated MSW for mouse Southwestern. acid residues 203-545) was used as antigen to immunize a When the amino acid sequence corresponding to the MSW rabbit and antiserum was obtained after four injections. open reading frame was used to search GenBank and EMBL Western Blotting. Human Al was purified from HeLa cells data banks (April 1991), we found that --90 amino acids were (2). After SDS/10% PAGE, proteins were transferred to 46% identical to a portion of Thermus thermophilus DNA nitrocellulose. After blocking with 5% bovine serum albumin ligase (20, 21). No significant similarity of MSW to DNA and 1% gelatin for 41wr, the membrane was incubated for 1 hr ligases was noted outside of this domain. Northern analysis with the primary antibodies at the concentrations indicated. revealed two of %-4.6 and ==5.4 kb in various were with a transcripts Antibody-antigen complexes detected biotin! mouse tissues with an unusually high level of expression in avidin-based, alkaline phosphatase-linked immunodetection the testis and moderate levels of expression in thymus, system (Vector Laboratories). spleen, and kidney (data not shown). The apparent DNA- Electrophoretic Mobility-Shift Assay. DNA binding to the the site and MSW fusion proteins was examined by a gel shift assay using binding specifi'city for CIV striking homology complementary double-stranded oligonucleotides. Oligonu- between a mammalian DNA-binding protein and that of cleotides were labeled with [y-32P]ATP and polynucleotide bacterial DNA ligase surprised us. For this reason, the kinase to similar specific activities. Reaction mixtures used in complete sequence of MSW was determined. Screening of the gel shift assay consisted of affinity-purified protein, both the oligo(dT)- and random-primed 14-day mouse em- labeled DNA (20,000 cpm/0.5 ng), buffer [10 mM Hepes, pH bryo libraries with the original 1.4-kb insert yielded eight 7.8/100 mM KCIl/ MM MgCl2/20% (vol/vol) glycerol]. overlapping clones containing the full MSW coding se- After incubation at room temperature for 10 min, the mixture quence. The composite sequence for MSW is 3980 bp long was resolved in a 4% polyacrylamide gel in 63 mM Tris-HCI/ and codes for an open reading frame of 1131 amino acids (Fig. 372mM glycine/2 mM EDTA, pH 8.5 for 1 hr at 150 V at 40C. 1). The nucleotides surrounding the initiation methionine codon are identical in both the human and mouse sequence (data not shown) and match the consensus ATG start codon RESULTS as proposed by Kozak (22). Both the human and murine Cloning of the Murine Al p145. Isolation ofmurine Al p145 proteins were 87% identical over the first 83 amino acids, and emerged from investigations of transcription factors that an in-frame stop codon was located 58 bp upstream of the interact with the bidirectional promoter for the murine al1 and translation initiation codon of the human gene. These results cr2 collagen IV chains (17, 18). This 130-bp bidirectional strongly suggested that the full coding regions of the murine promoter contains a large stem-loop structure (CIV) which MSW had been obtained and that the 5' untranslated regions has been shown to interact with several DNA-binding pro- are different in the two species.

M ]D I P K F G V I S S G K K P N E K H E T E 11" K C F E V N N K H D K K F Q H4 K 80 312 K K R I I Y D D E S S E T V Q K K E E L L 90 O Y K P G K V Q K D P V Y S E D E D D D CEK K A 120 A SK 8 K N G S N S Y L G T S H V K E N E N V K T K 150 981 P 121 N K P L S I K L T S V L D Y F G S v Q P S G K K H 1810 V T S K R F S K Q N T E D S R L N D I A K L Q L D E 210 D A E L E Q L H E D E E F A R L A L L G E E K I1K K A 240 241 REKD S E G E S F S V Q D D L S K A RE Q K PHN K A 270 271 E s LF S T R P K T S A K H G K G P A S E D A K Q P C K 5 301 30 A H EK E P C S S K S K L L M K KER E s Y N F T 330 P P V 33 E LL A A E K T E K G E K T T KFE T K O P T K 360 361 R E S V S E D) K K R T N Y A R S Y L N, R 8 G P K A E 390 391 L G SEKE P K~ A E5 C L I T G L5 S I E R5 420 421 D EARKS L T N E NDG D 450 4051 S GD0 SEK S I I L K T L L R T H A K P P. 480 481 GEERSEK A G E K K L E R T K H D K K K Q 510 511 GREETK A K K V K C K L T L L K N MEK A V 540 541 FEKE A S T P R G L D K T G N R S S N K E C L L W 570 571 V DRKY K P A S L K N I G D Q C A N K L L EMw L R 600 60 I N W H KS S P E E K K N A A K F K L A S KGD D C S S F K A 630 83 1 A L LESG P G G K T T T A S L V C 8 L G Y V E L N 6860 661 A S GTER S K N L K A V V A S L N N T S I K F Y T S G 890 691 A A P S V S A R A L I N D E D G N A G N E D R G G I E 720 721 L I G L I K N K I P I C N E N D N N P K P S L V Y 750 5 1 C F D L E F Q R P R E Q I K K A N L S I A F K E G L K I P 7 80 8SI P P AWMN 8 L G A N Q D V C Q L N L S N C A Q S K 810 811 AL T Y D Q K A D Q A K F D I TH PPF D T PR F 940 841 A ACGERE T A H S L M D K S L F F D Y S P 5 H A LOF 870 71 E N Y L H V K P A A G G D R K H L L L S R D S 2. A 9100 D G D L V D N Q R K H L P T AZ I S L. Q V L C 930 ELMERG Y H F P S N G K H S T G K H D P, v 960 Sr Q D L S L H L R T Y K T N N D Y L S H R D L 905 L 991 V R P L T S Q U V E A H 'T K H D T Y Y N E D A L L F E lG02O 1021 N I M E V S G G K P F K D P V K A A T R F Y 1050 1 0 51 HEREA H L D L Q K S R L G E V4 L D S E O 1080 C, S K 1021I REEF Q E D P Q E K Q D. F N I K K F T P O 1110 1 1 K P SK S E E 8 S K G K G K N 1131 FIG. 1. Deduced primary structure ofmurine Al p145 (MSW). The "ligase-like" domain is underlined. Potential CDC2 kinase phosphorylation sites are shown with a broken underline. The region with homology to the four small subunits of Al is shaded. Downloaded by guest on October 1, 2021 Genetics: Burbelo et al. Proc. Natl. Acad. Sci. USA 90 (1993) 11545

A DNA Ligase Domain MSW 9498 KGAENCLEGLTFVITGVLESIERDEAKSLIERYGGKVTGNVSKKTNYLVMGRDSGQSKSDKAAALGTKTLDEDGLLDLIR'TMPGKRSK 485 TrT 59 C KGGE -ALKGLTFVITGEL --SRPREEVKALLRRLGAKVTDSVSRKTSYLVVGENPG -SKLEKARALGVPTLTEEELYRLLEARTGKKA* ZMO 644 ELASSPLSGK I IV?TGSLQKI RDEAKRQAENLGAKVASSVSRKTNZVVAGEAAG- SKLSKAKELD__ISIDEDWWHRTVENGGQESIK SPO 275 GN- QSQNRK IGVIKR-LSSCEG-AEPKYLIRALEGKIRLQLAEKT.'VAL.ANA-AQYHADKNGEKLSQQDRIEGEQILRDVYCQL:.SY Co 5 c2 EE :}DPFA.{GKTV,VLGC-SLS QMSRDDAKARLVELC-AKVAGSVSKKDLD--LTVAGEA.mAGSKLAKAQWLC-'LIVIDEAEMLRT-LGS* H8R: 47 2 SRNKDEVKAMIEKLGGKLTG-TANKA B Al Accessory Protein Domain A-P b-ndirTg site MSEW; 5 *4 KEF:CL ;WvDKYKPASLKNT IGQQGDQSCANK-LRWLRNWHK-SPEEKKWAAKFGK-ASKDDGSS-K-ALLSGPPGVGKTTTASr cQ 650 Al- 40 2 (CH-hYELPW'VEKYWPVKLNEIVGNEDrl'MSRLEVFAREG(NVPWNI ---- - _I TAGPPGTGKTTSILCL AR 91 A1- 3 7 34 KAKPVPWVEKYR;PKCVDEVAFQEE\VAVLKKSLEGADL PNL ------LFYGPPGTGKTST3ILAAAR 93 Al-136 KIRNL?WVEKYRPVKLNEL.ISHQDILSTIQKFINE-DR-PHL ------LLYGPPGTGKTSTILACAK A- 38 GPSG-AGKKTEJrvMCTLRE ------LY -GV-GVEKLRIEHQTIT MEW 651 EL -GY ----SY -VELNASDTRSKNSLKAVVAESLNNTSIKG-FYTSGAAPSVSARH -ALIMDEVDGMAGNEDRGGIQELIGLTIKHTKI 730 Al 40 92 ALLGA --LKDAMLELNASNDR---- GIDVVRNKIKMFN ---- QQKVT-LPK --GREHKIILDEADSMTDGAQQALRRTMETIYSKTTRF ] 66 Al 17- 94 EL?GPELRFR.RVLELNASDER ---- GIQVVREKVKNFI/QLTVSGSWRSDGKP-5CCPPF'FK _VTIEDEADSMrSAAQAALRRTMEKESKTTRF l7b QLYKDKEC;SMVLELNASDDR ---- GI T-IC-PP --- SFAS RT'IT: - -GPLVI DEADAMTIQDGAQNWALRRVIEKFTETRF Al 16 r.P.CKKXK_EcST-:ASSNYHTEN- PS DVVIQEMLKT VAQSQQLElTNSQ --RDFKVVJ-LlTEVD--L:?-KDAQ-HALR?RTMIMKYMS lCC L 731 pT ICMCNDRNHPKIRSLVHYCFDLRFQRPRVEQIKSAMLSIAFKEGLKIPPPAMNEIILGANQDVRQVLIINL -S-MWCAQSKXAITYDQ 8 1 Al - 43 167 ALACNASDKIITEPIQSR---CAVLRYTKLTDAQILCPRLMNVIEKERVPYTDDGLEAIIPTAQGDMRQAL NNLQS-TFSGEGTINSEN 249 _A 7 TS7 T i CLICLYVERIC ICt.YVF,RI SNILTC{ CSKFRFK-P SDKI-QQQr-LLDIAKKENVPISHR--GIAFN q3 QI I NEVLWSGLRKITFQSA-'VSH K;..ST' EGDT RKAiTFLQSA-~TRLiGKIETGG KE I T EK 260; CLI C:4YLSK:- IPALQSR --- CTRFRFGPLTPELMVPRREHVV%!EEEKVDISEDGMKALVTLSSGDMRRALNILQS-TNMAFGKVTE'ET Al-38 LTCCNSTSKVIPPIRSR ---CL.AVRVPAPSIEDICHVLSTVCKKEGLNLPSQLAHRLAEKSCRNLRKALL-MCEACRVQQYPFTADQ FIG. 2. Domains within Al p145 (MSW) have homology with other DNA ligases and human Al accessory proteins. (A) Amino acid sequence comparison of Al p145 (MSW) with several DNA ligases. Identical residues are denoted by the shaded blocks. TTH, Thermus thermophilus (20, 21); ZMO, Zymomonas mobilis (23); ECO, Escherichia coli (24); SPO, Schizosaccharomycespombe (25). A short region ofhomology (HRI) is also found with human poly(ADP-ribose) polymerase (26-28). (B) Al p145 (MSW) and the four smaller subunits of Al aligned to show maximum homology. Identical residues are denoted by the shaded blocks. The protein sequence of Al p40 is from ref. 29; that of Al p37 from ref. 30, and those of Al p38 and Al p36 from ref. 31, in which the N termini of both proteins are still missing. MSW codes for a very basic protein with a predicted antiserum showed no such reactivity (Fig. 3, lanes 2 and 4). molecular mass of 127 kDa and predicted pI of 10.1. A search A monoclonal antibody to the Al p145 subunit also detected of the GenBank protein data (May 1993) with the full-length the same-sized protein (Fig. 3, lane 1). From these results, we MSW protein sequence revealed several interesting similar- conclude that MSW is most likely the p145 subunit ofAl and ities. Strong homology of amino acids 404-484 of MSW was will refer to it as Al p145 in the remainder of the text. These found with the very carboxyl end ofseveral prokaryotic DNA results also confirm that the Al p145 subunit is conserved ligases (Fig. 2A). The greatest similarity was seen with the between mouse and human. DNA ligases of T. thermophilus and Z. mobilis (23), while DNA-Binding Domain of Al p145. The Al p145 sequence less similarity was noted with E. coli DNA ligase (24). Weak did not resemble any characterized DNA-binding or dimer- similarity (22% identity) of this domain was detected within ization domains. The region responsible for DNA binding the yeast Sch. pombe DNA ligase (25). No significant simi- was determined by ligating various DNA fragments in frame larity was noted with human DNA ligase I. Human poly- in a position carboxyl-terminal to the segment of the PQE (ADP-ribose) polymerase (26-28) also showed a 25-amino vector encoding the polyhistidine tag. Although the cDNA acid segment of similarity within the ligase-like domain of for Al p145 codes for a large protein, we concentrated our MSW (Fig. 2A). This region ofpoly(ADP-ribose) polymerase efforts on the region spanning the original phage clone (amino corresponds to the automodification domain (32), which is acids 121-540), which had DNA-binding activity (Fig. 4A). thought to regulate binding to damaged DNA (33). These cDNAs were expressed in E. coli and bacterial extracts MSW also showed a region ofhomology spanning 200 amino acids with the four smaller subunits of Al (Fig. 2B). The Al containing recombinant proteins were purified by nickel- protein together with PCNA acts to tether DNA polymerase kDa 8 (e) to the DNA for rapid and highly processive synthesis 208 - (reviewed in ref. 34). The greatest similarity was seen with the - Al -p145 p40 subunit (29) with decreasing degrees of with the similarity wE,,. i...... p38 (31), p37 (30), and p36 (31) subunits. The regions of 101 - similarity include highly conserved ATP nucleotide and Mg2+ binding sites. Several other regions of similarity were also 71- identified that may be involved in protein-protein interactions of this family of proteins. For example, the sequence WV(D/ E)KY(K/R)P (at 570 of MSW) was found in all five subunits. 43- MSW also showed some homology (data not shown) with phage T4 g44 protein, an accessory protein for the T4 DNA polymerase. This similarity suggested that MSW might be a member of the Al family of proteins. 29- Western Blotting Identifies MSW as Al p145. Since MSW had significant amino acid homology with the four smaller subunits of Al and coded for a protein similar in size to the 1 2 3 4 5 large subunit, the possibility existed that MSW was the large subunit. Rabbit polyclonal antiserum was generated against FIG. 3. Anti-MSW (Al p145) antiserum reacts with the large subunit of Al. Human Al complex was purified as described (2). affinity-purified recombinant MSW (residues 203-545) to test Seventy-five nanograms of Al was electrophoresed in an SDS/10%6 whether MSW was part of the Al complex. Immunoblotting polyacrylamide gel and transferred to nitrocellulose. The Western of 75 ng of purified human Al protein complex revealed that blot was probed with a monoclonal antibody to the Al p145 protein the ~~~~~~~~~~~~~~~~~~~~~..... immune MSW antiserum reacted with the 145-kDa ...... : dalton (lane 1) or with preimmune serum (lanes 2 and 4) or immune serum component ofAl (Fig. 3, lanes 3 and 5), whereas preimmune to MSW (lanes 3 and 5) at 1:400 and 1:800 dilutions, respectively. Downloaded by guest on October 1, 2021 11546 Genetics: Burbelo et al. Proc. Natl. Acad. Sci. USA 90 (1993) C A B C D E F G

A DNA BINDING B PQE-102 203 --- 545 A B C D E F G +.. PQE-104 203 -379

PQE-1 10 364 707 -I-

PQE-1 08 364 _- 545 + PQE-1 1 2 485 - 545

PQE-1 18 364 -U 423 FIG. 4. DNA-binding activity ofvarious cloned domains ofAl p145. (A) Localization ofthe DNA-binding domain. Various cDNA fragments corresponding to the amino acid residues of Al p145 were expressed in E. coli and tested for their DNA-binding activity. Black box denotes the region ofsimilarity with the bacterial DNA ligases. (B) Bacterial expression ofvarious protein fragments ofAl p145. Various cDNA fragments of Al p145 were subcloned in frame in the PQE-10 bacterial expression vector. After large-scale culture, the fusion proteins containing the polyhistidine tag were purified by nickel-affinity chromatography. Purified recombinant proteins (4 p,g) were electrophoresed through an SDS/4-16%o polyacrylamide gel and stained with Coomassie brilliant blue. Lanes: A, molecular size markers (43, 29, 18.4, 14.3, 6.2, and 3 kDa); B, PQE-102; C, PQE-104; D, PQE-ll0; E, PQE-108; F, PQE-112; G, PQE-118. (C) Binding activity of various expressed regions of Al pl45. Gel shift reaction mixtures contained purified proteins (200 ng), buffer, and 32P-labeled double-stranded CIV (10,000 cpm) as the target. Lanes: A, no protein; B, PQE-102; C, PQE-104; D, PQE-108; E, PQE-110; F, PQE-112; G, PQE-118. After incubation for 10 min at room temperature, the reaction mixtures were electrophoresed in a nondenaturing 6% polyacrylamide gel at 4°C. affinity chromatography. An SDS/polyacrylamide gel ofthese the binding of PQE-108 to the CIV-2 double-stranded oligo- purified recombinant proteins is shown in Fig. 4B. These nucleotide. CIV-M1 contained the sequence GGTGG as a proteins were used in a gel shift assay using the duplex CIV substitution for TCCCC, and in oligonucleotide CIV-M2, oligonucleotide to delineate the DNA-binding domain. With GAGGA was replaced by AAATA. The CIV-M1 oligonucle- removal of 82 amino acid residues from the amino-terminal otides retained DNA-binding activity (Fig. 5, lane P), while no region, PQE-102 was still found to possess DNA-binding binding to the CIV-M2 oligonucleotide was detected (Fig. 5, activity (Fig. 4C, lane B), while the N-terminal region alone lane J). These results suggest that Al p145 has a preference for (PQE-104), amino acids 203-379, was found to lack DNA- the GAGGA motif (or CTCCT on the complementary strand) binding activity (Fig. 4C, lane C). Construct PQE-ll0 (Fig. 4C, within the CIV site, although additional higher-affinity sites lane D) containing the ligase-like domain and alarger carboxyl- may exist distinct from these sites. terminal region including the potential Mg2+ and ATP binding sites was also found to be active. Recombinant protein PQE- 112, representing the carboxyl-terminal region, amino acids DISCUSSION 485-545, also failed to show DNA-binding activity. Construct The Al complex (RF-C) is a multimeric protein complex PQE-118, representing a truncation in the ligase-like domain, five different subunits We have cloned the pl45 also failed to bind to the CIV oligonucleotide (Fig. 4C, lane G). containing (2). The smallest region retaining sequence-specific DNA-binding subunit of Al by Southwestern screening of a murine cDNA activity contained amino acids 364-545 (Fig. 4C, lane E). library with a C+T-rich double stranded oligonucleotide from Thus, at least part ofthe region of similarity between Al p145 the bidirectional promoter of the collagen IV gene. Our and the DNA ligases is important in the DNA-binding activity evidence supporting the conclusion that this cDNA codes for ofAl p145. Since both Al and DNA ligases require DNA ends the p145 subunit includes the following: (i) the MSW cDNA for their activities, this region of similarity may be involved in ABC DE H I J KLM NOP recognizing this structure. FG DNA-Binding Activity ofAl p145. A gel mobility-shift assay was used to assess the DNA-binding specificity ofAl p145. In these assays, a constant amount of PQE-108 was tested with a panel of different double-stranded oligonucleotides in the presence of plasmid DNA (pBluescript) as nonspecific competitor. Poly(dI-dC) was not used as a nonspecific competitor since it strongly inhibited the DNA-binding activity (data not shown). In these gel mobility-shift assays, the PQE-108 protein was found not to bind to AP-1, AP-2, AP-3, and CRE binding sites (Fig. 5, lanes B, D, F, and H, respectively). However, the binding of the PQE-108 protein was observed FIG. 5. Gel mobility-shift assay reveals that Al p145 has a pref- with the CIV-1 double-stranded oligonucleotide (Fig. 5, lane erence for certain DNA substrates. Assays were performed with 50 ng a variant of the CIV-1 site, was also of PQE-108 protein (lanes B, D, F, H, J, L, N, and P) or without L). CIV-2, 6-bp-shorter protein (lanes A, C, E, G, I, K, M, and 0). Double-stranded oligo- able to bind the PQE-108 protein (Fig. 5, lane N). Additional nucleotides (0.8 ng) were labeled to similar specific activities by using experiments under similar conditions revealed that the PQE- [y-32P]ATP and T4 polynucleotide kinase: AP-1 (lanes A and B), AP-2 108 protein had a much greater affinity for the CIV site than (lanes C and D), AP-3 (lanes E and F), CRE (lanes G and H), CIV-M2 other double-stranded oligonucleotides such as NF1, Spl, (lanes I and J), CIV-1 (lanes K and L), CIV-2 (lanes M and N), and GRE, or single-stranded DNA (data not shown). Two different CIV-Ml (lanes 0 and P). Specific binding of PQE-108 protein is mutated oligonucleotides were utilized to more precisely map indicated by the arrow. Downloaded by guest on October 1, 2021 Genetics: Burbelo et al. Proc. Natl. Acad. Sci. USA 90 (1993) 11547

codes foraprotein whose size is similar to the p145 subunit and system and expression in eukaryotic cells should define more whose amino acid sequence has considerable homology to the precisely the role of Al p145. other Al subunits; (ii) an antibody raised against a recombinant protein consisting ofamino acids 203-545 ofMSW reacts We are grateful to Drs. N. Nossal and M. Iadarola for critical with purified human Al p145; and (iii) recombinant MSW has reading of this manuscript. The studies carried out at Memorial binding activity for double-stranded DNA. Previously, DNA Sloan-Kettering Cancer Center were supported by National Insti- footprinting and UV crosslinking experiments demonstrated tutes of Health Grant GM34559 to Dr. J. Hurwitz. that Al binds to the primer-template junction through the 1. Kornberg, A. & Baker, T. A. (1991) DNA Replication (Free- 145-kDa subunit (4). In those studies, Al preferred target man, New York). sequences ofpoly(dA)/oligo(dT) to more complex sequences. 2. Lee, S.-H., Pan, Z. Q., Kwong, A. D. & Hurwitz, J. (1991) J. The data presented here support the finding that the large Biol. Chem. 266, 594-602. subunit has binding activity for double-stranded DNA and 3. Lee, S.-H., Kwong, A. D., Pan, Z. Q., Burgers, P. M. J. & Hurwitz, J. (1991) J. Biol. Chem. 266, 22707-22717. may have a preference for some DNA substrates. Further, the 4. Tsurimoto, T. & Stillman, B. (1991) J. Biol. Chem. 266, finding that the region required for Al p145 binding to DNA 1950-1960. has amino acid sequence similarity to a domain found in DNA 5. Nossal, N. G. (1992) FASEB J. 6, 871-878. ligases suggests that this region may be utilized by both 6. Young, M. C., Reddy, M. K. & vonHippel, P. H. (1992) Bio- proteins in recognizing DNA. chemistry 31, 8675-8690. The amino acid sequence of the 145-kDa subunit and the 7. O'Donnell, M. (1992) BioEssays 14, 105-111. 8. Wickner, S. H. (1976) Proc. Natl. Acad. Sci. USA 73, 3511- four smaller subunits of Al all show well-conserved, pre- 3515. dicted nucleotide and Mg2+ binding sites. This structural 9. Maki, S. & Kornberg, A. (1988) J. Biol. Chem. 263, 6561-6569. redundancy in all five of the subunits is consistent with the 10. Onrust, R., Stukenberg, P. T. & O'Donnell, M. (1991) J. Biol. DNA-dependent ATPase activity ofAl, which is essential for Chem. 266, 21681-21686. Al-dependent stimulation of polymerase 8 and E activity (3, 11. Kong, X.-P., Onrust, R., O'Donnell, M. & Kuriyan, J. (1992) 13, 15). Several other regions ofAl p145 show Cell 69, 425-437. homology with 12. Capson, T. L., Benkovic, S. J. & Nossal, N. G. (1991) Cell 65, the other subunits, including amino acids 333-456 and 456- 249-258. 666. These regions may be important in protein-protein 13. Tsurimoto, T. & Stillman, B. (1990) Proc. Natl. Acad. Sci. USA interactions with the four other subunits, PCNA, the single- 87, 1023-1027. stranded-DNA-binding protein HSSB, or the DNA polymer- 14. Lee, S.-H. & Hurwitz, J. (1990) J. Biol. Chem. 266, 5672-5676. ases. Recently, the Al p37 subunit was found to bind to 15. Burgers, P. M. J. (1991) J. Biol. Chem. 266, 22698-22706. primer ends, while the 16. Vinson, C. R., LaMarco, K. L., Johnson, P. F., Landschulz, Al p40 subunit showed no such W. H. & McKnight, S. (1988) Genes Dev. 2, 801-806. activity (35). Although no significant amino acid similarity 17. Burbelo, P. D., Martin, G. R. & Yamada, Y. (1988) Proc. Natl. was noted between the DNA-binding domain ofp145 and any Acad. Sci. USA 85, 9679-9682. of the other subunits of Al, there may be some cooperative 18. Kaytes, P., Woods, L., Theriault, N., Kurkinen, M. & Vogeli, DNA-binding activity among the subunits. G. (1988) J. Biol. Chem. 263, 19274-19277. Given the role ofAl p145 in DNA replication and similarity 19. Bruggeman, L. A., Burbelo, P. D., Yamada, Y. & Klotman, with P. E. (1992) Oncogene 7, 1497-1502. DNA ligases, one might expect Al p145 to have spe- 20. Barany, F. & Gelfand, D. H. (1991) Gene 109, 1-11. cialized specificity forthe 3' hydroxyl end ofthe primed DNA 21. Lauer, G., Rudd, E. A., McKay, D. L., Ally, A., Ally, D. & template. However, poly(dA)-poly(dT) primer-template Backman, K. C. (1991) J. Bacteriol. 173, 5047-5053. pairs were no more effective than double-stranded oligonu- 22. Kozak, M. (1988) J. Biol. Chem. 266, 19867-19870. cleotides ofthe similar size as competitors ofAl p145 binding 23. Shark, K. B. & Conway, T. (1992) FEMS Microbiol. Lett. 96, to the CIV oligonucleotides. The cloning of Al p145 using a 19-26. 24. Ishino, Y., Shinagawa, H., Makino, K., Tsunasawa, S., Saki- region of the bidirectional promoter of collagen IV may have yama, F. & Nakata, K. (1986) Mol. Gen. Genet. 204, 1-7. occurred because Al p145 prefers binding to sequences 25. Barker, D. G., White, J. H. M. & Johnson, L. H. (1987) Eur. within the CIV double-stranded DNA or, alternatively, be- J. Biochem. 162, 659-667. cause the nucleotide sequence of the CIV favors hairpins or 26. Cherney, B. W., McBride, W. O., Chen, D., Alkhatib, H., looping DNA structures. These results were substantiated in Bhatia, K., Hensley, P. & Smulson, M. E. (1987) Proc. Natl. the gel shift assay, in which Al p145 appeared to have a Acad. Sci. USA 84, 8370-8374. preference for the CIV site over other binding sites such as 27. Kurosaki, T., Ushiro, H., Mitsuuchi, Y., Suzuki, S., Matsuda, M., Matsuda, Y., Katunuma, N., Kangawa, K., Matsuo, H., AP-1, AP-2, CRE, NF-1, GRE, and Spl. The nucleotide Hirose, T., Inayama, S. & Shizuta, Y. (1987) J. Biol. Chem. sequence CTCCT (or GAGGA on the other strand) was part 262, 15990-15997. of this high-affinity binding site. Interestingly, several other 28. Uchida, K., Morita, T., Sato, T., Ogura, T., Yamashita, R., replication proteins have also been shown to have preferen- Noguchi, S., Suzuki, H., Nyunoya, H., Miwa, M. & Sugimura, tial DNA-binding activity. For example, human RP-A and T. (1987) Biochem. Biophys. Res. Commun. 148, 617-622. yeast RP-A bind to pyrimidine-rich strands with a 50-fold 29. Chen, M., Pan, Z. Q. & Hurwitz, J. (1992) Proc. Natl. Acad. higher affinity than purine-rich strands (36). A 90-kDa Sci. USA 89, 2516-2520. 30. Chen, M., Pan, Z.-Q. & Hurwitz, J. (1992) Proc. Natl. Acad. ATPase associated with the yeast DNA polymerase a-pri- Sci. USA 89, 5211-5215. mase complex also showed 3-fold greater activity with oli- 31. O'Donnell, M., Onrust, R., Dean, F. B., Chen, M. & Hurwitz, gopyrimidines than with oligopurines of the same size (37). J. (1993) Nucleic Acids Res. 21, 1-3. The preference for certain sequences, such as that within the 32. Kameshita, I., Masuda, Z., Taniguchi, T. & Shizuta, Y. (1984) collagen IV bidirectional promoter, may suggest that Al p145 J. Biol. Chem. 259, 4770-4776. has additional functions. For example, p145 may have aux- 33. Zahradka, P. & Ebisuzaki, K. (1982) Eur. J. Biochem. 127, iliary activity at replication origins, in DNA repair, and/or in 579-585. transcription. Another possibility is that there is some se- 34. Stiliman, B. (1989) Annu. Rev. Cell Biol. 5, 197-245. 35. Pan, Z.-Q., Chen, M. & Hurwitz, J. (1993) Proc. Natl. Acad. quence preference for the initial binding of Al to the nascent Sci. USA 90, 6-10. chain during replication. Alternatively, this preference for 36. Kim, C., Snyder, R. 0. & Wold, M. S. (1992) Mol. Cell. Biol. certain DNA sequences may be masked by the other domains 12, 3050-3059. of Al p145 and/or the other accessory proteins. Studies with 37. Biswas, E. E., Ewing, C. M. & Biswas, S. B. (1993) Biochem- full-length recombinant Al p145 using the in vitro replication istry 32, 3020-3026. 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