Biochemistry of DNA-Defective Amber Mutants of Bacteriophage T4

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1 JOURNAL OF VIROLOGY, Apr. 1974, p Copyright 1974 American Society for Microbiology Vol. 13, No. 4 Printed in U.S.A. Biochemistry of DNA-Defective Amber Mutants of Bacteriophage T4 IV. DNA Synthesis in Plasmolyzed Cells WILLIAM L. COLLINSWORTH AND CHRISTOPHER K. MATHEWS Department of Biochemistry, University of Arizona College of Medicine, Tucson, Arizona Received for publication 16 August 1973 Requirements for bacteriophage T4 DNA synthesis have been investigated in situ by use of plasmolyzed infected cells. When such cells are incubated with datp, dgtp, dttp, hydroxymethyldeoxycytidine triphosphate, and ratp, significant semiconservative synthesis of DNA occurs. This DNA hybridizes preferentially to T4 DNA. T4 amber mutants defective in genes 44 and 45, which display a DNA-negative phenotype in vivo, are unable to synthesize DNA in situ. By contrast, T4 amber mutants bearing lesions in genes 41 and 62, which also display a DNA-negative phenotype in vivo, do allow DNA synthesis in situ, the extent of synthesis being 8 to 9% that of the wild-type synthesis under the same conditions. Cells infected with gene 42 mutants (dcmp hydroxymethylase) are unable to synthesize DNA in situ even though exogenous nucleotides are provided. Also one gene 1 mutant (deoxynucleotide kinase) was found to synthesize DNA in situ, but two other gene 1 mutants did not. These results point to possible roles of hydroxymethylase and kinase in DNA metabolism, in addition to provision of essential DNA precursors, as has recently been suggested by Wovcha et al. (1973). A major interest of this laboratory is identification of the functions associated with T4 bacteriophage gene products essential to DNA replication, notably genes 41, 44, 45, and 62. According to the classification scheme of Warner and Hobbs (17), mutants in genes 44 and 45 are DNA negative because viral DNA replication is undetectable under nonpermissive conditions of infection. Mutants in genes 41 and 62 synthesize a small amount of DNA. The function of none of these gene products has been identified, although most of the gene products have been purified and their activity has been demonstrated in vitro (2). Our studies to date have involved experimentation on intact infected cells and have focused on parameters of RNA metabolism (11), sedimentation behavior of intracellular polydeoxynucleotides formed in abortive infection (16), and analysis of intracellular nucleotide pools (13). We started the present work in an attempt to ascertain whether the above-mentioned genes control hitherto unidentified steps in DNA precursor metabolism. Although mutants defective in genes 41, 44, 45, and 62 all gqnerate large pools of deoxyribonucleoside triphosphates (dntp's) in vivo (13), one could entertain the 98 notion, however remote, that the dntp's are not the proximal DNA precursors (19) or, perhaps, that the true precursors, whatever their nature, are compartmentalized within the cell, with the compartmentalization being somehow under phage genetic control. Accordingly, we sought a permeabilized infected cell preparation capable of allowing phage DNA replication in the presence of 5'-dNTP-s, so that we could ask whether DNA could be synthesized when the cells were infected with DNA-defective mutants. The sucrose-plasmolyzed cell system of Wickner and Hurwitz (21) gave satisfactory results and was used for these studies, which yielded some unexpected results. First, although mutants in all four genes display a DNA-negative phenotype in vitro in the lysedcell system of Barry and Alberts (2), we found substantial DNA synthesis in plasmolyzed cells infected with mutants in genes 41 or 62. Second, certain mutants defective in known steps of DNA precursor metabolism, which we had expected to synthesize DNA in situ, did not. Findings similar to these latter results have also been reported by Wovcha et al. (22) since the time we presented a preliminary communication of our work (W. L. Collinsworth and C. K. Downloaded from on October 5, 218 by guest

2 VOL. 13, 1974 T4 DNA SYNTHESIS IN SITU 99 Mathews, Fed. Proc. 32:52, 1973). MATERIALS AND METHODS Microorganisms and growth. Bacteria routinely used were Escherichia coli B, CR63, and D11 (polal -, Endl-, thy-), a derivative of P3478 (pola1 -, EndI+, thy-), which was kindly supplied by C. C. Richardson. Bacteria were grown on SM9, which is M9 (1) supplemented with.2% Casamino Acids. The cultures were routinely grown at 37 C with aeration. D11 was grown on SM9 supplemented with thymine (2 Ag/ml). The bacteriophages used were T4D and several amber or ts mutants which were present in our laboratory collection or came from the collection of H. Bernstein. All mutants came originally from the Cal Tech collection. Table 1 lists all phage strains used both by strain number and mutant type. Phage stocks were prepared in liquid media and purified by differential centrifugation. Chemicals. All nucleotides were from P-L Biochemicals except the labeled nucleotides, which were from New England Nuclear Corp. Hydroxymethyldeoxycytidine-5'-triphosphate (HM-dCTP) was prepared from 5'-dCMP. We first converted dcmp to 5'-hydroxymethyldeoxycytidylate (HM-dCMP) according to Flaks and Cohen (9) by using dcmp hydroxymethylase. Purified HM-dCMP was then phosphorylated with partially purified deoxynucleotide kinase from T2 (3) by using the phosphorylation conditions of Lehman et al. (1), except that phosphoenolpyruvate and pyruvate kinase were used to regenerate ATP. After purification by elution from DEAEcellulose by triethylammonium bicarbonate, the HMdCTP was checked for authenticity by thin-layer chromatography against standard HM-dCTP gener- TABLE 1. Bacteriophage T4 strains used Strain Mutant Mutant gene producta type B24 aml Deoxynucleotide kinase BU3b aml Deoxynucleotide kinase E957b aml Deoxynucleotide kinase N81b am4l Unknown N5 am4l Unknown N122 am42 Deoxycytidylate hydroxymethylase NG352b am42 Deoxycytidylate hydroxymethylase 434b am43 DNA polymerase P39 ts43 DNA polymerase N82 am44 Unknown E1 am45 Unknown E56b am56 dctpase-dutpase NG485 am62 Unknown E114 am62 Unknown a Identification of mutant gene products is reviewed elsewhere (12). bmutants which were not back-crossed to T4D in this laboratory. All other mutants were back-crossed at least twice. ously supplied by A. Kornberg. Thin-layer chromatography was carried out on PEI-cellulose.1-mm layers, backed by heavy aluminum foil, supplied by Brinkman Instruments. Elution in one dimension was carried out with 1. M LiCl saturated with boric acid and adjusted to ph 7.. Sucrose used for plasmolysis was the ultrapure form from Schwarz-Mann. Plasmolysis and DNA synthesis assay. D11 cells were grown to 5 x 1' cells per ml, harvested, and resuspended in SM-9 at 1' /ml. The cells were infected with 5 PFU/cell at 4 C for 5 min and then diluted threefold with warm medium (37 C) and incubated with aeration for 15 min. The cells were then harvested, washed, and plasmolyzed according to Wickner and Hurwitz (21) at a concentration of 1"l/ml. For the DNA synthesis assay, a 5-uliter sample of plasmolyzed cells was added to 25 Mliters of reaction mixture with components and concentrations as Wickner and Hurwitz (21), except that [CH,- 'H JdTTP (6.25 ACi/pmol) was used to label new DNA and HM-dCTP was used instead of dctp. Samples (5 uliters) were pipetted at appropriate times into.5 ml of.5 M sodium hydroxide-1% sodium dodecyl sulfate. After the addition of.2 ml of.1 M sodium pyrophosphate containing calf thymus DNA (1 mg/ ml), the tubes were incubated at 8 C for 15 min and cooled, and then 1 mg of bovine serum albumin and 5 ml of 1% trichloroacetic acid were added. The resulting precipitate was dispersed in 95% ethanol and then reprecipitated with 5 ml of 1% trichloroacetic acid and centrifuged. This was repeated again, and the resulting precipitate was dissolved in 1 ml of alcohol, added to a vial containing 5 ml of Aquasol (New England Nuclear Corp.), and counted. Preparation of bromouracil- and 32P-labeled bacteriophage. The grown conditions and labeling protocol were carried out as described in Murray and Mathews (16). The infection was allowed to go to complete lysis, and the resultant phage were purified by differential centrifugation. The phage were also treated with DNase and RNase at 1 gg/ml to clear the phage of contaminating nucleic acids. Pycnographic analysis. Plasmolyzed infected cells which had synthesized DNA were lysed by addition to a.3-ml reaction volume of.2 ml of.2 M EDTA,.2 ml of 1 M Tris-chloride buffer, ph 7.9, and 2 Ag of lysozyme, followed by incubation at 65 C for 2 min. Partial clearing occurred, and then 1 gliters of 2% sodium dodecyl sulfate and Pronase (2 mg/ml) were added and incubation continued for 1 h at 37 C. The lysate was then dialyzed against standard saline citrate (SSC) (.15 M NaCl-.15 M sodium citrate, ph 7.) overnight. Preparation of CsCl neutral and alkaline gradients was as described in Murray and Mathews (16). The gradients were formed by centrifugation at 35, rpm for 6 h for neutral gradients and 44, rpm for 44 h for alkaline gradients at 2 C in a Beckman SW5.1 rotor. Fractions (.13 ml) were collected on filter paper disks, washed three times with 5% trichloroacetic acid, and then washed three times with acetone, dried, and counted in 5 ml of Omnifluor (New England Nuclear Corp.). in Downloaded from on October 5, 218 by guest

3 91 COLLINSWORTH AND MATHEWS J. VIROL. DNA-DNA hybridization. In situ synthesized DNA was isolated from a 2.-ml reaction mixture after lysis as described above. The lysate was dialyzed against.1 x SSC overnight to reduce the salt content. The lysate was treated with heat-treated RNase (1 ug/ml) for 3 min at 37 C and then twice extracted with phenol which had been neutralized with 1. M phosphate buffer, ph 7.. The resultant solution was extracted with ether and dialyzed against.1 x SSC. After concentration by pressure dialysis, this solution was adjusted to.5 N NaOH, heated to 1 C for 5 min, neutralized, and then dialyzed against 3 x SSC. After sonic treatment for 2 min, this DNA was then hybridized with DNA-containing filters (1 jg/filter) according to Denhardt (6). The filters containing hybridized DNA were then washed, dried, and counted in Omnifluor. In vivo DNA synthesis assay. E. coli D11 were grown to 3 x 1' cells per ml on SM9 plus thymine (2 gg/ml) and uracil (2 gg/ml). The cells were harvested, washed with SM9, and resuspended in SM9 plus uracil (1 jg/ml). The cells were infected at 5 PFU/cell at 4 C for 5 min and then transferred to 37 C. [2-14C]uracil was immediately added to give a specific activity of 1.1 ;tci/4mol. Radioactivity incorporated into DNA was then determined after base hydrolysis of RNA according to Mathews (11). Because the labeling is carried out under conditions where exogenous uracil is the sole pyrimidine source, in vivo labeling data can be converted to amounts of DNA synthesis in terms of molecules of nucleotides incorporated per minute per cell. RESULTS In situ bacteriophage DNA synthesis. Plasmolyzed, bacteriophage-infected E. coli D11 could synthesize DNA, as shown by the incorporation of [3H]dTTP into acid-insoluble material. The synthesis took place in the presence of datp, dgtp, dttp, and HM-dCTP. The cytosine nucleotide, dctp, could replace HMdCTP in our system, but the extent of DNA synthesis in 3 min was usually considerably lower than when HM-dCTP was used (Fig. 1), possibly due in part to concomitant destruction of dctp by dctpase-dutpase. As described later in the paper, we found that deoxynucleoside-5'-monophosphates could substitute for the dntp's; Wovcha et al. (22) have reported similar findings. Interestingly, we found relatively late in the study that ratp did not appear to stimulate in situ DNA synthesis (Fig. 1), even though it is essential for DNA replication in plasmolyzed uninfected cells (21). However, ATP was present in all experiments reported in this paper. We also found that 4 mm N-ethylmaleimide could partially inhibit phage DNA synthesis in situ (Fig. 1), in line with the expectation that we were observing replicative DNA synthesis in situ. 6 x 4 ~C.>. 6 (1b) '4 z *dctp *+ATP FIG. 1. Incorporation of label from [3H]dTTP into alkali-resistant, acid-insoluble material. (a) Using plasmolyzed cells infected with bacteriophage T4Dam+ plus HM-dCTP; (b) using plasmolyzed cells infected by T4Dam+. As indicated, one incubation mixture contained no ratp, another contained 2 mm ratp, and another contained 2 mm ratp and 4 mm N-ethylmaleimide. The phage equivalent units of DNA per unit of radioactivity was calculated from a value for total nucleotides by assuming a value of 4.5 X 1O nucleotides per phage equivalent unit of phage T4 DNA. Total nucleotides incorporated is determined from nanomoles of dttp incorporated into alkali-resistant, acid-insoluble material. Each sample from the reaction mixture contained 8 x 18 plasmolyzed cells. The DNA synthesized in situ was mostly phage DNA, since it annealed specifically to purified T4 DNA and not to E. coli DNA (Table 2). There seemed also to be a small amount of E. coli DNA synthesized. Sedimentation analysis of DNA synthesized in situ showed that it sedimented closely with marker T4 DNA on both neutral and alkaline sucrose gradients, as shown in Fig. 2 for a 5 to 2% alkaline gradient. This indicated that DNA precursors were incorporated into DNA strands of almost mature DNA length. Infection of bacteria with bromodeoxyuridine [BUdR ]-labeled phage in the presence of [14C]BUdR and fluorodeoxyuridine (16) should give replicating DNA in which both strands at Downloaded from on October 5, 218 by guest

4 VOL. 13, 1974 TABLE 2. Specificity of in situ synthesized DNAa Input radioactivity bound In situ DNA source (%) T4 DNA E. coli DNA Uninfected cells T4-infected cells a The preparation of the in situ DNA added to the DNA-containing filters is described in Materials and Methods. The labeled DNA samples from uninfected and infected cells contained 1,577 and 22,17 counts per min per ml, respectively. The total volume of DNA sample added to each filter was 1. ml. The bound radioactivity in each case was corrected for nonspecific binding by subtraction of counts bound to blank filters treated otherwise identically. The relatively low efficiency of hybridization of DNA from infected cells to T4 DNA filters (19%) may have been due to partial saturation of binding sites on the filters with unlabeled parental and progeny DNA, the latter synthesized before plasmolysis. Fraction Number FIG. 2. Sedimentation analysis of in situ synthesized DNA on a 5 to 2% alkaline sucrose gradient. The DNA added to the gradient was a portion of a lysate of plasmolyzed cells in which in situ DNA synthesis had occurred. The lysate was prepared as described in Materials and Methods, except that ["4C]uracil-labeled phage was added to the mixture during lysis. The "4C-labeled DNA served as a sedimentation marker for mature T4 DNA. T4 DNA SYNTHESIS IN SITU the replication fork are BUdR-labeled. At the time of plasmolysis, which occurs at 15 min after the start of infection, most of the replication should be proceeding on DNA templates which have already undergone at least one round of replication such that most parental strands would be ["C ]BUdR-labeled. If in situ synthesis of DNA is a continuation of semiconservative replication (M. P. Oeschger and J. G. Files, Fed. Proc. 32:156, 1973), then one should observe the "C and 'H labels co-banding in a hybrid density position on neutral CsCl gradients, as was in fact observed (Fig. 3a). If similar in situ DNA were denatured, one would expect to see parental strands which are mostly ["C ]BUdR-labeled and thus would band at a dense position on an alkaline CsCl gradient. The daughter strands, on the other hand, would contain ["C ]BUdR label and [3H ]deoxythymidine label due to strand elongation by in situ replicative synthesis. Thus, for daughter strands the 14C and 3H labels should co-band in a hybrid density position. This also was observed (Fig. 3b). Presumably the "IC label in the dense region represented parental strands and the "4C and 3H radioactivity co-banding at a hybrid position represented the daughter strands. It is possible that synthesis of DNA in situ represents repair synthesis. However, our results tend to rule out this possibility. First, in situ repair synthesis would not be expected to generate a hybrid density molecular species (Fig. 3a), and second, repair would be expected to occur on either strand, such that one would not expect ["C`BUdR-labeled material which contained no 3H (dense material in Fig. 3b). Thus, DNA synthesis in situ seems to represent E Q z 'I ei Fraction Number 911 I i' O I! E Q FIG. 3. Isopycnic centrifugation of in situ synthesized DNA from plasmolyzed cells infected with BUdR-substituted T4 bacteriophage. DNA synthesized up to the point of plasmolysis was labeled with [4CClBUdR. In situ DNA was labeled by incorporation of label from [3H]ddTTP. (a) Neutral CsCI gradient. DNA was sheared before centrifugation by passage through a 21-gauge hypodermic syringe needle. (b) Alkaline CsCI. DNA was sheared before centrifugation by sonic oscillation for 5 min. The indicated reference points were determined by centrifugation of dense (HH) and light (LL) T4 DNA under identical conditions. z C a) co Downloaded from on October 5, 218 by guest

5 912 COLLINSWORTH AND MATHEWS J. VIROL. a continuation of the replication which was occurring at the time of plasmolysis. In asking whether genetic lesions in DNA synthesis due to faulty precursor metabolism could be corrected in situ, we observed that cells infected with an amber mutant in gene 56 could synthesize stable DNA in situ from exogenous dntp's (Fig. 4). Gene 56 codes for an enzyme of DNA precursor metabolism, namely, deoxycytidine and deoxyuridine di- and triphosphatase. Thus, a mutant which was DNA defective due to an error in precursor metabolism could synthesize DNA in situ. In this case, the defect involves (i) a deficiency of dcmp (from dctp breakdown), needed for HM-dCTP synthesis (18), and (ii) a limited synthesis of cytosinecontaining DNA, which is rapidly destroyed by phage-coded nucleases (2). A final general property of our system is its dependence upon T4 DNA polymerase, the product of gene 43. When we infected E. coli D11 with a gene 43 amber mutant (am434), we observed very little DNA synthesis (Fig. 4). This supports our conclusion that synthesis in situ represents DNA replication. Synthesis of DNA in situ with mutants in 6 (4a) *T4D b E c Do C > 46 w C 4... N 82 *E1O (4 b) T4D genes 41, 44, 45, and 62. By use of the in situ DNA assay system, we examined the DNA synthetic capacity of plasmolyzed cells infected with mutants in genes 41, 44, 45, and 62. Cells infected by mutants in gene 44 and 45 gave little if any DNA synthesis (Fig. 4), suggesting, in agreement with the results of Barry and Alberts (2), that these gene products participate in DNA replication at a post-precursor step. When we infected E. coli D11 with amber mutants in gene 41 (NO5) and gene 62 (NG485), we found that the plasmolyzed cells could synthesize DNA to approximately 8 to 9% of the extent seen at 3 min with wild-type T4- infected cells (Fig. 5). Both mutants displayed a small DNA synthesis phenotype in vivo in infection of E. coli D11 (Fig. 5). Thus, our in situ results were not due to suppression of the amber mutations in D11. We also tested other amber mutants in both gene 41 (N81) and gene 62 (E114) and found for N81 the same result seen with NO5 (Table 3). However, for E114, we found that this mutant did not give significant in situ synthesis (Table 3). Thus, only one of two tested amber mutants in gene 62 gave DNA synthesis in situ. Maximal rates of synthe- 8 6 I x 4 E a 2 ' x c D > c) LU -(5)L~.V~Y~Q a) n V iv o/t * T4D * NG485 * -_ *_ N Downloaded from on October 5, 218 by guest FIG. 4. Incorporation of label from [3H]dTTP into alkali-resistant material in situ. (a) Using plasmolyzed cells infected by T4Dam + anxd amber mutants E56 (gene 56), N82 (gene 44), and E1 (gene 45). (b) Using plasmolyzed cells infected with T4Dam+ and T4 am434 (gene 43) defective in T4 DNA polymerase. Q FIG. 5. (a) Incorporation of label from [2- l4c ]uracil into alkali-resistant, acid-insoluble material in vivo after infection by T4Dam+, N5(am4l), and NG485(am62). (b) Incorporation of label from [3H]dTTP into alkali-resistant, acid-insoluble material after plasmolysis of cells infected with T4D, N5, and NG485.

6 VOL. 13, 1974 TABLE 3. In situ synthesis of DNA by mutants in gene 41 and 62 Phage strain Phage straln units (x 18)a T4 DNA SYNTHESIS IN SITU Phage equivalent T4Dam NO5 (gene 41) N81 (gene 41) NG485 (gene 62) E114 (gene 62) a Expressed as number produced in 3 min with 5 x 19 cells per reaction mixture. sis in vivo and in situ are compared in Table 4. Although the efficiency of the in situ system was not very high for normal-infected cells, it was equally high for mutant-infected cells in situ and about 15-fold higher than corresponding rates for these mutants seen in vivo. DNA-DNA hybridization confirms that mutant-infected cells are synthesizing T4 DNA in situ (Table 4) Ġene 1 and gene 42 results. Early in our work, we tested the ability of T4amB24 (gene 1) to direct DNA synthesis in situ. Since gene 1 codes for deoxynucleotide kinase, essential for the synthesis of HM-dCTP, we expected that provision of this triphosphate in situ would circumvent the genetic block and permit DNA synthesis. Unexpectedly, we saw little if any DNA synthesis in situ. Of two additional gene 1 mutants tested, one (ambu3) also showed no DNA synthesis in plasmolyzed cells, whereas the other (ame957) showed the expected rescue of DNA synthesis in situ (Fig. 6). All three mutants display a DNA-defective phenotype in vivo (Fig. 6). Moreover, the synthesis seen in situ with E957-infected cells did not occur when DNA precursors were provided as deoxyribonucleoside monophosphates (Fig. 7), although T4D-infected cells were capable of such synthesis. Thus, E957 displayed the expected gene 1 phenotype, both in vivo and in sitli. That B24 and BU3 were also defective in gene 1 was TABLE 4. Rates of DNA synthesis in vivo and in situ Phage Rate of DNA synthesisa In vivo In situ T4Dam+. 2, N5 (gene 41) NG485 (gene 62) aexpressed as molecules of total nucleotide per minute per cell x 1-'. Rates are calculated from the periods of maximal incorporation observed in the experiment of Fig. 5. TABLE 5. Hybridization of DNA synthesized in situ in am41- and am62-infected cellsa DNA source Input radioactivity (%) bound to T4 DNA E. coli DNA T4-infected cells NO5-infected cell (am4l) NG485-infected cells (am62) aresults are expressed as in Table 2. The DNA samples added to the filters contained 4,625, 5,45, and 5,15 counts per min per ml for T4-, NO5-, and NG485-infected cells, respectively. The total volume of DNA sample added to each filter was 1. ml. 4v '3 (6a) In Vivo T4D E 2 1 ~~~~BU3 a) ell x 6- (6b) In Situ O~~~T41 in c ~~~~E LX w )~~~~~B3 Q_~ ~ * B FIG. 6. Incorporation of label into alkali-resistant, acid-insoluble material under: (a) in vivo conditions from [2-'4Cjuracil; and (b) in situ conditions from [3H]dTTP after infection by bacteriophage T4D and amber strains B24, BU3, and E957 in gene 1. confirmed by their failure to complement E957 in mixed infection. A similar result was observed in plasmolyzed cells infected by gene 42 mutants defective in deoxycytidylate hydroxymethylase. Again, since hydroxymethylase is involved in the pathway of synthesis of HM-dCTP, we expected that provision of this essential T4 DNA precursor in situ would allow DNA synthesis. However, no such synthesis was seen in plasmolyzed cells infected by either of two gene 42 mutants, N122 or NG352 (Fig. 8). Downloaded from on October 5, 218 by guest

7 914 COLLINSWORTH AND MATHEWS J. VIROL. 5o I x4-4) C: D +6 c 2. o-3 Q) L 1 2 FIG. 7. Incorporation of label from ["4C]dTMP, damp, dgmp, and HM-dCMP at 4 nmol/ml each, instead of dntp's. The other components of the reaction mixture were as described in Materials and Methods. Plasmolyzed cells infected by T4Dam+ and amber mutant E957 (aml) were used for the incorporation. DISCUSSION Several groups have now observed T4 DNA synthesis in vitro or in situ-barry and Alberts (2) with a lysed-cell system, Wovcha et al. (22) and ourselves with sucrose-plasmolyzed cells, and, quite recently, three groups with toluenetreated infected cells (7, 8, 14). Like us, none of the other workers have observed DNA synthesis in cells infected with gene 44 and 45 mutants. However, neither Barry and Alberts nor Wovcha et al. observed significant DNA synthesis in vitro with cells infected with gene 41 or 62 mutants, as did we. At present, we are not sure of the reason for this discrepancy, particularly that between our results and those of Wovcha et al. (22) in gene 41. However, as stated earlier, we have observed significant in situ DNA synthesis with two different gene 41 mutants. It may be significant, regarding the work of Barry and Alberts, that they used dctp in their in vitro system, whereas we used HM-dCTP. Perhaps cytosine-containing DNA can be synthesized in vitro but is subject to rapid degradation. Further study of the nature of the DNA synthesized in situ will be necessary for us to draw conclusions from our data regarding the functions of genes 41 and 62. It would be premature for us to conclude at this stage that these gene products control steps in DNA precursor metabolism, particularly since the apparent existence of the gene 62 product as a complex with the gene 44 product (2) would suggest a shared function for these genes. To date, comparison of the DNAs made in situ by T4am+, NO5, and NG458 mutants has revealed no significant differences when studied by. T4D / _*-- 9 E 957 DNA-DNA hybridization, banding patterns in CsCl gradients, or sedimentation analysis. The latter result suggests that the 41 and 62 defects do not involve the sealing of Okazaki fragments (M. P. Oeschger and J. G. Files, Fed. Proc. 32:156, 1973) into high-molecular-weight DNA, for if they did, one would expect to see most of the in situ DNA as small fragments, and this we did not see (data not shown). The failure of gene 1- and gene 42-infected cells to synthesize DNA after plasmolysis has also been reported by Wovcha et al. (22), who interpreted their gene 42 results in terms of a second function for dcmp hydroxymethylase -direct participation of the enzyme in DNA synthesis. However, since replication in all in vitro systems studied to date represents elongation of chains whose synthesis had been initiated in vivo (4), one could argue, as have Dicou and Cozzarelli (7), that the failure to observe DNA synthesis in situ with gene 1 or gene 42 mutants represents simply the failure of replication forks to have been initiated in vivo before plasmolysis. This conclusion is argued against by our results with T4amE957; a gene 1 mutant displaying a DNA-negative phenotype in vivo could nevertheless synthesize DNA at wild-type rates in situ (Wovcha et al. [24] did not observe DNA synthesis in E957-infected cells; presently we cannot explain this disagreement with our result). One possible conclusion from our gene 1 results is that the gene 1 product is a bifunctional protein. The amber fragments synthesized by BU3 and B24 may lack both activities of the complete protein, and hence the kinase block cannot be relieved in situ. However, the E957 amber fragment, by this argument, contains the presumed second function but not the kinase activity. Thus, provision of HM-dCTP in CD EoK6 D 4 o 4 K ~~T4 D c) t ~~~~~~N122 -o - - NG M i nutes FIG. 8. Incorporation of tritium from [3H]dTTP into alkali-resistant, acid-insoluble material in situ after infection by T4Dam+ and amber mutants N122 and NG352 in gene 42 (dcmp hydroxymethylase). Downloaded from on October 5, 218 by guest

8 VOL. 13, 1974 T4 DNA SYNTHESIS IN SITU 915 situ circumvents the only metabolic defect. One might expect from this that the E957 mutation maps far toward that end of the gene specifying the C-terminus. To date, however, ordering of the mutant sites within gene 1 has not been reported. Another possible explanation of our gene 1 results is that both B24 and BU3 are double amber mutants, each lacking kinase activity and some other function needed for DNA replication. This possibility is strongly countered by the fact that our B24 strain has been backcrossed four times to wild-type T4Dam+, so that a double mutant here would be quite unlikely. Also, both B24 and BU3 have reversion frequencies similar to that of E957 and considerably higher than those expected of double amber mutants (data not shown). The amng352 mutation maps farther, toward the C-terminus than any other in situ tested gene 42 mutation (W. L. Collinsworth and C. K. Mathews, Fed. Proc. 32:152, 1973). Thus, if the above reasoning applies both to gene 1 and gene 42, the putative second function of the hydroxymethylase protein requires a nearly complete polypeptide chain. Further analysis of these phenomena will require confirmation of the present results in an in vitro system permeable to macromolecules so that we can test the effects of purified kinase and hydroxymethylase and amber fragments thereof. ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant AI-823 from the National Institute of Allergy and Infectious Diseases and research grant from the American Heart Association. We thank Thomas North for his assistance in the synthesis of HM-dCTP. LITERATURE CITED 1. Adams, M. H Bacteriophages. John Wiley and Sons, New York. 2. Barry, J., and B. Alberts In vitro complementation as an assay for new proteins required for bacteriophage T4 DNA replication: purification of the complex specified by T4 genes 44 and 62. Proc. Nat. Acad. Sci. U.S.A. 69: Bello, L. J., and M. J. Bessman The enzymology of virus-infected bacteria. IV. Purification and properties of the deoxynucleotide kinase induced by bacteriophage T2. J. Biol. Chem. 238: Burger, R. M Toluene-treated E. coli replicate only that DNA which was about to be replicated in viuo. Proc. Nat. Acad. Sci. U.S.A. 68: Cornett, J. B., and M. Vallee The map position of the immunity (imm) gene of bacteriophage T4. Virology 51: Denhardt, D. T A membrane-filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23: Dicou, L., and N. R. Cozzarelli Bacteriophage T4-directed DNA synthesis in toluene-treated cells. J. Virol. 12: Elliot, J., C. Richter, A. Souther, and R. Bruner Synthesis of bacteriophage and host DNA in toluenetreated cells prepared from T4-infected Escherichia coli: role of bacteriophage gene D2a. J. Virol. 12: Flaks, J. G., and S. S. Cohen The enzymatic synthesis of 5-hydroxymethyldeoxycytidylic acid. Biochim. Biophys. Acta 25: Lehman, I. R., M. J. Bessman, R. S. Simms, and A. Kornberg Enzymatic synthesis of deoxyribonucleic acid. I. Preparation of substrates and partial purification of an enzyme from Escherichia coli. J. Biol. Chem. 233: Mathews, C. K Biochemistry of deoxyribonucleic acid-defective amber mutants of bacteriophage T4. I. Ribonucleic acid metabolism. J. Biol. Chem. 243: Mathews, C. K Bacteriophage biochemistry. Van Nostrand Reinhold Co., New York. 13. Mathews, C. K Biochemistry of deoxyribonucleic acid-defective amber mutants of bacteriophage T4. III. Nucleotide pools. J. Biol. Chem. 247: Miller, R. C., Jr., D. M. Taylor, K. MacKay, and H. W. Smith Replication of T4 DNA in Escherichia coli treated with toluene. J. Virol. 12: Moses, R. E., and C. C. Richardson Replication and repair of DNA in cell of Escherichia coli treated with toluene. Proc. Nat. Acad. Sci. U.S.A. 67: Murray, R. E., and C. K. Mathews Addition of nucleotides to parental DNA early in infection by bacteriophage T4. J. Mol. Biol. 44: Warner, H. R., and M. D. Hobbs Incorporation of uracil-c " into nucleic acids in Escherichia coli infected with bacteriophage T4 and T4 amber mutants. Virology 33: Warner, H. R., and M. D. Hobbs Nucleotide accumulations in Escherichia coli infected with some bacteriophage T4 amber mutants. Virology 36: Werner, R Nature of DNA precursors. Nature N. Biol. 233: Wiberg, J. S Amber mutants of bacteriophage T4 defective in deoxycytidine diphosphatase and deoxycytidine triphosphatase. J. Biol. Chem. 242: Wickner, R. B., and J. Hurwitz DNA replication in Escherichia coli made permeable by treatment with high sucrose. Biochem. Biophys. Res. Commun. 47: Wovcha, M. G., P. K. Tomich, C. Chiu, and G. R. Greenberg Direct participation of dcmp hydroxymethylase in svnthesis of bacteriophage T4 DNA. Proc. Nat. Acad. Sci. U.S.A. 7: Downloaded from on October 5, 218 by guest