Bacteriophage PRD1 DNA Polymerase: Evolution of DNA Polymerases (Lipid-Containing Phage/Protein-Primed DNA Replication) GUHUNG JUNG, MARK C

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Proc. Natl. Acad. Sci. USA Vol. 84, pp. 8287-8291, December 1987 Biochemistry Bacteriophage PRD1 DNA polymerase: Evolution of DNA polymerases (lipid-containing phage/protein-primed DNA replication) GUHUNG JUNG, MARK C. LEAVITT, JUI-CHENG HSIEH, AND JUNETSU ITO Department of Microbiology and Immunology, The University of Arizona Health Sciences Center, Tucson, AZ 85724 Communicated by Carl S. Marvel, August 10, 1987

ABSTRACT A small lipid-containing bacteriophage PRD1 sensitive to the drug aphidicolin, which is a specific inhibitor specifies its own DNA polymerase that utilizes terminal protein of eukaryotic DNA polymerases a and A (10, 16). as a primer for DNA synthesis. The PRD1 DNA polymerase In this communication, we report the nucleotide sequence gene has been sequenced, and its amino acid sequence has been and the deduced amino acid sequence of PRD1 DNA deduced. This protein-primed DNA polymerase consists of 553 polymerase.* We have compared the amino acid sequence of amino acid residues with a calculated molecular weight of this polymerase with those of other DNA polymerases and 63,300. Thus, it appears to be the smallest DNA polymerase found that PRD1 DNA polymerase, the smallest known DNA ever isolated from prokaryotic cells. Comparison of the PRD1 polymerase possessing a proofreading function, has partial DNA polymerase sequence with other DNA polymerase se- homology with many other DNA polymerases including quences that have been published yielded segmental but sig- phage T4 DNA polymerase. These results together with those hificant homologies. These results strongly suggest that many of others (17-22) suggest strongly that many prokaryotic and pi-okaryotic and eukaryotic DNA polymerase genes, regardless eukaryotic DNA polymerase genes, regardless of size, have of size, have evolved from a common ancestral gene. The evolved from a common ancestral gene. The results also results further indicate that those DNA polymerases that use suggest that DNA polymerases that use either an RNA or either an RNA or protein primer are related. We propose to protein primer have arisen from a common ancestral progen- classify DNA polymerases on the basis of their evolutionary itor. relatedness. MATERIALS AND METHODS Since DNA contains the genetic information of an organism, accurate DNA replication is one ofthe most important events DNA Sequencing. A recombinant plasmid (clone 3) that ofthe life cycle ofan organism. DNA polymerases are the key contains PRD1 genes 1 and 8 was kindly provided by L. enzymes catalyzing the accurate replication of DNA. How- Mindich (Public Health Research Institute ofthe City ofNew ever, none of the known DNA polymerases can initiate de York) (13). The plasmid DNA was first cleaved with Pst I, novo synthesis of a DNA chain without a primer (1). Gen- and the resulting DNA fragment containing genes I and 8 of erally, small RNA molecules are used as primers. These PRD1 was recloned into the bacteriophage vector M13mp9 provide a 3'-hydroxyl group for DNA chain elongation (1). (23). We then generated a nested set ofdeletions according to However, a different type of DNA polymerase has been the method of Dale et al. (24). Each set of deletions was identified in various systenis (2-7). These enzymes utilize sequenced by the dideoxy chain-termination method of proteins as primers for the initiation of DNA synthesis. Sanger et al. (25). For confirmation, some regions were Whereas these DNA polymerases are distinctive in the way sequenced by the method of Maxam and Gilbert (26). they initiate DNA synthesis, their mechanism for achieving Computation. Protein data base searchest were performed high fidelity of DNA replication appears to be similar to that by a FASTP microcomputer program (27). DNA polymerase of the RNA-primed DNA polymerases. Thus, all known nucleotide sequences were obtained from the GenBank data DNA polymerases acting at protein, primers also possess a baset and translated and screened against consensus se- 3'-+5' exonuclease activity, generally known as the editing quences with the use of the IBI (International Biotechnolo- function (8-10). gies, Inc.) sequence analysis system program. We have been studying bacteriophage PRD1 as a model RESULTS system for the initiation of linear DNA replication. PRD1 is a member of a group of lipid-containing phages that infect a Nucleotide Sequence and Predicted Amino Acid Sequence of wide variety ofGram-negative bacteria harboring PRD1 DNA Polymerase. In agreement with genetic studies by plasmids of McGraw et al. (13), nucleotide sequence analysis revealed the P, N, or W incompatibility type (11, 12). The genome of that there are only two major open reading frames at the left PRD1 is 14.7 kilobases long with terminal proteins covalently end of the PRD1 genome (Fig. 1). One open reading frame linked at both 5' ends (13). The linkage ofthe terminal protein corresponds to gene 8, the terminal protein gene consisting of to the PRD1 DNA involves a phosphodiester bond between 260 codons. The other one is gene 1, the DNA polymerase a tyrosine residue of the terminal protein and the 5'-terminal gene that consists of 554 codons (Fig. 1). The nucleotide se- dGMP (6). The PRD1 genome encodes a DNA polymerase quence of the PRD1 DNA polymerase gene along with the that catalyzes the formation of the terminal protein-dGMP complex as well as DNA chain elongation, analogous to the *The sequence reported in this paper is being deposited in the systems ofadenovirus (14, 15) and 429 (4,5). The PRD1 DNA EMBL/GenBank data base (Bolt, Beranek, and Newman Labora- polymerase has been isolated and shown to possess a tories, Cambridge, MA, and Eur. Mol. Biol. Lab., Heidelberg) 3'--5'exonuclease activity (10). It has also been shown to be (accession no. J03018). tProtein Identification Resource (1986) Protein Sequence Database (Natl. Biomed. Res. Found., Washington, DC), Release 9.0. T,he publication costs of this article were defrayed in part by page charge tNational Institutes of Health (1987) Genetic Sequence Databank: payment. This article must therefore be hereby marked "advertisement" GenBank (Research Systems Div., Bolt, Beranek, and Newman, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Cambridge, MA), Tape Release 48.0.

8287 Downloaded by guest on September 24, 2021 8288 Biochemistry: Jung et al. Proc. Natl. Acad. Sci. USA 84 (1987) PRD1 GENOME residues and a high percentage (41.2%) of hydrophobic residues. The initiation codon ATG for the PRD1 DNA polymerase gene is preceded by a region complementary to the 3' terminus of Escherichia coli 16S rRNA (29). Comparison of Amino Acid Sequences Between Phage PRD1 and Phage 429 DNA Polymerases. Our studies show that PRD1 and 429 DNA polymerases are the smallest known enzymes that possess 3'-5' exonuclease activity. Moreover, these two DNA polymerases utilize terminal proteins as primers for the initiation of DNA synthesis (4, 6, 9, 10). Because 429 infects Gram-positive bacteria and PRD1 grows on Gram-negative bacteria, the amino acid sequences of these two DNA polymerases were compared to discern the possible evolutionary relationship between these proteins. As Although the overall sequences are not strikingly homologous, three regions are highly conserved between these two er DNA polymerases (Fig. 3). To determine whether these localized homologies are specific for protein-primed DNA FIG. 1. Genetic and physical map of the region of PRD1 that polymerases, we compared these amino acid sequences with contains the genes for DNA polymerase (gene 1) and terminal protein that of phage SP02 DNA polymerase that has a similar (gene 8). The genetic map was adapted from that of McGraw et al. molecular weight. The Bacillus temperate phage SP02 spec- (13). Arrows indicate the direction of translation. The open circles at ifies its own DNA polymerase (31), and the gene encoding both ends of the genome indicate terminal protein. this polymerase has been cloned and sequenced by Raden and Rutberg (31). The SP02 DNA polymerase has a molec- predicted amino acid sequence and the flanking sequences ular weight of 72,000 and does not initiate DNA synthesis by are shown in Fig. 2. The calculated molecular weight ofPRD1 a protein-priming mechanism (31). No substantial amino acid DNA polymerase is 63,300, which is in rough agreement with homology was found when the sequence for SP02 DNA the reported size (28). This protein contains an approximately polymerase was compared with the sequence for either 429 equal nurpber of acidic (14.2%) and basic (15.7%) amino acid or PRD1 DNA polymerases (data not shown).

AAA CGC GGC TAT GGC AGC MG GGG GTT TAA GAT ATS| CCG CGC CGT TCC CGT AAA AAG GTG GAA TAT AA AT GCC LJ P R R S R K K V E Y K I A GCC '[T GAc 'Tr GM. ACT GAC CCT TTC AAG CAT GAC CGA ATC CCT AAA CCG TTT TCA TOG GGT 'FF TAT AAT GGC 15 A F D F E T D P F K H D R I P K P F S W G F Y N G GAA AT TAT MAA GAC TAT 'OG GOC GAT (;A TGC AlA GM CAG AvlT'FF1' '[AC'TG IT GAT ACC A'l'A GAA GAA CCG 40 E I Y K D) Y W G D D C I E Q F I Y W L D T I E E P CAC GOrr ATA [AC GmT CAT AAC GC GGC MG FTr GAT 'FITr CT[ r[T CrC ATG AMA TAC 1'IT CGC GGG AAA TG AAA 65 H V I Y A H N G G K F DI F L F L M K Y F R G K L K ATA G MT GGG CGT AAT IGT GAM GTA GAM CAC GGC ATO CAT AAA Ft CGC GAT AGT TAT GCA ATC CTG CCG GTG 90 I V N G K I L E V E H G I H K F R D S Y A I L P V CCG CrI' GC' GCC Gc, (;GAT GM MAG ATA GMAA NrIr GAT TAT ('C AAG ATG GMA AGG GM ACA CGC GAA CAG CAC AAG 115 P L A A S D E K I E I D Y G K M k R E 'r R k Q H K GCG GAA ANT 'WA GMAA 'Ac (G.A GG (GAT ''T G'rA ACC cTG CAT AM TG GTr TC 'rFA '-FI AMT1 GCT GAA T- 140 A E I L E Y L K G D C V 1' L H K M V S L F I A E F GGA MGlY. CGC ("'A ACI' MA GOC GGT AcG GCA ATG AAT GM- '[IA AM CAG 'rCAC CCr TAT GAC CCT GrG CG(C A 165 G M R L I G G T A M N E I, K Q F H P Y D P V R K GG(C 'FIT (;GM GM cC N[G cGC CCCc 'T[rr 'rAT 'F1' GGC GGA AGG 'TC CMA GA 'IrC GAG AAA GGA ATA AT'F GM GA'[ 190 G F 0 E A M R P F Y F G G R C Q A F E K G I I E D GAT ATA AAA (-T'' '[A GA'r GF[r AAT ACrT ATG 'AC CCCciCATGT ATG CGA MAT rcT CGC CAT ccI TiC AGC GAT GAA 215 D I K V Y D V N S N Y P H A M R N F R H P F S D E 'FFr[ AT GAA [GC'' sr[GM AA ACA GMA GAAxMCT TA' r'T AFi' GMA '1G (GM GGC GAG AT MC GGC GCG GTG CCT 240 F Y k A N E I T E E T Y F I E W F G k N N G A V P G'Fr AGG ACT AAA ACA GGT FrrA GAC 'FIrr AAT CAG CGr AG GGC ATr 'FT CAT ACG 'A At'V CAT GAA TGG CGG GCG 265 V R T K T G L I) F N Q R S G I F H T S I H E W R A GGT A'F[ GAT ACC GGC ACG A'F[T AA CCT MA'T' CGAG A'F ATA AGG ACA A'rC AAT TFrF ACT GAA ACA ACC ACT T'C GGC 290 G L I) T (; T I K P N R I I R T 1 N F T E T T T F G GCA T'M AlT GAC CA[r 'rF 'IT[ i\AG AAG CGT GAC G[wr CC AM MG OcG GG'[ GAT T'A I'Fi CAC AMr AI'T 'TI'TA'r 315 A F I I) H F F S K RD A A K K A G D L F H N I F Y AAA CTXAF[ 'rrA M' AGC AG(AT TAT GGG AAG '[M' CA CAA AMC CCC GMMl ''['ATlAAA GAG TOG ''[C AXA ACG GMA 340 K L I L. NS S Y G K F A Q N P E N Y K EW C I T E GGC GGCC A'Fr r'A '[rA GM GCC TAT GAc 0c GMA 000 'r[CC GM TA CA\G GAM CAT '[TA GAC TAT ITr 'ITA '[G0 GGT 365 G G I Y l, F G Y D G E G C K V Q F H L D Y I L W G AGO C(C(, GArMAAl'G 'F1' AA[ TN[ 'Irr AAC GrG WCA GMO GCG GCA AGT AF'r ACA G(C GCG 0cC CGO '[C GTT TTA 390 R P A E M F N Y F N V A V A A S I 'T' G A A R S V L T'rG ( GCOCA 'ITr OCG CM GCG GMA AGO CcG c[rr '[[ wTr GAC ACT GAT 'WI' AF'rl A'F '['c CGT OAT 'TA mA AAT 415 L R A L, A Q A H R P L 'Y C D T D S I' I C R D L K N (-IT ccG (Frr (GAG I' TrAC CAG C`TA 0CC GCG [GOA; GA'[ 'rTO GCA ACC QGC GAT AAA ATA 00 XrT C'C GT AAA 440 V P L I) A Y ( L G A W D' l, E A T G D K I A I A G K MA 'FA TA' CG cF[ '[Ac OTr GOr GA'[ AAMT TiC ([rr AAA A'F WCA AOr AAG 00G OT AGT (TG G[T CCG COr GAT 465 K L Y A L Y A G D N C V K I A S K G A S L V P R D AIT 000 Frrr 'F[A ATrG CCC CCG GAT AlT GAA CCG AAA GCC GCC AM AAG G'[A GCG CMA CAA AAG OT AAMN T AT'F 490 I G F L M P P V M E P K A A K K V A Q Q K A K N I GGT ('C GAG AMA AlT 'F[AG ('TO Owr M'[ Gca GOC '[rG 'r'AT OAT Trr' GTA AAM GAl' CC CCG W'A 'ITI AAG (TA 515 G G E K 1 L K V A G G0 V Y D F V N D A P S F K L MT GGC AAC GTG CM 'FF[ A'C MG CGC \CA AR' AM GGA ACA FMT GCA ATA TAC ACT 'TOGGA '['AT TAT CAG 540 N G N V Q F I K H T 1 K G TL

FIG. 2. The nucleotide sequence and predicted amino acid sequence (in the single-letter amino acid code) of the DNA polymerase gene. The Shine-Dalgarno sequence is underlined. Start and termination codons are boxed. The amino acid number is designated on the left side. Downloaded by guest on September 24, 2021 Biochemistry: Jung et al. Proc. Natl. Acad. Sci. USA 84 (1987) 8289

10 20 30 MPRR--SRKKVEYKIAAFDFETDP------F--KHDR---IPKPFS MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS----EYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLER 10 20 30 40 50 60 70 40 50 60 70 80 90 WGF------YN(;EIYKD---YWGDDCIEQFIYWLDT-IEEPH-VIYAHNGGKFDFLFLMKYFRGKLKIVNGRILEV NGFKWSADGLPNTYNTIISRMGQWYMIDICLG---YKGKRKI---HTVIYDS------LKKLPFPVKKIAKDFKLTV 80 90 100 110 120 130 100 110 120 130 140 150 160 170 EHG-1--HKFRDSYAILPVPLAASDEKIEIDYGKMERETREQHKAEILEYLKGDCVTLHKMVSLFIAEFGMRLTIGGTAM LRGDIDYHKER------Pk'GYKITPE--EYAYIKNDI----QIIAEAL--LIQFKQGLDRMTAGSDSLKGFKDIIT-T-- 150 160 170 180 190 200 180 190 200 210 220 2 230 NELKQFHPYDPVRK-GFDEAMRPFYFGG------RCQAF-EKGIIEDDI VYDVNSMYPHAMRNFRHP------FS------KKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDR---FKEKEIGEGM--fVFDVNSLYFAQMYSRLLPYGEPIVFEGKYV 210 220 230 240 250 260 270 240 250 260 270 280 290 300 -DEFYFANEITEET'YFIEWEGENNGAVPVRT---KTGLDFNQRSGIFHTSIHE----WRAGIDTGTIKP-----NRI-IR WD)IiDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYK-GNEYLKSSGG------EIADLWLSNVDLELMKEHYDLYNVEYIS 280 290 300 310 320 330 340 310 320 330 340 350 3 360 370 380 TINFTETTT-FGAFIDHFFSKRDAAKKAGDLFHNIF LILNSS'GKFAQN NYKEWCITEGGIYLEGYDGEGCEVQEH GLKF-ASTGLFKDFID----KWTYIKTTSEGAIKQLLMLNSLYGKFASN V------TGKVPYLKENGALGFRLGEE 350 360 370 380 390 400 410 390 400 410 420 430 1 440 450 LDYILWGRPAEMFNYFNVAVAASITGAARSVLLRALAQAE--RPLYCDTDSIICR-----DLKNVPLDAYQLGAWDLEAT ETKDPVYTPMGVF------1TAWARYTTITA-AQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWAHEST 420 430 440 450 460 470 480 460 470 480 490 500 510 520 (Gl)KIAIAGKKLYAL-YAGDNCVKIASK----GASLVPRDIGFLMPPD-MEPKAAKKVAQQKAKN IGGEKIL-KVAN--GG F-KRA---KYLRQKTYIQD IYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGG 490 500 510 520 530 540 550 560 530 540 550 VYDFVNDAPSFKLNGNVQF I KRT IKGT 'VL-VDDT.------F---TFIK 570 FIG. 3. Alignment of PRD1 (upper lines) and 429 (lower lines) DNA polymerase amino acid sequences (in the single-letter amino acid code). The amino acid sequence of the 029 DNA polymerase was deduced from DNA sequence data reported from this laboratory (30). Dashed lines indicate gaps. Each consensus region is shadowed and numbered.

Homology to the Other DNA Polymerases. Several labora- our knowledge, this is the smallest DNA polymerase ever tories have noted partial homologies among various viral isolated from prokaryotic cells. Though small in size, the DNA polymerases (17-22). When the amino acid sequence of PRD1 DNA polymerase is a multifunctional enzyme (10). It PRD1 DNA polymerase was compared with those of other has at least three enzymatic activities: (i) terminal protein- viral DNA polymerases, three partially homologous regions dGMP covalent complex formation, (ii) a DNA chain- are again evident. Fig. 4 shows the amino acid sequence elongation activity, and (iii) a 3'->5' exonuclease activity. In alignment ofthe three conserved regions. As noted by Larder addition, this polymerase is expected to have a DNA binding et al. (22), the order and spatial relationship of the three site, an association site for terminal protein, and a binding site homologous regions appear to be conserved in all DNA for both a dNTP and a pyrophosphate. polymerases compared, regardless of size (Fig. 5). Our Several groups have reported partial amino acid homolo- computer search revealed that phage T4 DNA polymerase gies among various virus-encoded DNA polymerases (17- also contains three conserved regions (Figs. 4 and 5). 22). Here, we have also undertaken a comparative amino acid Since the DNA polymerases of T4, herpesvirus, and sequence analysis between the PRD1 DNA polymerase and vaccinia virus are not protein-primed DNA polymerases, other DNA polymerases. Our results are consistent with these conserved regions are clearly not unique to the DNA others and show further that PRD1 DNA polymerase as well polymerases that use protein primers. Rather, it is likely that as T4 DNA polymerase share conserved homologous regions these three conserved regions represent domains that per- with the other viral DNA polymerases. These results suggest form important functions common to many DNA polymer- that neither insertion nor frameshift mutations have occurred ases. These functions could include DNA chain elongation, within these regions since the separation of eukaryotes from the 3'->5' exonuclease activity, DNA binding, nucleotide prokaryotes more than 2 billion years ago. This also implies triphosphate binding, or pyrophosphate binding. that both protein-primed and RNA-primed DNA polymerases share a common ancestor. DISCUSSION It must be stressed here that E. coli DNA polymerase I, which is the most completely studied DNA polymerase, does We have determined the nucleotide sequence of the PRD1 not share any significant homology with any DNA polymer- DNA polymerase gene. The predicted amino acid sequence ase described above (34). Bacteriophage T7 DNA polymer- indicates that this polymerase contains 553 amino acids. To ase, on the other hand, has extensive homology with DNA Downloaded by guest on September 24, 2021 8290 Biochemistry: Jung et al. Proc. Natl. Acad. Sci. USA 84 (1987)

REGION 1

PRD1 421 A E R P L Y C D T D S I I C R D L K 029 449 Y D R I I YC D T D S I H L T G TE T4 611 E D F I AA G D T D S V Y V C V DK Vaccinia 719 RF R S V Y G D T D S V F T E I D S EBV 748 Q L R V I Y G D T D S L F I E CR G Herpes 879 S M R I I Y G D T D S I F V L C R G Ad -2 863 PL K S V Y G D TD S L F V T E R G

REGION 2 PRD1 198 Y F G G R C Q A F E K G I I E D D I K V Y D V N S N Y P H AN 029 227 G G F T W L N D R F K E K E I G E G MV F D V N S L Y P A CN T4 386 S F P G A F VFEP K P I A R R Y I M SF D L T S L Y P S I I Vaccinia 502 Y E G G K V F AP K Q K M F S NN V L I F D Y N S L Y P N V C EBV 563 Y Q G A T V I Q PL S G F Y N S P V L V V D F A SL Y P S I I Herpes 695 Y Q G A K V L D P TS G F H V N PV V V F D F A S L Y P S I I Ad-2 521 I R G G RC Y P T Y L G I L R E F L Y V Y D I C G M YA S A L

REGION 3

PRD1 330 A G D L F H N I F Y K L I L N S S Y G K F A Q N P 029 373 T S E G A I K Q L A KL M L N S L Y G K F A SN P T4 547 TL A N T N Q L N R K I L I N S L Y G A L G N I H Vaccinia 626 A I Y D S M Q Y T Y K I V A N S V Y G L M G F RN EBV 671 T I L D K Q L A I K CTC N A V Y G F T G V A N Herpes 801 V L L D K Q 9 A A I K V V C N S V Y G F T G V Q H Ad -2 685 D K N Q TL R S I A K L L S N A L Y G S F A T K L

FIG. 4. Regions of homology between the PRD1 DNA polymerase and other DNA polymerases. Regions were designated 1, 2, and 3 according to Larder et al. (22). Three-digit numbers indicate the first residue in each sequence. Residues that are shared by four or more of the seven polymerases are shadowed. The amino acid sequences of the DNA polymerase of each virus were obtained from the sources indicated: T4 (GenBank), vaccinia virus (20), Epstein-Barr virus (EBV) (32), herpes simplex virus (17), and adenovirus type 2 (Ad-2) (33).

polymerase I (35). Therefore, these two DNA polymerases considerable homology. This prediction is based on evidence appear to have evolved by way of a different evolutionary that several vaccinia genes share significant sequence ho- route. The SPO2 DNA polymerase does not have any mology with their eukaryotic cellular counterparts (37-39). If significant homology with E. coli DNA polymerase I or T7 this proves to be the case, it can be expected that PRD1 DNA DNA polymerase. polymerase will have some segmental homology with eu- Based on evolutionary relatedness, we propose to classify karyotic DNA polymerase a. DNA polymerases as follows. Adopting the terminology of The small size of PRD1 DNA polymerase makes it espe- Doolittle (36), we designate E. coli DNA polymerase I and T7 cially amenable to studies on structure-function relationships DNA polymerase to be DNA polymerase family A. All DNA and on the mechanism by which high fidelity is achieved polymerases carrying the three conserved regions described during DNA replication. Information on the nucleotide se- above are designated family B. It is possible that there are quence of the PRD1 DNA polymerase gene should provide a other families of DNA polymerases (i.e., a family including basis for carrying out precise genetic alterations at each ofthe SPO2 DNA polymerase) that do not have any homology with conserved regions using site-specific mutagenesis. family A or B enzymes. Such DNA polymerases can be We thank Dr. L. Mindich for supplying phage PRD1 and bacterial classified in families C, D, and so on. strains. We also thank Drs. H. Bernstein and R. Friedman for their Earl et al. (20) have predicted that vaccinia virus DNA critical reading of this manuscript. This research was supported by polymerase and cellular DNA polymerase a should share Grant GM 28013 from the National Institutes of Health (to J.I.).

PROI 2 31 (553)

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