Genetic Evidence for Two Protein Domains and a ... - Semantic Scholar

Copyright 0 1990 by the Genetics Society of America

Genetic Evidence for Two Protein Domains and a Potential New Activity in 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 ( t s ) mutator mutants and more carboxy (C)-terminal mutants lacking DNA polymerase polymerizing functions. Protein sequences surrounding N-terminal mutation sites are similar to sequences found in Escherichiacoli ribonuclease H (RNase H) and in the 5’ + 3’ exonuclease domain of E. coli DNA polymerase I. These observations suggest that T 4 DNA polymerase, like E. coli DNA polymerase I, . . contains a discrete N-terminal domain. ”

ANY DNA polymerases from diverse organisms (e.g., human; yeast; viruses-herpes, adeno, vaccinia; and bacteriophage-T4, 429, PRD1) share several regions of colinear protein sequence homology et al. 1987; JUNG et al. (SPICERet al. 1988; BERNARD 1987; WANG,WONGand KORN 1989). DNA polymerase I (pol I) from Escherichia coli may contain just one of the conserved sequences, a part of the most Nterminal conserved region (REHA-KRANTZ 1988a,b; 1989; SPICERet al. 1988). Although the protein sequence similarity involves only a few amino acids, these amino acids in E . coli DNA pol I form part of the 3’ + 5’ exonuclease active site (JOYCE and STEITZ et al. 1988). Thus, the most N1987;DERBYSHIRE terminal conserved region in the related DNA polymerases, called region IV by WANG,WONGand KORN (1 989)is likely the location of active or cryptic 3’ + 5’ exonuclease function(REHA-KRANTZ1988a, b; 1989). The C-terminal conserved sequences are predicted to encode polymerizing functions such as deoxynucleoside triphosphate bindingand DNA binding (GIBBSet al. 1985, 1988; SPICERet al. 1988; WANG, WONGand KORN 1989;REHA-KRANTZ1988a, b). Polymerizing functions are also located in the C terminus of E. coli DNA pol I (JOYCE and STEITZ1987). Thus, although there is little proteinsequence homology shared between E . coli DNA pol I and the above DNA polymerases, many DNA polymerases appear to have the same relative organization of 3’ + 5 ’ exonuclease and polymerase activities. (REHAKRANTZ1988a, b; BERNAD et al. 1989; LEAVITTand IT0 1989). Arguments are presented here that extend comthe

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parisons between E. coli DNA pol I and bacteriophage T 4 DNA polymerase, and, by inference, toother DNA polymerases. Many DNA polymerases contain N-terminal protein sequences upstream from the proposed 3’ += 5’ exonuclease and polymerase domains. In E . coli DNA pol I, the N terminus encodes a discrete protein domain with 5’ + 3‘ exonuclease activity that can be separated by mild proteolysis from the larger Klenow fragment (3’ + 5’ exonuclease and polymerase domains) (LEHMAN and CHIEN1973; JACOBSEN, KLENOW andOVERGAARD-HANSEN1974). Some Nterminal T 4 DNA polymerase protein sequences are similar to sequences in the E. coli DNA polymerase I 5’ + 3’ exonuclease domain; there is also some protein sequence similarity with E. coli RNase H. Furthermore, intragenic complementation has been detected between T 4 N-terminal DNA polymerase mutants and DNA polymerase C-terminal mutants. Intragenic complementation has also been observed with some herpes (HSV-1) N-terminal DNA polymerase mutants (CHARTRAND et al. 1980; WELLERet al. 1983; A. I. MARCYand D.M. COEN,personal communication). T h e positive complementation results suggest that T 4 and HSV-1 DNA polymerases may have discrete, N-terminal domains while the protein sequence similarities suggest that the N terminus may encode a 5’ + 3’ exonuclease or RNase H-type activity; an RNase H-type activity has recently been detected in the HSV-1 DNA polymerase (CRUTEand LEHMAN 1989; MARCYet al. 1989). The main prediction arising fromthese studies is that many DNA polymerases may share the same structural-functional organization as found in E . coli DNA polymerase I: a distinct N-terminal domain that may encode a 5’ + 3’ exonuclease or RNase H activity adjacenttoa Klenow-type domain with3’ + 5’ exonuclease and polymerase activities.

214

L. J. Reha-Krantz MATERIALS AND METHODS

Phage and bacterial strains: Phage T 4 and bacterial et al. 1986; strainshavebeendescribed (REHA-KRANTZ REHA-KRANTZ 1988a). Complementationtests: Single and mixed infections at a were doneat multiplicity of five foreachphagestrain nonpermissiveconditions:hightem erature(43") which restricts ts phage andhost E. coli B (su ) which does not have a nonsense suppressortRNA andrestricts phage with amber mutations. Many gene 43 amber mutants aresuppressed at low levels on suo hosts at 30" by translationalambiguity (KARAM and O'DONNELL 1973). At 43", however, there is little suppression (see discussion on transmission coefficients below). At 10 min post infection, cultures were diluted 100fold into 43" broth to diluteaway from unadsorbed phage. Chloroform was added at 40 min and the progeny yields were determined by plating on CR63 (su+) at 30". Progeny are expectedonly if the infecting phage can mutually supply in trans the function missing in the other coinfecting phage. T h e burst size is equal to the total number of progeny divided by the total number of infected bacteria. T h e infective center titer of the wild-type control was used to calculate all burst sizes. Parental phage were recovered in all cases of positive complementation. Transmission coefficients: In order to determine the level of suppression due to translational ambiguity at 43" in E. coli B, dilutions of the amber phage-E. coli B infective centers weremixed at 10 min postinfection with the su+ host CR63 in soft agar, then overlaid on hard agar and incubated overnight at 43". Any viable phage produced in the amber phage-E. coli B infective centers infect the surrounding CR63 bacteria and produce plaques. No plaques were observed from amE4314, amE4306 and amE4302 infective centers, but 10% of the amB22-E. coli B infective centers produced plaques. Thus, low levels of suppression cannotaccount for the positive complementation results observed with amE4314, amE4306 and amE4302 infections. Suppression is also probably not a factor in the positive complementation results observed with amB22. For complementation tests, only 40 min was allowed to produce viable phage and few progeny were detected in amB22 infections (Table 1). Thus, apparently a growth cycle longer then 40 min is required to produce progeny from amB22-infected E. coli B cells. Alkaline sucrose gradients: Pulse-labeled DNA was prepared as described (KONRADandLEHMAN1974). E. coli K12SH28 (FANGMAN 1969), which has reduced thymidine phosphorylase acitivity, was grown at 43" to 2 X lo8 bacteria/ml in M 9 mediumsupplemented with 0.4% glucose, 0.5% casamino acids, and 0.01% MgS04. At to, cells were infected with phage at a multiplicity of ten. At t 5 min, the infected cells were diluted tenfold into 10ml of prewarmed M9 medium. At t7 min, 200 p1 of ["]thymidine (0.3 mCi/ ml were added. Cultures were aerated by shaking througho u t infection and labeling procedures. Incorporation was quenched at 10 sec, 45 sec, 2 min and 10 min by adding an equalvolume of 75%ethanol solution containing 2% phenol, 20 mM sodium acetate (pH 5.2), and 2 mM EDTA. Cells were centrifuged at 15,000 X g for 10 min and the pellet was resuspended in 0.3 mlof 0.2 M NaOH-10 mM for 1 hr. The solution was EDTA and incubated at 40" clarified by centrifugation at 14,000 X g. Samples, 100 PI, 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 over a 0.5 r n l shelf of 80% sucrose. T h e gradients were centrifuged for 120nrin at 40,000 rpm at 5". Marker DNA, 2.7 kb, was prepared by 3'-endlabeling EcoRI digested

B

TABLE 1 Complementation betweenT4 DNA polymerase (g43) alleles -

~~

~~

Phage

Mutation and site"

Single phage infections Wild type amE4314 Tamber r p 202 T r pamber 20.004 13 amE4306 0.005 Gln amber 731 amB22 amber 844Trp amE4302 Gly 82 Asp tsmeA62 tsL56 Ala 89 T h r , Asp 363 Asn Asp 112 Asn tsPS7 tSPN Y 123 Pro Leu tsmel5 Asp 131 Cly tsA6O 0.07 Ser Gly 172 tsA 7 3 225 Arg His Pro Leu 340 tsMl9 tsSY Met 671 Ile Mixed infections: tsmel62 amB22 tsL56 amB22 tsPS7 + amB22 tsmel5 tsL56 tsmel5 tsme162 tsPS7 tsmel62 tsmel5 tsPS99 tsA60 amE4314 tsA60 amB22 tsA6O amE4302 tsA73 amE4314 tsA73 amE4302 tsmel5 tsA60 tsPS99 tsA60 tsmel5 tsA73 tsPS99 tsA73 tsPS99 + tsMlY tsPSY9 tsS9 tsPS99 amE4314 tsPS99 amE4306 tsPS9Y + amB22 tsPS99 amE3402 tsmel5 amE4314 tsme15 amE4306 tsmel5 + amB22 tsmel5 amE4302 Extragenic complementation: amB22 amN122 (gene 4 2 )

+ + + + + + + + + + + + + + + + + + + + + + +

Burst sile'

64.0 0.06

0.01 0.03 0.04 0.01

0.09 0.08 0.02 0.01 0.03

0.06 0.05 0.5 0.6 0.5 0.3 0.8 0.2 0.7 0.1 1.4 0.1 70.0 11.0 31.0 8.0 5.0 30.0 28.0 19.0 11.0 10.0 13.5 11.0 11.0 8.0 46.0

Standard three letter amino acid code is used. T h e wild-type amino acid is given first followed by the codon position and then the mutant aminoacid. Mutation identifications are given in REHAKRANTZ( 1 988a) and REHA-KRANTZ (1989). *Burst size is equal tothe total number of progenyphage produced in one cycle of replication divided by the total number of infected bacteria. All infections were at 43" in host E. coli B (see MATERIALS AND METHODS).

pUCl8 plasmid DNA; the peak of activity was recovered in fraction 22. Gradient samples, 200 pl, were collected from the bottom and precipitated with 10% trichloroacetic acid. Precipitates were collected on Whatman GF/A filters and radioactivity was determined in a liquid scintillation counter. RESULTS

Phage T4 N-terminal mutations: Mutational analyses of the T4 DNA polymerase gene (gene 4 3 ) have

T4 DNA Polymerase Structure-Function hm I

N

-1

I

rsme162 fsPs99

-

\ I 1

-

5’ -> 3 e

tsme/5, PS43

\

H

/

7” ”\

I I

amE4314 x

o

h

,

215

I

I r l I I I I I I l

I

I

I ‘ I I I I I I I I

I

“-

amE4306

3‘-> 5’ e1xo-

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-KRANTZet al. 1986) or that affect U V mutagenesis (DRAKE 1988). 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; REHAKRANTZ 1989).

with more C-terminal (codon #172 and above) conbeen used to study DNA polymerase structure-funcditional lethal mutations (Table 1). All of the DNA tion relationships (REHA-KRANTZ1988a,b;1989). polymerase fragmentsproduced in gene 43 amber DNA polymerase mutations that confer strong mutainfections lack polymerizing activity, and 3’ + 5’ extor phenotypes have been identified throughout gene onuclease activity has been detected only inthe amB22 43, but many of the mutations are located in the N fragment (NOSSAL1969; NOSSALAND HERSHFIELD terminus(Figure1)(REHA-KRANTZ1988a,1989). 197 1). The tsPS99 and tsmel5 strains have N-terminal The most N-terminal mutation sites are located upmutations (Figure 1) and must be deficient onlyin stream to the predicted 3’ + 5’ exonuclease active some essential, N-terminal activity, because these site and C-terminal polymerase functions (Figure 1) DNA polymerases apparently supply DNA polymer(REHA-KRANTZ 1988a, b; 1989). Mutator activity may ase functions that are missing in the amber fragments. be produced by a reduction in 3‘ -+ 5‘ exonuclease T h e apparent positive complementation results are activity thatfunctions to “edit” newly synthesized DNA (MUZYCZKA,POLAND and BESSMAN 1972); hownot likely due to productionof wild-type DNA polymerase molecules by recombination. In mixed infections ever, the tsmel62-DNA polymerase, the only mutant when few progeny were produced, e.g., tsA6O DNA polymerase with a mutation from this region to 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 growth at 43” on E. coli B. When positive comple1987). Reduced accuracy in base selection can also KARAMand mentation was observed, up to2.5% of the total produce the mutator phenotype (SPEYER, LENNY 1966; HERSHFIELD 1973; GILLENand NOSSAL progeny were wild type (e.g., tsmel5 amB22). 1976), but DNA polymerase functions predicted to Protein sequence similarities: Because some probe important for nucleotide selection are located aptein sequence similarities have been detected in the parently in the C-terminal half of the protein (REHA3‘ + 5’ exonuclease domains ofE. coli DNA pol I KRANTZ 1988a, b; SPICER1988). Thus, the most Nand T 4 DNA polymerase (REHA-KRANTZ 1988a, b; terminal mutations may identify a previously undeSPICERet al. 1988),thecorresponding N-terminal tected T 4 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 T 4 DNA polymerase encodes an essential funcwere found (Figure 2). Some protein sequence simition, perhaps in a domain separate from the 3’ -+ 5‘ larity was also detected in E. coli RNase H (Figure 2). exonuclease and polymerase activities, complementaAlthough it is difficult to assess the significance of the tion tests were performed, limited sequence similarities, the regions are colinear Complementationtests: Extensive tests for intraand threeregions contain sites ofT 4 DNA polymerase genic complementation in gene 43 were conducted mutations (Figure 2). In addition, one T 4 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 T 4 and ( t s ) phage (DRAKE et al. 1969); however the ts mutant E. coli DNA sequences in region3(Figure 2); this collection for those studies didnotcontain 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). Four E. coli DNA pol I collection of ts strains, but onlyin 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

T4 pol

5 16

V EF IT D ( 1 6R )Y R G R E K T

. .. .. . .

. . pol I

.. ..

.. ..

V E R Y D1 E N G K E R T R E V E

115

G D D T LV Al R E A E

T4 mutants

K

S hrn

I3!3mu T4 pol

77

pol I

210

G L E A L G M N D F K L A Y I S

.. .. . . .

. . .

. .

G L D T L Y A E P E KAI G L S

T4 mutants

D fsrne162

T

E€!mu RNase

H

64

L S TE D V S I Q Y V R

.. ..

. . . T4 pol

123

P M K A E Y E ID A IT H Y D S ID D R

. . HSV

248

pol I

251

-

. . . . .

F E A E V V E R T D V Y Y Y E

-

T KI T D V E L E L T- C

L

T4 mutants

-

T

-

-

G,N tsmel5, PS43

tSPS99

REGION 4 RNase H

137

A R A A A

. . 161 T4 pol pol I

.. ..

A K L A A K - L D C E G G D E V P Q E I L D R V l Y M P F D N E R D M L

.. ..

302

.. .. ..

. . .

. .. .. . .

A K P A A K P Q E T S V A D E A P E V T A T V 3 2 4 .............

.. ..

354

F D T E T D S L

FIGURE2.-Protein sequence similarities in the N-terminal DNA polymerase domains of E. coli DNA pol I (JOYCE,KELLEY and GRINDLEY 1982), phage T 4 (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 1988a). T h e hm mutation has been polymerase mutant strains and the strain designations are given and discussed in the text (REHA-KRANTZ described (DRAKE1988). Identical amino acids are indicated by (:) and conservative amino acid substitutions by (.). In region 3, similar amino acids i n T 4 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. T h e C-terminal end of region 4 corresponds to region 1 in REHA-KRANTZ (1988a), to region 2 i n SPICERet al. (1988) and to region IV in WANG,WONGand KORN(1989).

T 4 and herpes DNA polymerase N-terminal sequences were also searched for homologies. Although there are extensive T 4 and herpes DNA polymerase protein sequence similarities in regions predicted to encode polymerase and 3' + 5' exonuclease activities (SPICERet al. 1988; REHA-KRANTZ 1988a, b),only a little weak sequence similarity was detected in the Nterminus (region 3, Figure 2). This protein sequence (region 3) is also similar to a sequence found in E. coli RNase H and in the predicted RNase H domains of retroviral polymerase genes (JOHNSON et al. 1988). Thus, therecently discovered RNase H activity in the herpes DNA polymerase could reside in the N-termi-

nal domain. Some sequence similarity was also found in this region between the herpes (residues 210-260) and human DNA polymerases (residues 368-4 18) (sequences not shown). DNA synthesis in wild-type and tsmeZ5 infections: DNA synthesized in vivo by wild-type and tsmel5-DNA polymerases at 43" was monitored in alkaline sucrose gradients (Figure 3). DNA synthesis was examined for intervals of 10 sec, 45 sec, 2 min and 10 min. At all times, less DNA and smaller DNA was synthesized in tsmel5 infections (Figure 3). Mutator activity of the tsmeZ5 and tsPS99-DNA polymerases: T h e tsPS99 and tsmel5 mutations in-

T4 DNA Polymerase Structure-Function Wild type

Wild type 8000

217

I

I

10

0

20

10

0

30

tsmel5

tsmel5 I

600 500 400 300 200

100

o ! 0

30

Fraction number

Fraction number

700

20

1

1

10

20

0 30

Fraction number FIGUREJ.-Newly

i 0

10 rnin

A 10

20

30

Fraction number

synthesized DNA in wild type andtsmel5 infections at 43": analysis by sedimentation through alkaline sucrose gradients. T h e procedures are describedin MATERIALS A N D 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 100fold (REHA-KRANTZ1988a).Mutations produced in these mutator strains seem to occur preferentially at potential RNA priming sites and in tracts of purine residues. Six of tenmutationssequenced in these strains are within potential RNA primer sites which occur every 36 nucleotides on average in T 4 DNA (Figure 4) (CHAand ALBERTS1986). Two independent mutations were isolated in one sequence that may form asmall, stable hairpin structure similar to unusually stable hairpinstructuresthatare stabilized by et al. certain tetra loop sequences (Figure 4) (TUERK 1988). The sequenceproducesasequencing gel compression, likely due to hairpin formation, in 7 M urea at 50" (L. J. REHA-KRANTZ, unpublished observations). Thus,thestructure has anexpected free et al. energy value less than -8 kcal/mol (SHINEDLING 1987). T w o RNA primer sequences are utilized in in uitro priming assays withnon-T4 DNA (Figure 4) (CHA and ALBERTS1986); however, only the pppACNNN

primer is detected in T4 infections (KUROSAWAand OKAZAKI 1979)inor in vitro assays with T 4 DNA (LIU and ALBERTS1981). T 4 DNA is modified with glucosylated 5-hydroxylmethyl cytosine that is thought to block priming at 5' GCT sites (LIU and ALBERTS 1981).Thus, if the small collection of sequenced mutations is representative of themutational spectrum for the N-terminal mutant DNA polymerases, thenthere may be atypical RNA priming in these strains. DISCUSSION

Complementationwithinthephage T4 DNA polymerasegene: Allelic complementation was observed between strains with certain N-terminal mutations, tsPS99 (codon #123) and tsmel5 (codon #131), and strains with more C-terminal DNA polymerase conditional lethal mutations (Table 1). Only a few molecules of T 4 DNA polymerase are required to produce at least one viable phage per infected cell

L. J. Reha-Krantz

218 RNA

3’ N N N C A / G p p p

DNA

5’ N N N G T / C T

tivities, but a protein with homology to the 5’ + 3’ exonuclease domain of E. coli pol I is encoded on a separate gene (LEAVITT and ITO 1989). Similarly, it is Mutation imagined that the tsPS99- and tsmel5-DNA polymer1. 5’ G C T G T T sites ases provide Klenow-type activities while the amber fragments and C-terminal mutant DNA polymerases A provide an essential activity encoded in the N-terminus. 2. 5 ’ G A T G T T Function of the T4 DNA polymerase N-terminal domain: Many questions about a potential T 4 5‘ + C 3’ exonuclease activity remain unresolved. The pu3 . 5 ’ G A T GC T rified T 4 DNA polymerase cannot “nick translate” (NOSSAL and HERSHFIELD 1971; COZZARELLI, KELLY A and KORNBERC1969) and thus does not appear to have a powerful 5‘+3‘ exonuclease activity. Strand 4 . 5 ’ A T T GC T displacement synthesis is observed in T 4 DNA replication assayswith the addition of gene 32 protein T A - T T (helix-destabilizing protein) and DNA polymerase acC G 5 . 5 ’ A C C GC T cessory proteins, the products of genes 4 4 , 62 and 45 A e G C 6. (NOSSALand ALBERTS1983).It is not known how C G A A G G S A C RNA primers are removed during T 4 DNA replicaFIGURE4.-Mutations produced in T 4 D N A polymerase strains tion, although an RNase H activity has been detected with N-terminalmutations.While determiningtheamino acid in T4-infected cells (NOSSALand ALBERTS1983). An substitutions responsible for the mutator phenotype of T 4 D N A attractive model consistent with some data presented polymerase mutants, several “other” mutationswere detected in the here is that the DNA polymerase, perhaps stimulated DNA polymerase gene in various subcultures that did not seem to by accessory proteins or other proteins, has an RNase be required for the mutator phenotype and may be a result of the endogenousmutator activity of themutator DNA polymerase H-type activity. If the T 4 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 thatat high temperature, ts T 4 DNA polymerase priming sites (CHAand ALBERTS1986). T h e DNA sequence for mutants deficient in RNAprimer removal activity initiation of RNA priming is 5’ G T T or 5’ GCT and the initiation would be defective in joining nascent DNA fragments. sequence is underlined in each of themutation sites (CHA and ALBERTS1986). T w o mutation sites, 5 and 6, are within a sequence Alkaline sucrose gradient analyses of DNA synthethat may form a small hairpin structure. sized by wild-type and tsmel5 phage do not support this model. At 43” (Figure 3) and30” (datanot (KARAMand O’DONNEL 1973), but relatively the high shown) high molecular weight DNA was synthesized burst sizes observed underthe restrictive growing in tsmel5 infections, but at a slower rate than in wildconditions used here suggest that each of the phage type phage infections. No accumulation of short Okastrains provides essential DNA polymerase functions zaki fragments was observed as expected if removal in trans. T h e T 4 DNA polymerase in vivo compleof RNA primers was prevented. mentation may be achieved analogously to the in vitro Another puzzling question ishow can presumed complementation observed for the large and small deficiencies in 5’ + 3’ exonuclease or RNase H activfragments of E. coli DNA pol I (LEHMANand UYEity reduce the fidelity of DNA replication? Although MURA 1976). E. coli DNA pol I can be cleaved by mild removal of RNA primers does not appear to be a proteolysis into two discrete domains: the large, Cmajor problem in infections with these mutant phage terminal “Klenow” fragment that contains DNA po(Figure 3), mutations may occur preferentially at polymerase and 3’ + 5’ exonuclease activities, and a tential RNA priming sites (Figure 4). smaller, N-terminal fragment containing 5’ + 3‘ exModel: A working model to explain the properties onuclease activity (LEHMAN and CHIEN 197 ;1JACOBof N-terminal DNA polymerase mutations follows. NSON, KLENOWand OVERCAARD-HANSEN 1974). When terminal mutator mutants are located in a discrete both fragments are added toin vitro assays, full DNA domain that is essential for phage T 4 viability. Protein polymerase activity is restored (LEHMAN and UYEsequence comparisons indicate some similarities with MURA 1976). The bacteriophage T 5 DNA polymerase the 5’+ 3’ exonuclease domain of E . coli DNA pol I may provide an exampleof in vivo interaction between and with E. coli RNase H. RNA primer removal is not a DNA polymerase and a separate protein with 5’ + altered significantly in the mutant strains, but perhaps 3 ’ exonuclease activity. The bacteriophage T 5 DNA modest decreases in RNase H activity or in some other polymerase gene encodes aKlenow-type DNA polymaspect of 5’ + 3’ exonuclease activity, such as strand erase with 3’ + 5’ exonuclease and polymerase ac-

c

+

c

+

c +

219

Structure-Function Polymerase T 4 DNA

displacement, is responsible for the slow and reduced DNA synthesis. Forexample,strand displacement may require the DNA polymerase N-terminal domain in combination with DNA replication accessory proteins. Alternatively, some aspect of RNApriming during lagging strand synthesis may be affected. This idea is supported by a yeast N-terminal DNA pol I mutation thataffects primase-polymerase complex stability (LUCCHINI et a l . 1988). If the mutants are defective inDNA polymerase-primase interactions, only leading strand synthesis may occur under nonpermissive conditions. T4 primase-deficient strains synthesize significant amount of DNA by displacement synthesis (MOSIGet a l . 1981; NOSSAL and ALBERTS 1983). DNA synthesized in DNA polymerase N-terminal mutant strains may also be due primarily to displacement synthesis (Figure 3). Thus, the slow DNA synthesis may be due to “pauses” when RNA primers and/or secondarystructures in the DNA template are encountered or as a consequence of inadequate priming and reduction of lagging strand synthesis. In any case, lethality in these strains at high temperature is likely due to insufficient production of very high molecular weight,concatemeric DNA requiredfor packaging (BLACK and SHOWE1983). Mutations may be induced as a consequence of extended “pauses” in DNA replication. For example, persistant DNA discontinuities may interfere with the repair of damaged DNA and increase the probability of bypass replication (replication past nontemplating DNA lesions). Bypass replication has been detected in vitro for the tsmel62-DNA polymerase (REHA-KRANTZ 1987) and another N-terminal mutant, hm, increases ultraviolet mutagenesis presumably also by some type ofbypass replication (DRAKE 1988). Future prospects: One application of these finding is to use genetic engineering to produce a phage T 4 “Klenow”fragment with 3’ + 5’ exonuclease and polymerase activities. This project could succeed because T4 protein sequences with similarities to E . coli DNA pol I flank both sides of the E . coli Klenow N terminus at Val324(Region 4, Figure 2). The engineeringandcloning of E. coli DNA pol I Klenow fragment (JOYCE and GRINDLEY 1983) were invaluable for subsequent structural and biochemical studies (JOYCE and STEITZ1987). I thank A. MARCY,D. COEN,N. NOSSAL, L. BLANCOand G. M~SIG for helpful discussions; I . R. LEHMAN, W. KONIGSBERGand P. HASTINGS for critical reviews of the manuscript; and A. CHILDSSMITHand T. CHUDY-BRYAN for preparing the manuscript. This work was supported by theNatural Sciences andEngineering ResearchCouncil, Canada and the National CancerInstituteof Canada. The author isa Scholar of the Alberta Heritage Foundation for Medical Research.

Note Added in Proof. A monoclonal antibody directed against the a subunit of E. coli pol 111 holoenzyme cross-reacts with the N-terminal, 5’ + 3’ exo-

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