Copyright 0 1990 by the Genetics Society of America

Genetic Evidence for Two Protein Domains and a Potential New Activityin Bacteriophage T4 DNA

Linda J. Reha-Krantz

Department of Genetics, University of Alberta, Edmonton, Alberta, Canada T6G 2E9 Manuscript received June 22, 1989 Accepted for publication October 7, 1989

ABSTRACT Intragenic complementation was detected within the bacteriophage T4 DNA polymerase gene. Complementation was observed between specific amino (N)-terminal,temperature-sensitive (ts) mu- tator mutants and more carboxy (C)-terminal mutants lacking DNA polymerase polymerizing func- tions. Protein sequences surrounding N-terminal mutation sites are similar to sequences found in Escherichiacoli (RNase H) and in the 5’ + 3’ domain of E. coli DNA polymerase I. These observations suggest that T4 DNA polymerase, like E. coli DNA polymerase I, .. ” contains a discrete N-terminal domain.

ANY DNA from diverse organisms parisons between E. coli DNA pol I and bacteriophage M (e.g., human; yeast; viruses-herpes, adeno, vac- T4 DNA polymerase, and, by inference, toother cinia; and bacteriophage-T4, 429, PRD1) share sev- DNA polymerases. Many DNA polymerases contain eral regions of colinear protein sequence homology N-terminal protein sequences upstream from the pro- (SPICERet al. 1988; BERNARDet al. 1987; JUNG et al. posed 3’ += 5’ exonuclease and polymerase domains. 1987; WANG,WONG and KORN 1989). DNA polym- In E. coli DNA pol I, the N terminus encodes a discrete erase I (pol I) from may contain just protein domain with 5’ + 3‘ exonuclease activity that one of the conserved sequences, a part of the most N- can be separated by mild proteolysis from the larger terminal conserved region (REHA-KRANTZ 1988a,b; (3’ + 5’ exonuclease and polym- 1989; SPICERet al. 1988). Although the protein se- erase domains) (LEHMAN andCHIEN 1973; JACOBSEN, quence similarity involves only a few amino acids, KLENOW andOVERGAARD-HANSEN 1974). Some N- these amino acids in E. coli DNA pol I form part of terminal T4 DNA polymerase protein sequences are the 3’ + 5’ exonuclease active site (JOYCE and STEITZ similar to sequences in the E. coli DNA polymerase I 1987;DERBYSHIRE et al. 1988). Thus, the most N- 5’ + 3’ exonuclease domain; there is also some pro- terminal conserved region in the related DNA polym- tein sequence similarity with E. coli RNase H. Fur- erases, called region IV by WANG,WONG and KORN thermore, intragenic complementation has been de- tected between T4 N-terminal DNA polymerase mu- (1 989)is likely the location of active or cryptic 3’ + tantsand DNA polymerase C-terminal mutants. 5’ exonuclease function(REHA-KRANTZ 1988a, b; Intragenic complementation has also been observed 1989). The C-terminal conserved sequences are pre- with some herpes (HSV-1) N-terminal DNA polym- dicted to encode polymerizing functions such as de- erase mutants (CHARTRANDet al. 1980; WELLERet al. oxynucleoside triphosphate bindingand DNA binding 1983; A. I. MARCYand D.M. COEN,personal com- (GIBBSet al. 1985, 1988; SPICERet al. 1988; WANG, munication). The positive complementation results WONG and KORN 1989;REHA-KRANTZ 1988a, b). suggest that T4 and HSV-1 DNA polymerases may Polymerizing functions are also located in the C ter- have discrete, N-terminal domains while the protein minus of E. coli DNA pol I (JOYCE and STEITZ1987). sequence similarities suggest that the N terminus may Thus, although there is little proteinsequence ho- encode a 5’ + 3’ exonuclease or RNase H-type activ- mology shared between E. coli DNA pol I and the ity; an RNase H-type activity has recently been de- above DNA polymerases, many DNA polymerases tected in the HSV-1 DNA polymerase (CRUTEand appear to have the same relative organization of 3’ + LEHMAN 1989;MARCY et al. 1989). The main predic- 5’ exonuclease and polymerase activities. (REHA- tion arising fromthese studies is that many DNA KRANTZ1988a, b; BERNADet al. 1989; LEAVITTand polymerases may share the same structural-functional IT0 1989). organization as found in E. coli DNA polymerase I: a Arguments are presented here that extend com-the distinct N-terminal domain that may encode a 5’ + 3’ exonuclease or RNase H activity adjacentto a The publication costs of this article were partly defrayed by the payment of page charges. This article must therefore be hereby marked “advertisement” Klenow-type domain with3’ + 5’ exonuclease and in accordance with 18 U.S.C. $1734 solely to indicate this fact. polymerase activities.

Genetlcs 124: 213-220 (February, 1990) 214 L. J. Reha-Krantz

MATERIALS AND METHODS TABLE 1

Phage and bacterial strains: Phage T4 and bacterial Complementation betweenT4 DNA polymerase (g43) alleles strainshave been described (REHA-KRANTZet al. 1986; - ~~ ~~ REHA-KRANTZ 1988a). Phage Mutation and site" Burst sile' Complementation tests: Single and mixed infections at a Single phage infections multiplicity of five foreach phage strain were doneat Wild type 64.0 nonpermissiveconditions: high tem erature(43") which amE4314 Trpamber 202 0.06 restricts ts phage andhost E. coli B (suB ) which does not have amE4306 Trpamber 20.004 13 a nonsense suppressortRNA andrestricts phage with amber amB22 0.005Glnamber 731 mutations. Many gene 43 amber mutants aresuppressed at amE4302 amber844 Trp 0.01 low levels on suo hosts at 30" by translationalambiguity tsmeA62 Gly 82 Asp 0.03 (KARAM and O'DONNELL 1973).At 43", however, there is tsL56 Ala 89 Thr, Asp 363 Asn 0.04 little suppression (see discussion on transmission coefficients tsPS7 Asp 112 Asn 0.01 below). At 10 min post infection, cultures were diluted 100- tSPN Y 123 Pro Leu 0.09 fold into 43" broth to diluteaway from unadsorbed phage. tsmel5 Asp 131 Cly 0.08 Chloroform was added at 40 min and the progeny yields tsA6O 0.07 SerGly 172 were determined by plating on CR63 (su+) at 30". Progeny tsA 73 225 Arg His 0.02 are expectedonly if the infecting phage can mutually supply tsMl9 ProLeu 340 0.01 in trans the function missing in the othercoinfecting phage. tsSY Met 671 Ile 0.03 The burst size is equal to the total number of progeny Mixed infections: divided by the total number of infected bacteria. The infec- tsmel62 + amB22 0.06 tive center titer of thewild-type control was used to calculate tsL56 + amB22 0.05 all burst sizes. Parental phage were recovered in all cases of tsPS7 + amB22 0.5 positive complementation. tsmel5 + tsL56 0.6 Transmission coefficients: In order to determine the tsmel5 + tsme162 0.5 level of suppression due to translational ambiguity at 43" in tsPS7 + tsmel62 0.3 E. coli B, dilutions of the amber phage-E. coli B infective tsmel5 + tsPS99 0.8 centers weremixed at 10 min postinfection with the su+ tsA60 + amE4314 0.2 host CR63 in soft agar, then overlaid on hard agar and tsA60 + amB22 0.7 incubated overnight at 43". Any viable phage produced in tsA6O + amE4302 0.1 the amber phage-E. coli B infective centers infect the sur- tsA73 + amE4314 1.4 rounding CR63 bacteria and produce plaques. No plaques tsA73 + amE4302 0.1 were observed from amE4314, amE4306 and amE4302 in- tsmel5 + tsA60 70.0 fective centers, but 10% of the amB22-E. coli B infective tsPS99 + tsA60 11.0 centers produced plaques. Thus, low levels of suppression tsmel5 + tsA73 31.0 cannotaccount for the positive complementation results tsPS99 + tsA73 8.0 observed with amE4314, amE4306 and amE4302 infections. tsPS99 + tsMlY 5.0 Suppression is also probably not a factor in the positive tsPSY9 + tsS9 30.0 complementation results observed with amB22. For comple- tsPS99 + amE4314 28.0 mentation tests, only 40 min was allowed to produce viable tsPS99 + amE4306 19.0 phage and few progeny were detected in amB22 infections tsPS9Y + amB22 11.0 (Table 1). Thus, apparently a growth cycle longer then 40 tsPS99 + amE3402 10.0 min is required to produce progeny from amB22-infected tsmel5 + amE4314 13.5 E. coli B cells. tsme15 + amE4306 11.0 Alkaline sucrose gradients: Pulse-labeled DNA was pre- tsmel5 + amB22 11.0 pared as described (KONRADand LEHMAN 1974). E. coli tsmel5 + amE4302 8.0 K12SH28 (FANGMAN 1969),which has reduced thymidine Extragenic complementation: phosphorylase acitivity, was grown at 43" to 2 X lo8 bacte- amB22 + amN122 (gene 42) 46.0 ria/ml in M9 mediumsupplemented with 0.4% glucose, 0.5% casamino acids, and 0.01% MgS04. At to, cells were Standard three letter amino acid code is used. The wild-type amino acid is given first followed by the codon position and then infected with phage at a multiplicity of ten. At t5 min, the the mutant aminoacid. Mutation identifications are given in REHA- infected cells were diluted tenfold into 10ml of prewarmed KRANTZ(1 988a) and REHA-KRANTZ(1 989). M9 medium. At t7 min, 200 p1 of ["]thymidine (0.3 mCi/ *Burst size is equal tothe total number of progenyphage ml were added. Cultures were aerated by shaking through- produced in one cycle of replication divided by the total number of out infection and labeling procedures. Incorporation was infected bacteria. All infections were at 43" in host E. coli B (see quenched at 10 sec, 45 sec, 2 min and 10 min by adding an MATERIALS AND METHODS). equalvolume of 75%ethanol solution containing 2% phenol, 20 mM sodium acetate (pH 5.2), and 2 mM EDTA. pUCl8 plasmid DNA; the peak of activity was recovered in Cells were centrifuged at 15,000 X g for 10 min and the fraction 22. Gradient samples, 200 pl, were collected from pellet was resuspended in 0.3 ml of 0.2 M NaOH-10 mM the bottom and precipitated with 10% trichloroacetic acid. EDTAand incubated at 40" for 1 hr. The solution was Precipitates were collected on Whatman GF/A filters and clarified by centrifugation at 14,000 X g. Samples, 100 PI, radioactivity was determined in a liquid scintillation counter. were layered on to 5 ml alkaline sucrose gradients: 5-20% sucrose with 0.1 M NaOH, 0.9 M NaCl and 2 mM EDTA RESULTS over a 0.5 rnl shelf of 80% sucrose. The gradients were centrifuged for 120nrin at 40,000 rpm at 5". Marker DNA, Phage T4 N-terminal mutations: Mutational anal- 2.7 kb, was prepared by 3'-endlabeling EcoRI digested yses of the T4 DNA polymerase gene (gene 43) have T4 DNA Polymerase Structure-Function 215

hm rsme162 fsPs99 tsme/5, PS43 I \ - \/ H I IrlIIIIIIl I “- N I I1 - II 7” ”\ I I‘IIIIIIII I amE4314 amE4306

1- 1- 5’ -> 3 exoh, 3‘ -> 5’ exo-1

FIGURE1 .-Two clusters of mutation sites in the N terminus of phage T4 DNA polymerase. Intervals of 100 amino acids are indicated (0).The mutations, indicated by (I) were isolated by genetic selection techniques that enriched for mutants with strong mutator phenotypes (REHA-KRANTZ 1988a;REHA-KRANTZ et al. 1986) or that affect UV mutagenesis (DRAKE1988). Two amber mutants (X) are located in this region. Residues in the T4 DNA polymerase similar to the E. coli DNA pol I 3’ + 5’ exonuclease domain are centered around amino acid

#200 and are indicated by a bar (1-1). Potential 5’ --f 3’ exonculease and 3’ --f 5’; exonuclease domains are indicated; polymerase functions are located in the C terminus. The unnamed site near the tsmel62 site is one of two mutation sites found in the tsL56 strain (Table 1; REHA- KRANTZ1989).

been used to study DNA polymerase structure-func- with more C-terminal (codon #172 and above) con- tion relationships (REHA-KRANTZ1988a, b; 1989). ditional lethal mutations (Table 1). All of the DNA DNA polymerase mutations that confer strong muta- polymerase fragmentsproduced in gene 43amber tor phenotypes have been identified throughout gene infections lack polymerizing activity, and 3’ + 5’ ex- 43, but many of the mutations are located in the N onuclease activity has been detected only inthe amB22 terminus(Figure 1) (REHA-KRANTZ 1988a, 1989). fragment (NOSSAL1969; NOSSALAND HERSHFIELD The most N-terminal mutation sites are located up- 197 1). The tsPS99 and tsmel5 strains have N-terminal stream to the predicted 3’ + 5’ exonuclease active mutations (Figure 1) and must be deficient onlyin site and C-terminal polymerase functions (Figure 1) some essential, N-terminal activity, because these (REHA-KRANTZ 1988a, b; 1989). Mutatoractivity may DNA polymerases apparently supply DNA polymer- be produced by a reduction in 3‘ -+ 5‘ exonuclease ase functions that are missing in the amber fragments. activity thatfunctions to “edit” newly synthesized The apparent positive complementation results are DNA (MUZYCZKA,POLAND and BESSMAN1972); how- not likely due to productionof wild-type DNA polym- ever, the tsmel62-DNA polymerase, the only mutant erase molecules by recombination. In mixed infections DNA polymerase with a mutation from this region to when few progeny were produced, e.g., tsA6O + be characterized biochemically, has nearly wild-type amE4314, tsA73 + amE4302,0.2-1.2% of the progeny levels of 3’ -+ 5’ exonuclease activity (REHA-KRANTZ were detected as wild-type recombinants as judged by 1987). Reduced accuracy in base selection can also growth at 43” on E. coli B. When positive comple- produce the mutator phenotype (SPEYER,KARAM and mentation was observed, up to2.5% of the total LENNY 1966;HERSHFIELD 1973;GILLEN and NOSSAL progeny were wild type (e.g., tsmel5 + amB22). 1976), but DNA polymerase functions predicted to Protein sequence similarities: Because some pro- be important for nucleotide selection are located ap- tein sequence similarities have been detected in the parently in the C-terminal half of the protein (REHA- 3‘ + 5’ exonuclease domains of E. coli DNA pol I KRANTZ1988a, b; SPICER1988). Thus, the most N- and T4 DNA polymerase (REHA-KRANTZ 1988a, b; terminal mutations may identify a previously unde- SPICERet al. 1988),the corresponding N-terminal tected T4 DNA polymerase function important for sequences of both DNA polymerases were scanned by fidelity. In order to determine if the N terminus of eye for similarities. Four regions ofweak similarity the T4 DNA polymerase encodes an essential func- were found (Figure 2). Some protein sequence simi- tion, perhaps in a domain separate from the 3’ -+ 5‘ larity was also detected in E. coli RNase H (Figure 2). exonuclease and polymerase activities, complementa- Although it is difficult to assess the significance of the tion tests were performed, limited sequence similarities, the regions are colinear Complementation tests: Extensive tests for intra- and threeregions contain sites ofT4 DNA polymerase genic complementation in gene 43 were conducted mutations (Figure 2). In addition, one T4 mutation previously and no complementation was detected in that produces an alanine to threonine substitution at mixed infections with various temperature-sensitive codon #126 increases the similarity between T4 and (ts) phage (DRAKEet al. 1969); however the ts mutant E. coli DNA sequences in region3 (Figure 2); this collection for those studies didnot contain many mutation was isolated as a second-site mutation that strains with N-terminal mutations(HUGHES et al. partially compensates for thetsmel5 mutation at codon 1987). Complementationwas detected with our larger #131 (REHA-KRANTZ 1988a). FourE. coli DNA pol I collection of ts strains, but only in mixed infections missense mutations have been found in the 5’ --., 3‘ under nonpermissive conditions with phage carrying exonuclease domain: Y(77)C, G( 103)E, G( 184)D and either the N-terminal tsPS99 mutation, codon #123, G( 192)D (JOYCE et al. 1985), but these mutations are or thenearby tsmel5 mutation, codon #13 1, and phage outside regions shown in Figure 2. 216 L. J. Reha-Krantz BEGl!uJ RNase H 5 VEIFTD(16) RYRGREKT .. .. T4 pol 16 VERY1DENGKERTREVE ...... pol I 115 GTLAREAEDDVl

T4 mutants K S hrn I3!3mu T4 pol 77 GLEALGMNDFKLAYI S ...... pol I 210 GLDTLYAEPEKIAGLS

T4 mutants D T fsrne162 E€!mu RNase H 64 LSTDSQYVREVI - - ... .. T4 pol 123 PMKAEYEIDAI THYDSI - DDR ...... HSV 248 FEAEVVERTDVYYYE- - pol I 251 TIKTDVELEL- TC -

T4 mutants L T G,N tSPS99 tsmel5, PS43

REGION 4

RNase H 137 A R A A A .. .. pol 161T4 pol AKLAAK- LDCEGGDEVPQEI LDRVl YMPFDNERDML ...... pol I 302 AKPAAKPQETSVADEAPEVTATV324...... FDTETDSL 354 FIGURE2.-Protein sequence similarities in the N-terminal DNA polymerase domains of E. coli DNA pol I (JOYCE,KELLEY and GRINDLEY 1982), phage T4 (SPICERet al. 1988), and herpes simplex virus type 1 (HSV) (GIBBSet al. 1985), and in E. coli RNase H (MAKI,HORIUCHI and SEKIGUCHI 1983). The standard,single-letter amino acid code is used and the residue numberof the first amino acid in each sequence is given. Sequences are aligned to optimize similarity; a dash (-) indicates the absence of an amino acid. The amino acid changes in T4 DNA polymerase mutant strains and the strain designations are given and discussed in the text (REHA-KRANTZ1988a). The hm mutation has been described (DRAKE1988). Identical amino acids are indicated by (:) and conservative amino acid substitutions by (.). In region 3, similar amino acids in T4 DNA polymerase and in E. coli DNA pol I and in RNase H and HSV DNA polymerase are underlined. In region 4, 30 amino acids in the E. coli DNA pol I sequence between Val"', the N terminus of the KIenow fragment, and the start of the conserved 3' "* 5' exonuclease sequence (#354) are not shown. The C-terminal end of region 4 corresponds to region 1 in REHA-KRANTZ(1988a), to region 2 in SPICERet al. (1988) and to region IV in WANG,WONG and KORN(1989).

T4 and herpes DNA polymerase N-terminal se- nal domain. Some sequence similarity was also found quences were also searched for homologies. Although in this region between the herpes (residues 210-260) there are extensive T4 and herpes DNA polymerase and human DNA polymerases (residues 368-4 18) (se- protein sequence similarities in regions predicted to quences not shown). encode polymerase and 3' + 5' exonuclease activities DNA synthesis in wild-type and tsmeZ5 infections: (SPICERet al. 1988; REHA-KRANTZ 1988a, b),only a DNA synthesized in vivo by wild-type and tsmel5-DNA little weak sequence similarity was detected in the N- polymerases at 43" was monitored in alkaline sucrose terminus (region 3, Figure 2). This protein sequence gradients (Figure 3). DNA synthesis was examined for (region 3) is also similar to a sequence found in E. coli intervals of 10 sec, 45 sec, 2 min and 10 min. At all RNase H and in the predicted RNase H domains of times, less DNA and smaller DNA was synthesized in retroviral polymerase genes (JOHNSON et al. 1988). tsmel5 infections (Figure 3). Thus, therecently discovered RNase H activity in the Mutator activity of the tsmeZ5 and tsPS99-DNA herpes DNA polymerase could reside in the N-termi- polymerases: The tsPS99 and tsmel5 mutations in- T4 DNA Polymerase Structure-Function 217

Wild type Wild type 8000 I I

0 10 20 30 0 10 20 30 Fraction number Fraction number

tsmel5 tsmel5

700 I

600 10 rnin 500

400

300

200

100 A

o! 1 1 0 0 10 20 30 0 10 20 30 Fraction number Fraction number FIGUREJ.-Newly synthesized DNA in wild type andtsmel5 infections at 43": analysisi by sedimentation through alkaline sucrose gradients. The procedures are describedin MATERIALS AND METHODS. Gradient samples, 200 PI,were collected from the bottom. Thus, thefirst samples collected contain the highest molecular weight DNA. A 2.7-kh marker DNA was recovered in fraction 22. crease spontaneous mutation frequencies about 100- primer is detected in T4 infections (KUROSAWAand fold (REHA-KRANTZ1988a). Mutations produced in OKAZAKI 1979)in orin vitro assays with T4 DNA (LIU these mutator strains seem to occur preferentially at and ALBERTS1981). T4 DNA is modified with glu- potential RNA priming sites and in tracts of purine cosylated 5-hydroxylmethyl cytosine that is thought residues. Six of tenmutations sequenced in these to block priming at 5' GCT sites (LIU and ALBERTS strains are within potential RNA primer sites which 1981).Thus, if the small collection of sequenced occur every 36 nucleotides on average in T4 DNA mutations is representative of themutational spec- (Figure 4) (CHAand ALBERTS1986). Two independ- trum for the N-terminal mutant DNA polymerases, ent mutations were isolated in one sequence that may thenthere may be atypical RNA priming in these form asmall, stable hairpin structure similar to unusu- strains. ally stable hairpinstructures that are stabilized by certain tetra loop sequences (Figure 4) (TUERKet al. DISCUSSION 1988). The sequenceproduces asequencing gel Complementationwithin the phage T4 DNA compression, likely due to hairpin formation, in 7 M polymerasegene: Allelic complementation was ob- urea at 50" (L. J. REHA-KRANTZ,unpublished obser- served between strains with certain N-terminal muta- vations). Thus,the structure has anexpected free tions, tsPS99 (codon #123) and tsmel5 (codon #131), energy value less than -8 kcal/mol (SHINEDLINGet al. and strains with more C-terminal DNA polymerase 1987). Two RNA primer sequences are utilized in in conditional lethal mutations (Table 1). Only a few uitro priming assays withnon-T4 DNA (Figure 4) (CHA molecules of T4 DNA polymerase are required to and ALBERTS1986); however, only the pppACNNN produce at least one viable phage per infected cell 218 L. J. Reha-Krantz

RNA 3’ N N N C A/Gppp tivities, but a protein with homology to the 5’ + 3’ DNA 5’ N N N G T/CT exonuclease domain of E. coli pol I is encoded on a separate gene (LEAVITTand ITO 1989). Similarly, it is Mutation imagined that the tsPS99- and tsmel5-DNA polymer- sites 1. 5’ G C T G T T ases provide Klenow-type activities while the amber c fragments and C-terminal mutant DNA polymerases A provide an essential activity encoded in the N-termi- nus. 2. 5’G A T G T T Function of the T4 DNA polymerase N-terminal + domain: Many questions about a potential T4 5‘ + C 3’ exonuclease activity remain unresolved. The pu- 3.5’GATGCT rified T4 DNA polymerase cannot “nick translate” c (NOSSALand HERSHFIELD 1971; COZZARELLI,KELLY A and KORNBERC1969) and thus does not appear to 4.5’ATTGCT have a powerful 5‘+3‘ exonuclease activity. Strand displacement synthesis is observed in T4 DNA repli- + cation assayswith the addition of gene 32 protein T A-T T CG (helix-destabilizing protein) and DNA polymerase ac- 5.5’ACCGCT AeG C cessory proteins, the products of genes 44, 62 and 45 6. c+ CG (NOSSALand ALBERTS1983). It is not known how AA GG SAC RNA primers are removed during T4 DNA replica- FIGURE4.-Mutations produced in T4 DNA polymerase strains tion, although an RNase H activity has been detected with N-terminalmutations. While determiningthe amino acid in T4-infected cells (NOSSALand ALBERTS1983). An substitutions responsible for the mutator phenotype of T4 DNA attractive model consistent with some data presented polymerase mutants, several “other” mutationswere detected in the DNA polymerase gene in various subcultures that did not seem to here is that the DNA polymerase, perhaps stimulated be required for the mutator phenotype and may be a result of the by accessory proteins or other proteins, has an RNase endogenousmutator activity of themutator DNA polymerase H-type activity. If the T4 DNA polymerase is respon- (REHA-KRANTZ1988a). Six of ten such mutations detectedin tsPS99 sible for removing RNA primers, then it is predicted and tsmel5 culturesare within potential,pentanucleotide RNA priming sites (CHAand ALBERTS1986). The DNA sequence for thatat high temperature, ts T4 DNA polymerase initiation of RNA priming is 5’ GTT or 5’ GCT and the initiation mutants deficient in RNAprimer removal activity sequence is underlined in each of themutation sites (CHA and would be defective in joining nascent DNA fragments. ALBERTS1986). Two mutation sites, 5 and 6, are within a sequence Alkaline sucrose gradient analyses of DNA synthe- that may form a small hairpin structure. sized by wild-type and tsmel5 phage do not support (KARAMand O’DONNEL 1973), but relativelythe high this model. At 43” (Figure 3) and30” (datanot burst sizes observed underthe restrictive growing shown) high molecular weight DNA was synthesized conditions used here suggest that each of the phage in tsmel5 infections, but at a slower rate than in wild- strains provides essential DNA polymerase functions type phage infections. No accumulation of short Oka- in trans. The T4 DNA polymerase in vivo comple- zaki fragments was observed as expected if removal mentation may be achieved analogously to the in vitro of RNA primers was prevented. complementation observed forthe large and small Another puzzling question ishow can presumed fragments of E. coli DNA pol I (LEHMANand UYE- deficiencies in 5’ + 3’ exonuclease or RNase H activ- MURA 1976). E. coli DNA pol I can be cleaved by mild ity reduce the fidelity of DNA replication? Although proteolysis into two discrete domains: the large, C- removal of RNA primers does not appear to be a terminal “Klenow” fragment that contains DNA po- major problem in infections with these mutant phage lymerase and 3’ + 5’ exonuclease activities, and a (Figure 3), mutations may occur preferentially at po- smaller, N-terminal fragment containing 5’ + 3‘ ex- tential RNA priming sites (Figure 4). onuclease activity (LEHMAN andCHIEN 197 ; 1 JACOB- Model: A working model to explain the properties SON, KLENOWand OVERCAARD-HANSEN 1974). Whenof N-terminal DNA polymerase mutations follows. N- both fragments are added toin vitro assays, full DNA terminal mutator mutants are located in a discrete polymerase activity is restored(LEHMAN and UYE- domain that is essential for phage T4 viability. Protein MURA 1976). The bacteriophage T5 DNA polymerase sequence comparisons indicate some similarities with may provide an exampleof in vivo interaction between the 5’ + 3’ exonuclease domain of E. coli DNA pol I a DNA polymerase and a separate protein with 5’ + and with E. coli RNase H. RNA primer removal is not 3 ’ exonuclease activity. The bacteriophage T5 DNA altered significantly in the mutant strains, but perhaps polymerase gene encodes aKlenow-type DNA polym- modest decreases in RNase H activity or in some other erase with 3’ + 5’ exonuclease and polymerase ac- aspect of 5’ + 3’ exonuclease activity, such as strand D N A Polymerase Structure-FunctionPolymerase T4 DNA 219 displacement, is responsible for the slow and reduced nuclease domain of E. coli pol I and with T4 DNA DNA synthesis. Forexample, strand displacement polymerase (M. A. FRANDENand C. S. MCHENRY, may require the DNA polymerase N-terminal domain 1988 Detection of an epitope, not required for po- in combination with DNA replication accessory pro- lymerization, that is conserved between E. coli DNA teins. Alternatively, some aspect of RNApriming polymerases I and 111 and bacteriophage T4 DNA during lagging strand synthesis may be affected. This polymerase. Nucleic Acids Res. 16: 6353-6360). idea is supported by a yeast N-terminal DNA pol I mutation thataffects -polymerase complex sta- LITERATURECITED bility (LUCCHINIet al. 1988). If the mutants are defec- BERNAD, A,,L. BLANCO,J. M. LAZARO,G. MARTIN and M. SALAS, tive inDNA polymerase-primase interactions, only 1989 A conserved 3’ + 5’ exonuclease active site in prokar- leading strand synthesis may occur under nonpermis- yotic and eukaryotic DNA polymerases. Cell 59: 219-228. sive conditions. T4 primase-deficient strains synthe- BERNAD,A,, A. ZABALLOS,M. SALAS and L. BLANCO, size significant amount of DNA by displacement syn- 1987 Structural and functional relationship between prokar- yotic and eukaryotic DNA polymerases.EMBO J. 6 4219- thesis (MOSIGet al. 1981 ; NOSSALand ALBERTS1983). 4225. DNA synthesized in DNA polymerase N-terminal mu- BLACKL. W., and M. K. SHOWE,1983 Morphogenesis of the T4 tant strains may also be due primarily to displacement head, pp. 219-245 in Bacteriophage T4, edited by C. K. MATH- synthesis (Figure 3). Thus, the slow DNA synthesis EWS, E.M. KUTTER, G. MOSIGand P. B. BERGET.American Society for Microbiology, Washington, D.C. may be due to “pauses” when RNA primers and/or CHA,T.-A,, and B. ALBERTS,1986 Studies of the DNA - secondarystructures in the DNA template are en- RNA primase unitfrom bacteriophage T4. A trinucleotide countered or as a consequence of inadequate priming sequence on the DNA template starts RNA primer synthesis. and reduction of lagging strand synthesis. In any case, J. Biol. Chem. 261: 7001-7010. lethality in these strains at high temperature is likely CHARTRAND, P.,C. S. CRUMPACKER,P. A. SCHAFFERand N. M. WILKIE,1980 Physical andgenetic analysis of theherpes due to insufficient production of very high molecular simplex virus DNA polymerase locus. Virology 103: 31 1-326. weight,concatemeric DNA requiredfor packaging COZZARELLI,N. R., R. B. KELLYand A. KORNBERG,1969 Enzymic (BLACKand SHOWE1983). Mutations may be induced synthesis of DNA. XXXIII. Hydrolysis of a 5’-triphosphate- as a consequence of extended “pauses” in DNA repli- terminated polynucleotide in the active center of DNA polym- cation. For example, persistant DNA discontinuities erase. J. Mol. Biol. 45: 513-531. may interfere with the repair of damaged DNA and CRUTE,J. J., and 1. R. LEHMAN,1989 Herpes simplex-1 DNA polymerase: identification of an intrinsic 5’ + 3’ exonuclease increase the probability of bypass replication (replica- with ribonuclease H activity. J. Biol. Chem. 264: 19266-19270. tion past nontemplating DNA lesions). Bypass repli- DERBYSHIRE,V., P. S. FREEMONT,M. R. SANDERSON,L. BEESE,J. cation has been detected in vitro for the tsmel62-DNA M. FRIEDMAN,C. M. JOYCE andT. A. STEITZ,1988 Genetic polymerase (REHA-KRANTZ 1987)and another N-ter- and crystallographic studies of the 3’,5’-exonucleolytic site of DNA polymerase I. Science 240: 199-20 1. minal mutant, hm, increases ultraviolet mutagenesis DRAKE,J. W.,1988 Bacteriophage T4 DNApolymerase deter- presumably also by some type of bypass replication mines the amount and specificity of ultraviolet mutagenesis. (DRAKE 1988). Mol. Gen. Genet. 214: 547-552. Future prospects: One application of these finding DRAKE, J. W.,E. F. ALLEN,S. A. FORSBERG,R.-M. PREPARATA and is to use genetic engineering to produce a phage T4 E. 0. GREENING,1969 Spontaneous mutation. Nature 221: 1128-1131. “Klenow”fragment with 3’ + 5’ exonuclease and FANGMAN,W. L., 1969 Specificity and efficiency of thymidine polymerase activities. This project could succeed be- incorporation in Escherichia coli lacking thymidine phosphoryl- cause T4 protein sequences with similarities to E. coli ase. J. Bacteriol. 99: 681-687. DNA pol I flank both sides of the E. coli Klenow N GIBBS,J. S., H. C. CHIOU,J. D. HALL, D. W. MOUNT, M. J. terminus at Val324(Region 4, Figure 2). The engi- RETONDO,S. K. WELLERand D. M. COEN, 1985Sequence neeringand cloning of E. coli DNA pol I Klenow and mapping analyses of the herpes simplex virus DNA polym- erase gene predicta C-terminal substrate binding domain. Proc. fragment (JOYCE and GRINDLEY 1983)were invalua- Natl. Acad. Sci. USA 82: 7969-7973. ble for subsequent structural and biochemical studies GIBBS,J. S., H. C. CHIOU,K. F. BASTOW,Y.-C. CHENG andD. M. (JOYCE and STEITZ1987). COEN, 1988 Identificationof amino acids in herpes simplex virus DNA polymerase involved in substrate and drug recog- I thank A. MARCY,D. COEN,N. 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