Copyright 0 1990 by the Genetics Society of America Genetic Evidence for Two Protein Domains and a Potential New Activityin Bacteriophage T4 DNA Polymerase 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 Escherichia coli ribonuclease H (RNase H) and in the 5’ + 3’ exonuclease 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 polymerases 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 Escherichia coli 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; Klenow fragment (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 with 3’ + 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 were mixed 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. EDTA and incubated at 40" for 1 hr.