Studies on Polynucleotides, Xc.* Dna with Single

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STUDIES ON POLYNUCLEOTIDES, XC.* DNA POLYMERASE-CATALYZED REPAIR OF SHORT DNA DUPLEXES WITH SINGLE-STRANDED ENDSt BY N. K. GUPTA AND H. G. KHORANA

INSTITUTE FOR ENZYME RESEARCH, UNIVERSITY OF WISCONSIN, MADISON Communicated July 11, 1968 Short chemically synthesized deoxyribopolynucleotides containing appropriate 5'-phosphate and 3'-hydroxyl end groups can be enzymatically joined end to end, when they are properly aligned, to form bihelical complexes.l1 2 In this way, several short DNA's (Fig. 1, DNA-III-DNA-VI) corresponding to a part of the gene for yeast ala-tRNA have been prepared.2 The products (Fig. 1) are not perfect duplexes; the strands at the 3'-hydroxyl termini are shorter by one, a few, or as many as ten nucleotide units. To pursue our interest in the chemistry of DNA repair and to further characterize the above products of DNA-joining enzymes,2 we have studied the incorporation of nucleotides catalyzed by the DNA polymerase of E. coli by using conditions under which repair of single- stranded ends has previously been demonstrated.' At the start of the present work, we were particularly encouraged by the recent results of Wu and Kaiser4 who have successfully used the DNA polymerase for structural analysis of the cohesive ends of XDNA. Our present results with the short DNA's of Figure 1 show that: (1) the DNA polymerase does indeed bring about repair of the strands shorter at the 3'-hydroxyl ends; (2) the repair reaction continues essentially to completion except for the very terminal nucleotide unit; and (3) there is no evidence of any further synthesis, i.e., any nucleotide incorporation beyond that required for the repair reaction. Furthermore, there was no manifestation of any exonucleolytic activity under the conditions used for the repair reactions and, again, this conclusion is in agreement with that of Wu and Kaiser.4 Materials and Methods.-All of the DNA's used in the repair reactions are shown in Figure 1: DNA-Il is simply a mixture of equivalent amounts of the two synthetic icosanucleotides Icosa-I and Icosa-II;2 DNA-I is a mixture of Icosa-I and the complementary heptanucleotide P12-C-T-A-A-G-G-G (Hepta-I) ;2 DNA-III-DNA-VI all origi- nated from the DNA ligase-catalyzed joining of short complementary oligonucleotides to one or both of the icosanucleotides of DNA-Il, as described previously.2 Thus, DNA-III resulted from Icosa-I + Icosa-II + the nonanucleotide P12-T-C-T-C-C-G-G-T-T (Nona- II); DNA-IV resulted from the two icosanucleotides + Hepta-I (see above) and Hepta-II (P32-T-C-T-C-C-G-G); DNA-V resulted from the two icosanucleotides + Hepta-I + Nona-II, and DNA-VI resulted from the two icosanucleotides + Nona-I (P'2-C-T-A-A G-G-G-A-G) + Hepta-Il. The joining reactions used 5'-P12-labeled (weakly) hepta- and nonanucleotides and were carried out with the T4-induced ligase as described before,2 except that the shorter components (hepta- and nonaiiucleotides) were used ill amounts somewhat in excess (25-75%) of those estimated to be stoichiometric with the icosanueleotides. In some experiments, the ligase products were freed from the excess of the short oligonucleotides by gel filtration on a Sephadex G-50 column; very often, however, the incubation mixtures after the joining reactions served directly as stock solutions for the repair reactions. In the latter case, the gel filtration method used for isolation of the DNA-polymerase products separated the DNA's both from the excess of the 5'-P'2-oligonucleotides and the excess of the deoxyribonucleoside 5'-triphosphates. 215 Downloaded by guest on September 29, 2021 216 BIOCHEMISTRY: GUPTA AND KHORANA -PRO.c. N. A. S. HI-labeled dATP, dCTP, and dTTP were commercial products and had the following specific activities: dATP, 5.66 c/mole; dTTP, 4.0 c/mmole; dCTP, 26.0 c/mmole. a-P12-labeled dATP, dTTP, and dCTP were obtained from International Chemical and Nuclear Corporation. The triphosphates were checked for purity by paper chromatography with: solvent A, isobutyric acid-conc. ammonium hydroxide-water (66:1:33), and solvent B, ethanol-1 M ammonium acetate (7:3, v/v), pH 7.5. The preparation of E. coli DNA polymerase was a generous gift from Drs. M. Deutscher and A. Kornberg. The enzyme had a specific activity of approximately 20,000 units/mg protein. Dilutions (40-fold or higher) of this preparation were made with the standard diluent,5 and the diluted solution was stored in ice. Conditions for repair reactions and isolation of the products: The reaction mixture (0.05- 0.1 ml) contained 120 mM potassium phosphate buffer, pH 6.9, 12 mM MgCl2, 12 mM dithiothreitol, 0.02 mM a-P"2-labeled or HI-labeled deoxynucleoside triphosphate, 0.02 mM of other unlabeled deoxynucleoside triphosphates, where indicated, an aliquot (0.005-0.01 ml) of the standard DNA-joining enzyme incubation mixture2 and 50-200 units/ml of E. coli DNA polymerase. The concentrations of the enzyme used in different experiments are indicated in the legends. Incubation was performed at 4-5O for 3.5-4 hr. Experiments in which the incorporation of labeled nucleotides was measured as a function of time showed that a plateau was reached within 2 hr even when the lower level (50 units/ml) of the enzyme was used. The solution was then heated in a boiling- water bath for 2 min, applied onto a Sephadex G-50 (superfine) column (a 2-ml graduated pipet) pre-equilibrated with 0.05 M triethylammonium bicarbonate buffer, pH 7.5, and eluted with the same buffer. The first radioactive peak (after about 1.5 ml) corresponded to the repaired DNA, which also contained weak P12-radioactivity introduced from the ligase reaction. This was followed by an excess of the 5'-P12-oligonucleotide and the radioactive deoxynucleoside triphosphates. Examples of separation of the products are in Figure 2 (see below). The first peak was repeatedly evaporated first with pyridine to remove triethylammonium bicarbonate and then with added ammonia. The product was then degraded to 3'-nucleotides and nucleotides for nearest-neighbor analysis2 and the products were separated by paper chromatography. Paper chromatography and tritium counting: When HI-labeled deoxynucleoside triphosphates were used in the repair reaction mixture, the nuclease-digested material was chromatographed on Whatman no. 40 paper in solvent C, isopropanol-NH3-H2O (7:1:2). The chromatograms were cut into 1-cm wide strips along the length, and after being further cut into smaller pieces, the strips were shaken in 0.5 ml of 0.1 N ammonium hydroxide overnight in scintillation vials. This was necessary for complete elution of the nucleotides from the paper, since the HI-counting efficiency of the nucleotides otherwise was very poor. The ammoniacal solution in the vials was mixed with the aqueous scintillation medium and counted for radioactivity. When a_-P2-labeled deoxynucleoside triphosphates were used, the mixture of 3'-nucleotides was analyzed by paper chromatography in either of two solvent systems: solvent A, as above, or solvent D, ammonium sulfate (60 gm)-0.1 M sodium phosphate, pH 6.8, (100 ml)-n-propanol (2 ml). Sometimes, the more readily accessible deoxynucleoside-5'-phosphates were used as markers; al- though the Rf's are not completely identical with those of 3'-nucleotides, the relative positions are essentially the same. Preparation and characterization of 5'-P_2-labeled DNA-V: The 5'-OH end groups of DNA-V were labeled with p32, and the product was isolated by gel filtration.' Degrada- tion with venom phosphodiesterase showed that the only radioactive product (32,000 cpm) was d-pG. Results.-(A) Repair reactions using a-P32-deoxynucleoside 5'-triphosphates: In these experiments, one deoxynucleoside triphosphate carried P32-label in the a-phosphate group, and this labeled triphosphate was used either alone or, most often, in the presence of the other three triphosphates, which were cold. In a typical experiment, the incorporation of a-P32-dATP in the presence of cold Downloaded by guest on September 29, 2021 VOL.VBIOCHEMISTRY:61, 1968 GUPTA AND KHORANA 217 dTTP + dCTP + dGTP was studied with DNA-I and DNA-II (Fig. 1). The results in Figure 2, which are illustrative of the method of separation used throughout in the present work, show that (1) incorporation of P32-dATP did occur; (2) incorporation requires the presence of both strands of DNA; and (3) the extent of incorporation with DNA-II was much higher than that with DNA-I. For repair at the 3'-hydroxyl end, the addition of the sequence A-G-C may be expected in the case of DNA-I, whereas with DNA-II, both arms would be filled in and the sequence showing incorporation of P32-dATP would be C-T-A-A-G-G- G-A-G-C. The actual incorporation of label in DNA-I, as seen in Figure 2, was less than the expected one third of that in DNA-II and it therefore appears that the repair of a structure such as DNA-I was not very efficient under the conditions used. The P32-labeled products obtained in the experiment shown in Figure 2 were characterized by nearest-neighbor analysis (Table 1). (Some radioactive inorganic phosphate traveling at the front was observed in this experiment.) Similarly, the incorporations of a-P32-dTTP and of a-P32-dCTP in the presence of the other three cold triphosphates were studied with DNA-II, and the results of the nearest-neighbor analyses performed on the products are shown in Table 1. All these results showed that the incorporations were, on the whole, as expected for extension of the shorter complementary chain by growth at the 3'-hydroxyl SYNTHETIC SHORT DNA!S WITH SNGLE-STRANDED ENDS STRUCTURE DESIGNATION (HEPTA-I) 3'- G-G-G-A-A-T-C (5')-DEOXY 111111l lDNA-I S'-G-C-T-C-C-C-T-T-A-G-C-A-T-G-G-G-A-G-A-G (3')-DEOXtY (ICOSA-D

(ICOSA-I) 3'- G-T-A-C-C-C-T-C-T-C-A-G-A-G-G-C-C-A-A-G (5')-DEOXY I DNA-I[ 5'-G-C-T-C-C-C-T-T-A-G-C-A-T-G-G-GA-G-A-G (3')-DEOXY C ICOSA-U)

3'- G-T-A-C-C-C-T-C-T-C-A-G-A-G-G-C-C-A-A-G (5')-DEOXY | 11II 1 1 DNA-_ 5'-G-C-T-C-C-C-T-T-A-G-C-A-T-G-G-G-A-G-A-G-T-C-T-C-C-G-G-T-T (3')-DEOXY

3'- G-G-G-A-A-T-C-G-T-A-C-C-C-T-C-T-C-A-G-A-G-G-C-C-A-A-G (5')-DEOXY I I I I DNA-1Zi 5'-G-C-T-C-C-C-T-T-A-G-C-A-T-G-G-G-A-G-A-G-T-C-T-C-C-G-G (3')-DEOXY

3'- G-G-G-A-A-T-C-G-T-A-C-C-C-T-C-T-C-A-G-A-G-G-C-C-A-A-G (5)-DEOXY I DNA- Y 5'-G-C-T-C-C-C-T-T-A-G-C-A-T-G-G-G-A-G-A-G-T-C-T-C-G-G-G-T-T (3')-DEOXY

3'- G-A-G-G-G-A-A-T-C-G-T-A- C-C-C-T-C-T-C-A-G-A-G-G-C-C-A-A-G (5')-DEOXY I I I I I I I I I lI I I DNA-YI 5'-G-C-T-C-C-C-T-T-A-G-C-A-T-G-G-G-A-G-A-G-T-C-T-C-C-G-G )-DEOXY FIG. 1-Synthetic DNA's corresponding to nucleotide sequence 21-50 of yeast ala-tRNA. All of the DNA's with polynucleotide chain lengths greater than 20 were obtained by enzymatic joining of the appropriate short deoxyribopolynucleotides to the two icosanucleotides shown, as described previously.2 Downloaded by guest on September 29, 2021 218 BIOCHEMISTRY: GUPTA AND KHORANA P.ROc. N. A. S.

HEPTA-I IC^O 1 1 I FIG. 2.-Separation of DNA polymerase re- a -oANEM tl pair reaction product from the of the l labeled deoxyribonucleoside 5'-triphosphate. Gel filtration technique 0-800 I I 11 as described in Ma- w _ terials and Methods was used. The first peaks n \ [ B from the left correspond to the polynucleo- a. s tidic material and are followed by the excess I \ of the labeled triphosphate. The incubation X- I rl for the repair reaction was under standard 4 1 1 conditions and used 200 units/ml of the DNA 400- 1polymerase. The labeled triphosphate wasa- P'2-dATP, the other three triphosphates being cold. The polynucleotides used in different tubes are shown in the figure.

10 I' 2 30 FRACTION NUMBER ends. Thus, with a-P32-dTTP, the radioactivity distribution in 3'-nucleotides on complete repair of the two arms should be d-Ap: d-Gp: d-Tp: d-Cp = 0:2:1:2. No radioactivity was, in fact, found in d-Ap, and the radioactivity in d-Gp and d-Cp was equal, as expected. The radioactivity in d-Tp was less than half of that in d-Gp or d-Cp, and this result agrees with the conclusion (see below) that the addition of the nucleotides at or close to the terminus is incomplete. For the

a-P32-dCTP experiment, the radioactivity in the 3'-nucleotides should be: d- Ap: d-Gp: d-Tp: d-Cp = 0:2:3:1. Again, no radioactivity was found in d-Ap. The ratio of radioactivity in d-Cp and d-Tp was also as expected, but that in d-Gp seemed to be more than was expected. The reason for this is not understood (see also below). Further experiments with a-P32-deoxynucleoside triphosphates were carried out extensively with DNA-III and DNA-IV (Fig. 1), and the manner of incor- TABLE 1. Nearest-neighbor analyses of incorporated nucleotides in repair reactions with a-P"2-deoxyribonucleoside triphosphates. a-P32-deoxy- Radioactivity Found(cpm) DNA triphosphate d-Ap d-Gp d-Tp d-Cp Pi DNA-I dATP 0 780 0 0 0 DNA-II dATP 772 773 840 0 340 dTTP 0 5,563 1,762 5,829 62 dCTP 0 5,548 5,028 1,616 0 DNA-III dATP 1,054 918 908 0 0 dTTP 0 0 0 1,148 0 dCTP 0 1,047 202 0 0 DNA-IV dATP 0 1,844 0 0 0 dTTP (a) 0 1,730 1,350 0 0 dTTP(b) 1,620 1)045 0 0 dCTP O 1,120 240 0 0 Incubation conditions were standard: the level of DNA polymerase in repair reactions with DNA-I and DNA-IL was 220 units/mi, whereas with DNA-IIL and DNA-LV, it was 45 units/ml. All of the repair reactions contained one a-P32-deoxynucleoside triphosphate as shown and the other three cold triphosphates, except in experiment (b) with DNA-LV when a-P32-dTTP alone was used. Aliquots of the repair products were degraded to 3'-nucleotides, and the products were separated in solvent D. Downloaded by guest on September 29, 2021 VOL. 61, 1968 BIOCHEMISTRY: GUPTA AND KHORANA 219

porations was studied by nearest-neighbor analyses. The results are all included in Table 1. Thus, with DNA-III, when a-P82-dATP was used together with the three cold triphosphates, the radioactivity was distributed essentially equally between d-Ap, d-Gp, and d-Tp, none being found in d-Cp. When a-P32-dTTP was used in the presence of dATP + dGTP + dCTP with DNA-III, all of the radioactivity was found in d-Cp. In addition to proving that only repair of the single-stranded arm occurred, the results of this experiment show conclusively that the exonucleolytic activity from the 3'-hydroxyl end was completely nonfunctional under the conditions of the present experiments. Thus, DNA-III contains the nucleotide sequence T-T at the right-hand (in Fig. 1) 3'-OH terminus. If the exonucleolytic activity were at all functional, then the first consequence would be the loss of the terminal T-nucleotide. This would then be restored by the DNA polymerase. In the present experiment with a-P32-dTTP plus three cold triphosphates, some radioactivity would be found in d-Tp upon subsequent degradation of the repaired product. No radioactivity was actually found in this nucleotide. In repair experiments with DNA-IV, when a-P32-dATP + dTTP + dCTP + dGTP were used, all of the radioactivity was specifically found in d-Gp. With a-P32-dTTP + dATP + dCTP + dGTP, all of the radioactivity was found in d-GP and d-TP (Table 1). Theoretically, for complete repair of the T-T-C end in DNA-IV, the radioactivity in the a-P32-dTTP experiment should be divided equally between d-Tp and d-Gp. However, as seen in Table 1, the radioactivity in d-Gp was somewhat higher than that in d-Tp (56% in d-Gp and 44% in d-Tp). The introduction of the penultimate T-unit in the repair reaction was therefore less than quantitative, about 80 per cent of the one preceding it. The insertion of the penultimate T was evidently reduced further when a-P32- dTTP alone was used in repair reaction, the radioactivity in d-Gp being even higher than that in d-Tp (Table 1). It should again be pointed out that the incorporations in these experiments are all plateau incorporations, for a further check of the kinetics of incorporation of a-P32-dTTP for up to four hours in the above experiment showed that the incorporation was already maximal at about one hour. The lack of complete repair at the ends was further evident in experiments using a-P32-dCTP + dTTP + dGTP + dATP. With DNA-III, the radioactivity was found as expected in d-Gp and d-Tp only. However, the radioactivity in d-Gp was much higher than that in d-Tp (Table 1). (For complete repair, the ratio of radioactivity should be d-Gp:d-Tp = 2:1). With DNA-IV, where the C units to be introduced are at the two 3'-OH termini and the ratio of radioactivity in d-Gp: d-Tp should be equal, there was much more radioactivity in d-Gp than in d-Tp (Table 1). As noted above in a similar con- nection, this feature of the incorporation with a-P32-dCTP is not understood. (B) Repair experiments with H3-deoxynucleoside 5'-triphosphates: The completion of the repair reactions with regard to the very terminal nucleotides was further studied with H3-deoxynucleoside triphosphates. In the first experiment of this set, DNA-IV was used and H3-dATP was the only triphosphate provided in the repair reaction. Radioactivity was incor- Downloaded by guest on September 29, 2021 220 BIOCHEMISTRY: GUPTA AND KHORANA PRoc. N. A. S.

porated, and upon degradation to 3'-nucleotides and nucleosides, all of the radioactivity was released, as expected, in the form of the nucleoside deoxyadenosine (Fig. 3C). When the other three cold triphosphates were also provided along with H3-dATP and the repair product was degraded, most of the radioactivity was now in the form of the nucleotide d-Ap (Fig. 3D). The result thus shows that in the insertion of the sequence AGC, lacking in the left-hand 3'-OH terminus of DNA-IV (Fig. 1), the residue A, the first to be inserted, is almost completely covered by G, the penultimate nucleotide at the terminus. In the second experiment, the insertion of the sequence T-T-C at the right-hand terminus in DNA-IV was investigated by using H3-dTTP alone and in the presence of cold dCTP. Here, if the addition of the T-T sequence were to be complete with H3-dTTP alone as the triphosphate, then the ratio of the radioactivity in d-Tp: d-T upon degradation should be 1:1. Further, in this experiment, if cold dCTP is also provided and the penultimate labeled T is completely covered by the terminal C, then all of the radioactivity in the product should now be in d-Tp. The presence of the radioactive nucleoside would, correspondingly, reveal incomplete addition of the terminal C unit. In the experiment in which H3-dTTP alone was used, the H3 counts found in d-Tp and d-T, respectively, were 917 and 1,895; when the three cold triphosphates were also present, the counts in d-Tp and d-T were 1,699 and 1,041, respectively. The results in a further experiment of this type with DNA-IV were similar. Thus, it appears again that there is difficulty in complete addition of the nucleotide units at the termini.

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()Q B DNA-!+H3d-CTP+3 COLD DEOXY D: DNA-t+H3d-ATP+3 COLD DEOXY o 7RPHOSFHATES O C=) TRIPHOSPHATES ° 20C d-Cp dC d- d-A 15C

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I U fluIfJ zu1.3f MillX0------00 10 20 3 DISTANCE FROM ORIGIN (CM) FIG. 3.-Analysis of the incorporation of H3-nucleotides in repair reactions with DNA-V. The incubation conditions were standard and used mixtures of H3-labeled and cold triphosphates as shown. The levels of DNA polymerase were: (A) 90 units/ ml; (B) 225 units/ml; (C and D) 45 units/ml. For A, C, and D, the time of incubation was 4 hr at 50, whereas for B, the time was 10 hr even though the incorporation showed a leveling off at 2 hr. Isolation of the products and degradation to nucleotides and nucleosides were by the standard method. Paper chromatography was in solvent C, in which the nucleosides move well away from the mononucleotides. Downloaded by guest on September 29, 2021 VOL. 61, 1968 BIOCHEMISTRY: GUPTA AND KHORANA 221

In the third experiment, H3-dCTP was used with DNA-V. With H3-dCTP alone or in the presence of the three cold triphosphates, most of the radioactivity that was incorporated was in the terminal position. Thus as seen in Figure 3A and B, chromatography after degradation released most of the radioactivity in the form of the nucleoside. The pattern in Figure 3B showed the release of a small amount of the radioactivity as the nucleotide d-Cp. The reason for this is not clear. It certainly cannot be due to synthesis beyond repair, for its amount was constant even after a 10-hour reaction with the DNA polymerase (the incorporation had leveled off in less than two hours). Test for 5'-exonuclease activity: DNA-V (Fig. 1) labeled with p32 at the 5'- termini (16,000 cpm) was incubated with the DNA polymerase under conditions identical to those used in the repair reaction (50). No radioactive d-pG, which is the expected product of 5'-exonuclease,6 was detected with paper chromatography. However, at 370 a significant part (18%) of the radioactivity was present in the mononucleotide area. Discussion.-The results described show that the E. coli DNA polymerase brings about nucleotide incorporations into the short DNA's shown in Figure 1 so that the shorter strands bearing 3'-hydroxyl groups are extended to essentially complete the bihelical structure. The nucleotide incorporations were specific and mostly as expected for the repair reactions. Under the conditions used, there was no indication of replication beyond repair. The repair reactions were evidently complete except for the terminal one or two nucleotides. The reason for the incomplete insertion of the very terminal nucleotides is not clear. The incorporations in the terminal positions were rapid and had leveled off in the time periods used. The lack of completion does not appear to be due to any competition from the exonucleolytic activity operating from the 3'-hydroxyl end, for this activity was found to be completely nonfunctional under the conditions used. Nor is the lack of complete insertion of the 3'-terminal nucleotide likely to be due to the exonucleolytic removal of the G units at the 5'-hydroxyl end. At least when tests were made after the 5'-hydroxyl ends were labeled with p32_ phosphate group, there was no evidence of the exonucleolytic removal of the labeled end. The possibility that the exonucleolytic activity might be working on the DNA's (Fig. 1) carrying 5'-hydroxyl groups cannot be ruled out. Per- haps a more likely possibility for the incomplete insertion of the 3'-terminal nucelotides is the facile removal of the last unit by pyrophosphorolysis. Despite the incomplete addition of the nucleotide at the 3'-terminus, the incorporations were exactly as expected for the repair reactions, and the present results therefore demonstrate that the DNA polymerase can be used as an addi- tional tool for monitoring the step-by-step and coordinated synthesis of double- stranded DNA. As pointed out earlier,2 the total synthetic strategy involves chemical synthesis of short deoxypolynucleotides and subsequent end-to-end joining of these when they are aligned to form bihelical complexes. Careful checks would be required, first, at the level of chemical synthesis and, second, at the level of enzymatic joining of the segments in a stepwise fashion. At every alternate addition of a polynucleotide segment, the 3'-hydroxyl end of the de- veloping DNA duplex would be shorter by, for example five units (the length of the Downloaded by guest on September 29, 2021 222 BIOCHEMISTRY: GUPTA AND KHORANA PRIM. N. A. S.

sticky end). It should be possible to prove by repair reactions at the sticky ends that the joining reactions were proceeding quantitatively and as expected. Thus there would be three types of analyses that would monitor the joining reactions catalyzed by the DNA ligases: (1) conversion of the 5'-P32-labeled end of the incoming polynucleotide segment to a diester form as shown by insensitivity to phosphomonoesterase; (2) nearest-neighbor analysis of the products after every joining reaction to confirm that the joining reactions involved the expected terminal nucleotide residues, and (3) pattern of nucleotide incorporations as catalyzed by the repair property of the DNA polymerase at every step when the strand bearing the 3'-hydroxyl group is shorter than the complementary strand bearing the 5'-end. Summary.-E. coli DNA polymerase catalyzed the incorporation of deoxynucleoside 5'-triphosphates into DNA-I-VI (Fig. 1) at pH 6.9 and 5°. The patterns of nucleotide incorporations are all uniformly consistent with the repair of the single-stranded ends to form essentially complete duplexes. No evidence of any polynucleotide synthesis beyond the repair reaction or of any exonucleolytic activity was obtained under the conditions used. The following abbreviations are used: dATP, dCTP, dTTP, and dGTP stand for the deoxynucleotide 5'-triphosphates of adenine, cytosine, thymine, and guanine, respectively. * Paper LXXXIX of this series is by N. K. Gupta, J. Biol. Chem., in press. t This work has been supported by grants from the National Science Foundation, Washing- ton (GB-7484X), the National Cancer Institute of the National Institutes of Health, U. S. Public Health Service (CA-05178), and the Life Insurance Medical Research Fund (6544). 1 Gupta, N. K., E. Ohtsuka, H. Weber, S. Chang, and H. G. Khorana, these PROCEEDINGS, 60, 285 (1968). 2 Gupta, N. K., E. Ohtsuka, V. Sgaramella, H. Buchi, A. Kumar, H. Weber, and H. G Khorana, these PROCEEDINGS, 60, 1338 (1968). 3 Richardson, C. C., R. B. Inman, and A. Kornberg, J. Mol. Biol., 9, 46 (1964). 4Wu, R., and D. Kaiser, J. Mol. Biol., in press, and private communication. 5 Richardson, C. C., C. L. Schildkraut, H. V. Aposhian, and A. Kornberg, J. Biol. Chem., 239, 222 (1964). 6 Klett, R. K., A. Cerami, and E. Reich, Federation Proc., 27, 396 (1968). Downloaded by guest on September 29, 2021