THE JOURNAL OF BIOLOGICAL CHEMIBTRY Vol. 249, No. 12, Issue of June 25, PP. 36&3688, 1974

Printed in U.S.A.

Covalent Attachment of Polyribonucleotides to Polydeoxyribonucleotides Catalyzed by

Deoxyribonucleic Acid Ligase*

(Received for publication, December 13, 1973)

KABIALENDU NATHI AND JERARD HURWITZ

From the Division of Biology, Department of Developmental Biology and Cancer, Albert Einstein College of Medicine, Bronx, New York 10461

SUMMARY

DNA ligase isolated from Escherichia coli or phage T4-in- fected E. coli catalyzes the covalent joining of the 5’-phos- phate termini of polydeoxyribonucleotides and 3’-hydroxyl termini of polyribonucleotides. This reaction occurs with poly(dA) and poly(A) in the presence of poly(dT). The 5’-phosphate terminus of poly[d(A-T)] can be linked by an intramolecular reaction to its 3’-hydroxyl terminus substi- tuted with a ribonucleotide. Phage T4-induced DNA ligase also joins poly(dT) to poly(U) in the presence of poly(dA).

The joining of 5’-phosphate termini of polyribonucleotides to 3’-hydroxyl termini of polydeoxyribonucleotides was de- tected only with phage T4-induced DNA ligase. This reac- tion, however, occurred to a limited extent with poly(dA) sub- strates containing AMP residues at the 5’ terminus in the presence of poly(dT).

These results suggest that although DNA ligase from E. coli is incapable of joining RNA to RNA, it is capable of join- ing 5’-phosphate terminus of a DNA to the 3’-hydroxyl termi- nus of RNA in addition to the well known joining reaction between DNA to DNA. The phage T4-induced DNA ligase on the other hand joins DNA to DNA, RNA to RNA, and RNA to DNA in all combinations.

The over-all reaction catalyzed by DNA ligase’ results in the formation of a phosphodiester bond between juxtaposed 5’- phosphate and 3’-hydroxyl termini in duplex DNA (l-9). This process occurs with the formation of a covalent DNA-ligase- AMP complex which then transfers the AMP moiety to the 5’-phosphate termini of the DNA. The subsequent sealing of single strand breaks results in the liberation of AMP. Two

* This investigation was supported by grants from the National Institutes of Health, the National Science Foundation, and the American Cancer Society.

$ Postdoctoral Fellow of National Cancer Institute. Present address, Cell Biology Unit, Veterans Administration Hospital, 4500 South Lancaster Road, Dallas, Texas 75216.

1 In view of the recent findings of a RNA ligase (20) the term DNA ligase has been used in place of the name polynucleotide ligase.

distinct classes of DNA ligase have been isolated, one utilizing DPN as a cofactor (isolated from Escherichia coli) while the other utilized AT1 (first observed with the phage T4-induced enzyme) (l-9). In addition to these, other differences have been noted in the specificity of these two systems. The phage T4-induced DNA ligase catalyzes the joining of polyribonuclro- tides (10, 11) and DNA duplex structures at their base-paired ends (12, 13). It also carries out the linkage of polydeoxyribo- nucleotides possessing a mispaired base (14) and utilizes poly- ribonucleotides as template for the joining of polydeosyribo- nucleotides (10, II, 15). In contrast, none of these reactions were catalyzed by DNA ligase from E. coli. We report here that, both E. coli and T4-DNA ligase join the 5’.phosphate ter- mini of polydeoxyribonucleotides to the 3’-hydroxyl cuds of polyribonucleotides. T4 DNA ligase, in addition, joins 5’. phosphate termini of polyribonucleotides to the 3’-hydroxyl ends of polydeoxyribonucleotides.

EXPERIMEZTAL PROCEDURE

Materials

Enzymes-Electrophoretically purified pancreatic DNase I (free of RNase), nuclease-free bacterial alkaline phosphatase inorganic pyrophosphatase, and snake venom phosphodiesterase were purchased from Worthington Corp. Snake venom phos- phodiesterase was further treated by the method of Sulkowski and Laskowski (16) to remove contaminating 5’.nucleotidase activity. Calf thymus terminal deoxynucleotidyltransferase (17) was obtained from General Biochemicals and 5’-nucleotidase (acid Dhosnhatase from potato (18)) was from Miles. Nuclease- iree DNAIdependent R-NA polymerase (19) from E. coli was kindly provided by Dr. L. Yarbrough of this Department, while T-4-induced 5’-hydroxyl polynucleotide kinase was prepared by Drs. R. Silber and V. G. Malathi (20), exonuclease III by Dr. Et. Wickner and E. coli DNA polymerase I by Dr. B. Ginsberg (21). T4 DNA ligase and E. coli DNA ligase were prepared in this lab- oratory by Drs. J. P. Leis and L. Finer, respectively as previously described (4, 8, 22). DNA ligase preparations from E. coli (23) and T4 (9) were also obtained as generous gifts of Drs. I. It. Leh- man of Stanford University and C. C. Richardson of Harvard University, respectively.

Nucleotides and Oligonucleotides-Adenosine[r - 32P]triphos- phate and 3H-labeled deoxyribonucleoside triphosphntes were purchased from New England Nuclear Corp., and 3H-labeled ribonucleoside triphosphates were from New England Nuclear Corp. and Schwarz-Mann. Oligo(dT)d was obtained from Col- laborative Research, Inc., Waltham, Mass. Oligo[(A)a(dA),] of sequence pApApApdApdA was a generous gift of Dr. P. T. Gilham

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of Purdue University. Synthetic polynucleotides, poly(dT), poly(dA), and poly[d(A-T)] were obtained from General Bio- chemicals; poly(A) and poly(U) containing 5’- and 3’.hydroxyl termini were kindly provided by Drs. R. Silber and V. G. Malathi (20).

Introduction of s2F’ at 5’-Terminus of Polynucleotides-The 5’- phosphate ends of polynucleotides were removed by the action of alkaline phosphatase. Reaction mixtures (0.25 ml) containing 20 mM Tris.HCI, pH 8.0, 100 to 150 nmoles of polynucleotides and 1 to 2 units of bacterial alkaline phosphatase were incubated at 80” for 30 min. This reaction was either stopped by addition of phenol (see below) or when the introduction of 32P at 5’-hydroxyl termini was desired the reaction mixture was supplemented with 10 mM MgC12, 17 mM 2-mercaptoethanol, 1.7 mM sodium phosphate buffer, pH 7, 24 #M [Y-~~P]ATP and 30 to 50 units of 5’-hydroxyl polynucleotide kinase (24). The reaction mixture (0.6 ml) was incubated at 37” for 45 min at which time an equivalent amount of [Y-~~P]ATP and polynucleotide kinase was added and the reac- tion mixture incubated for an additional 30 min. In most cases, the extent of phosphorylation was complete after the first 45-min incubation. The reaction mixture was treated with 100 pmoles of EDTA and 1 to 2 ml of redistilled aqueous phenol which was freshly neutralized with Tris.HCl, pH 8.0. After incubation at 37” for 30 min the aqueous phase was collected by centrifugation. The phenol phase was re-extracted twice with 0.2 to 0.4 ml of 20 mM Tris, pH 8.0. The aqueous portions were pooled, extracted twice with 2 to 4 volumes of ether, and reduced to a volume of 0.5 to 1 ml with a stream of air. The polynucleotides were finally isolated by filtration through a column of Sephadex G-25 or G-50 (0.8 X 24 cm), concentrated, dialyzed three times against a solu- tion (125 ml) containing 10 rnM Tris. HCl, pH 8.0 and 10 mM NaCl, and stored frozen till needed.

The average chain length of various polynucleotides were be- tween 50 to 300 nucleotides as measured by the number of 5’ termini. Only one lot of commercial poly(dA) was found to be nearly 1700 nucleotides long; it was partly digested with pan- creatic DNase I to an average chain length of 150 to 200 nucleo- tides.

Covalent Attachment of [JMP to PoZy[d(A-T)]-[3H]UMP was covalently linked to the 3’.hydroxyl termini of 5’.32P-labeled poly[d(A-T)] with DNA-dependent RNA polymerase isolated from E. coli (25). A reaction mixture (0.75) containing 15 rnl\l Tris.HCl, pH 8.0, 0.67 mM dithiothreitol, 13.3 mM MgClz, 0.1 M KCl, 10 PM [3H]UTP (8900 cpm per pmole), 0.84 nmole of ZZP linked to [5’-32P]poly[d(A-T)], and 0.75 nmole of recycled core RNA polymerase (25) was incubated at 37” for 2 hours. The [5’-32P]- poly[d(A-T)].[3H]UMP product was isolated after treatment with phenol and collected from a Sephadex G-50 column as de- scribed above.

Synthesis of Polynucleotides Using Terminal Deoxynucleotidyl- transjerase-Primer oligo[(A),(dL4)z] was labeled with 32P at 5’ terminus with polynucleotide kinase, extracted with phenol and eluted from a DEAF,-cellulose column (0.5 X 100 cm) that was equilibrated with a solution containing 7 M urea, and 20 mM Tris. HCl, pH 7.5. Using a 0 to 0.5 hl NaCl gradient, [5’-32P]-oligo- [(A),(dA)2] was eluted in a volume of 22 ml at 0.2 M NaCl, dialyzed five times against a solution (21) containing 0.1 mM Tris.HCl, pH 8.0, and concentrated under vacuum. Poly(dA) was added to [5’-““P]oligo[(A),(dA),1 as follows: a reaction mixture (0.4 ml) containing 100 mM potassium cacodylate, pH 6.8,1 mM 2-mercapto- ethanol, 5 mM MgC&, 1 mM [3H]dATP, 0.45 nmole of 5’-32P linked to oligo[(A)a(dA)Z], 50 units of calf thymus terminal deoxynucleo- tidyltransferase, and 1 rg of inorganic pyrophosphatase was incubated for 7.5 hours at 35”. This resulted in the formation of polynucleotides of average chain lengt,h of 300 nucleotides (Fig. 1). The product [““PI (A) 3 (dA)a,, was extracted with phenol and iso- lated from a Sephadex G-50 column as described above.

Synthesis of poly(dT) of an average chain length of 8000 nucleo- tides was carried out similarly by incubating primer (dT)r with terminal deoxynucleotidyltransferase for 24 hours.

Methods

Measurement of Joiuing of Polynucleotides--DNA ligase reac- tions were performed as depicted in Scheme 1. Formation of a phosphodiester bond between 5’-s2P terminus of a polynucleotide and 3’-hydroxyl terminus of another polynucleotide converted

9.6

6.4

3.2

TIME (hrs)

FIG. 1. Synthesis of poly(dA) on [5’-32P]oligo[(A)3(dA)21 primer by terminal nucleotidyltransferase. The conditions used were as described under “Experimental Procedure.”

dA A A dA

I poly(dT)+

DNA ligase

(alkaline phosphotase

resistant =P)

NaOH trsatment

(alkaline phosphotase (alkaline photphatose

sensitive =P I resistant ‘*PI

SCHEME 1. Measurement of linkage in products formed by DNA ligase.

the phosphatase-sensitive 32P into a phosphatase resistant form (2, 6). If one of the two polymers were RNA then further treat- ment with alkali would have the following effects. Alkali stable 32P at a 5’ terminus of DNA when joined to the 3’-hydroxyl end of RNA would be rendered acid-soluble, while alkali labile 5’-32P attached to RNA when joined to the 3’.hydroxyl end of DNA would remain acid-insoluble after alkali treatment.

DNA ligase assay mixtures (0.1 ml) containing 15 mM Tris.HCl, pH 8.0, 5 PM dithiothreitol, 5 pg of dialyzed bovine serum albumin (26), 0.1 mM NAD in the case of E. coli ligase or 53 /IM ATP in the case of T4 ligase, and indicated amounts of polynucleotides were preincubated at 37” for 15 min. Mixtures were then transferred to 30” and reactions initiated by the addition of 2 mM MgClz and DNA ligase. After specified periods (as indicated), reactions were terminated by heating at 100” for 90 s. To determine alka- line phosphatase resistant 3zP generated, reaction mixtures were treated with 0.1 ml of 20 mM Tris.HCl, pH 8.0 and incubated with 0.3 unit of bacterial alkaline phosphatase at 80” for 30 min. This was followed by addition of 0.5 rmole of EDTA, 0.1 ml of 0.1 M sodium pyrophosphate, 0.2 ml of a 30% Norit suspension, and 3 ml of 1 N HCl. The Norit was collected on a glass fiber filter (GF/C), washed with 1 N HCl, dried, and radioactivity deter- mined with a toluene based scintillator.

To determine the joining of RNA to DNA, after incubation

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with bacterial alkaline phosphatase, reaction mixtures were t,reated with0.1 ml of 1 N NaOH and O.Bpmole of EDTA and heated at 100” for 10 min. Acid-insoluble radioactivity was determined after cooling by the addition of 0.1 ml of 0.1 M sodium pyrophos- phate, 50 Fg of bovine serum albumin, 10 pg of denatured salmon sperm DNA, and 4 ml of 10% trichloroacetjic acid followed by filtration through Millipore filters (type HA). Filters were washed three times with 1% trichloroacetic acid. rinsed with 95% ethanol, dried, and radioactivity determined with a toluene based scintillator.

Assay of DNA Ligase Activity with Exonuclease III-This as- say measured the formation of circular poly[d(A-T)] (27). After incubating pol.y[d(A-T)] with DNA ligase, as above, the reac- tion mixture (O:l ml) was treated with 0% to.2 units of exonuclease III and 9 mM 2-mercaotoethanol. This mixture was incubated at 37” for 30 min afte; which time acid-insoluble radioactivity was determined.

Exonuclease IZ Assay-The inability of exonuclease II activity of DNA polymerase I to degrade polynucleotides bearing a 3’- phosphomonoester group (28,29) was utilized to confirm the struc- ture 5’. OH dAp--pdA 32pAp (see below, Scheme 3). Inctl- bations were carried out as described by Lehman and Richardson (28), and loss of acid-soluble 32P was monitored in the presence of 0.24 unit of DNA polymerase I.

Identification of Nucleoside Monophosphate-Nucleoside mono- phosphates containing 32P were isolated after incubation with alkali or venom phosphodiesterase and adsorbed to Norit in the presence of 1 N HCI. After washing with water, nucleotides were eluted from Norit with 50yo ethanol-1.5yo NHIOH. The 32P eluted from Norit was completely hydrolyzed to 32P; with bac- terial alkaline phosphatase. The susceptibility of 32P-labeled mononucleotides to the action of potato acid-phosphatase at pH 5.0 (5’. and 2’(3’)-nucleotidase) and at pH 9.0 (5’.nucleotidase alone) (18) was used to differentiate 5’-mononucleotides from 2’(3’)-mononucleotide. The identity of mononucleotides was also verified by migration on paper after high voltage electro- phoresis (5000 volts) in 50 mM sodium citrate, pH 4.0.

Zone Sedimentation Analysis-Products of ligase reactions (0.1 ml) were dialyzed four times against a solution (125 ml) con- taining 1 M NaCl, 10 mM NaCl, 10 mM Tris.HCI, pH 8.0, and 1 mM EDTA, and twice against 50 mM NaCl and 10 mM Tris.HCl, pH 8.0. The dialyzed sample was mixed with 100 mM sodium phosphate, pH 7.0 and denatured with 127, neutralized formaldehyde at 69” for 2.5 min followed by rapid chilling in ice water (30). The final sample (0.2 ml) was layered on a 5 to 25% sucrose gradient (5 ml) containing 0.7 M NaCl, 25 mM sodium phosphate, pH 7.0, and 6% formaldehyde. All gradients contained a cushion of BOY0 sucrose (0.15 ml). Sedimentation was performed in a Spinco SW 50.1 rotor at 42,000 rpm for 22 to 48 hours at 4”. Fractions (15 drops) were collected by piercing the bottom; fractions were diluted with 1 ml of water and radioactivity determined with a dioxane based scintillation fluid.

RESULTS

Linkage of Poly(dA) with Poly(A)

E. coli DNA Ligase-As previously described (23) this en- zyme catalyzed the covalent joining of [5’J2P]poly(dA) but did not catalyze the linkage of [5’-32P]poly(A) (Table I; Experiments I and II). In Experiment III, Table I, 20% of the input [5’J2P]- poly(dA) was converted to an alkaline phosphatase resistant form in the presence of 3’-OH, 5’.OH poly(A). Virtually all of this 32P was rendered acid-soluble after alkaline hydrolysis indicating the transfer of 32P from poly(dA) to poly(A) (see Scheme 1). This reaction was dependent on NAD, poly(dT) and ~01~ (A).2 As shown in Experiment IV, E. coli DNA ligase did not catalyze the joining of [5’-32P]poly(A) to the 3’.OH ter- minus of poly(dA).

2 The discrepancy between Experiments I and III of Table I is due to the difference in the ratio of poly (dA) to poly (dT). In Experiment I, this ratio was approximately 1:l while in Experi- ment III, the ratio was about 1:4.

TABLE I

Joining of poly(dA) and poly(A) by Escherichia coli DNA ligase

Reaction mixtures (0.1 ml) contained 15 mM Tris.HCl, pH 8.0, 5 MM dithiothreitol, 0.1 mM NAD, 5 pg of bovine serum albumin, and polynucleotides. After incubation at 37” for 15 min the re- action was initiated at 30” by addition of 2 mM MgC12 and indi- cated amounts of E. coli ligase. After 30 min, the reaction was terminated by heating at 100” for 1.5 min. Incubation with alkaline phosphatase was carried out at 80” for 30 min after dilu- tion with 0.1 ml of water and addition of 0.15 unit of bacterial alkaline phosphatase. The phosphatase-resistant 32P was deter- mined by adsorbing polynucleotides to Norit in Experiments I and II (except for NaOH treatment) by addition of 0.1 ml of 0.1 M sodium pyrophosphate, 0.2 ml of 30% Norit suspension followed by 3 ml of cold 1 N HCl. After standing for 5 min in ice, the Norit was collected on a glass fiber filter (GF/C), washed two times with 1 N HCl, the filter dried, and radioactivity determined with a toluene based scintillation fluid.

In experiments where NaOH treatment was carried out reac- tion mixtures were made 0.3 N with NaOH, incubated 10 min at loo”, and cooled in ice. The acid-insoluble 32P was determined by addition of 0.1 ml of 0.1 M sodium pyrophosphate, 50 fig of bovine serum albumin, 10 pg of denatured salmon sperm DNA, and 5O/c trichloracetic acid. The precipitate was collected on a Millipore membrane filter (type HA), washed three times with lYc trichloracetic acid, once with ethanol, dried, and radioactivity determined.

The polynucleotide concentrations were as follows: Experi- ment I, 230 pmoles of [32P]poly(dA) plus 250 pmoles of poly (dT); Experiment II, 250 pmoles of [32P]poly(A) plus 250 pmoles of poly(dT); Experiments III, 230 pmoles of [32P]poly(dA) plus 210 pmoles of poly (A) plus 990 pmoles of poly(dT); Experiment IV, 250 pmoles of [32P]poly(A) plus 230 pmoles of poly(dA) plus 990 pmoles of poly(dT).

Experi- ment

I A B C

II A B

III

A B C D E

IV

A

Additions

poly(dA) (1.22 pmoles of 5’-32P) 1 unit of E. coli DNA ligase 0.2 unit of E. coli DNA ligase As in B, followed by NaOH treatment Poly(A) (4.48 pmoles of 5’-32P) 1 unit of E. coli DNA ligase 0.2 unit of E. coli DNA ligase Poly(dA) (1.69 pmoles of 5’-32P) +

3’-OH, 5’-OH poly(A) 0.2 unit of E. coli DNA ligase As in A; omit poly(A) As in A; omit NAD As in A; omit poly(dT) As in A; followed by NaOH treatment Poly (A) (1.98 pmoles of 5’-32P) + 3’.OH,

5’-OH poly (d A) 0.2 unit of E. coli DNA ligase

Alkaline phospha- tax resistant a@’

Imole/ sin al 30

0.24 0.13 0.18

0.03 0.00

0.33 0.01 0.02 0.01 0.04

0.00

% nput =P

20 11 15

<l <l

20 <l

1 <l

2

<1

a One unit of E. coli DNA ligase catalyzed the conversion of 1 pmole of 5’-32P terminus of poly(dT) in 1 min to a form insuscep- tible to the action of bacterial alkaline phosphatase (2).

* The amount of alkaline phosphatase-resistant 3zP observed in the absence of DNA ligase in Experiments I to IV was 120,93, 53, and 52 cpm, respectively; the input radioactivity was 1120, 6420, 1550, and 2840 cpm, respectively.

The sedimentation behavior of various products formed with

poly(dA) and poly(A) in formaldehyde-sucrose gradients con- firmed the results of Table I. While [32P]poly(dA) and poly(A)

after incubation with E. coli DNA ligase sedimented faster than

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TIME (min)

FIG. 2. Rate of joining of [5’-3*P]poly(dA) to 3’-OH poly(A) by Escherichia coli DNA ligase. The reaction conditions are as described in Table I except the reaction volume was increased to 0.52 ml and preincubation was carried out at 37” for 15 min fol- lowed by 15 min at 30”. The reaction was initiated with 2 mM MgClz and 1.5 units of E. cola’ DNA ligase. At designated in- tervals, 25-/r] aliquots were added to 0.11 ml of 20 mM Tris.HCl, pH 8.0 and the mixture was heated at 100” for 90 s. The samples were then divided in two portions of 50 ~1 and both fractions were incubated with 0.2 unit of alkaline phosphatase at 80” for 30 min. Subsequently, one portion was made 0.7 N with NaOH and incu- bated 10 min at 100”. Acid-insoluble radioactivity present in both portions was measured as previously described. An aliquot of the reaction mixture was withdrawn at 30 min, indicated by the arrou~ and incubated in presence of an additional 1.5 units of E. coli DNA ligase. The polynucleotide concent,rations were 0.75 nmole of [32P]poly(dA) plus 2.G nmoles of 3’.OH, 5’.OH, poly(A) plus 3.25 nmoles of poly(dT) in the case of [32P]poly(dA) plus poly (A), and 0.75 nmole of [32P]poly(A) and 0.75 nmole of [32P]poly(dA) plus 0.65 nmole of poly(dT) for [32P]poly(A), re- spectively. The symbol X represents alkaline phosphatase- resistant and l alkali-resistant 3zP; t,he symbols enclosed in circles indicate results obtained after addition of more DNA ligase. The inset is the alkali-labile radioactivity representing the transfer of 32P from 5’ terminus of poly(dA) to the 3’ terminus of poly (A).

the starting materials, [32P]poly(A) and poly(dA) after incuba- tion with DNA ligase did not.

The rate of joining of 5’J*P termini of poly(dA) to 3’-OH ter- mini of poly(A) in Fig. 2 was deduced from the phosphatase resistant 32P that subsequently became susceptible to alkali. In the inset of Fig. 2, the yield of joining of input [32P]poly(dA) to poly(A) was almost complete within 20 min; the addition of more enzyme at 30 min did not increase the final yield of 50%. The joining of [32P]poly(dA) was almost complete in 10 min and the addition of more enzyme at 30 min did not affect the final yield of 20%.

When the alkaline phosphatase resistant product poly(A- [32P]poly(dA) was treated with NaOH, acid-soluble 32P appeared as 2’(3’)-AMP. The 32P migrated as AMP in high voltage elec- trophoresis and was resistant to the action of 5’-nucleotidase.

2’4 DNA Ligase-This enzyme not only joined poly(dA) and poly(A) (10, 11) but also 5’-phosphate termini of poly(dA) to 3’-OH termini of poly(A) (Table II). The enzyme did not link the 5’-phosphate terminus of poly(A) to the 3’-OH terminus of poly(dA). In Experiment III, approximately 30% of the input [5’-32P]poly(dA) was converted to a covalent linkage with 3’-OH

3683

TABLE II Joining of poly(dA) and poly(A) by T4 DNA ligase

Reaction conditions were as in Table I except that NAD was replaced with 0.05 mM ATP and E. coli DNA ligase was replaced with T4 DNA ligase. Experiment I was performed without pre- incubation at 37” and the reaction was run for 15 min at 30”; in Experiment II the preincubation period at 37” was 5 min. The polynucleotide concentrations used in Experiments I and II were 110 pmoles of [32P]poly(dA) plus 250 pmoles of poly(dT) and 250 pmoles of [32P]poly(A) plus 500 pmoles of poly(dT), respectively; the concentration of nucleotides used in Exneriments III and IV were the same as those reported in Table I.’

Experi- ment

I A B

II A B

III

A B C D E

IV

A B C 1) I?:

Addition9

Poly (dA) (0.53 pmole of 5’-32P) 0.3 unit of T4 DNA ligase 1.5 units of T4 DNA ligase Poly(A) (5.22 pmoles of WFP) 0.3 unit of T4 DNA ligase 1.5 units of T4 DNA ligase Poly(dA) (169 pmoles of 5’-3’P) +

3’-OH, Y-OH poly(A) 0.05 unit of T4 DNA ligase

As in A; omit poly(A) As in A; omit ATP As in A; omit poly(dT) As in A; followed by NaOH treatment Poly(A) (2.58 pmoles of 5’-32P) + 3’-OH,

5’-OH poly(dA) 0.05 unit of T4 DNA ligase As in A; omit poly(dA) As in A; omit ATP As in A; omit poly(dT) As in A; followed by NaOH treatment

-

i -

1 m

Alkaline phospha- .ase resistant a@

0.02 0.04

0.25 1.11

0.57 0.05 0.02 0.02 0.04

0.89 0.86 0.00 0.00 0.00

-

% rjwi '2P

4 8

5 21

34 3 1 1 2

35 33

<l <l <l

G One unit of enzyme catalyzed the conversion of 1 nmole of 32P to an alkaline phosphatase-resistant form in 20 min (6).

b Alkaline phosphatase-resistant 32P obtained in the absence of T4 DNA ligase in Experiments I to IV was 38, 76, 39 and 37 cpm, respectively; the input radioactivity was 1,782, 24,617, 1,409, and 3361 cpm, respectively.

poly(A) as judged by the sensitivity of the product to alkali and

In Experiment. IV of Table II, the conversion of [5’-32P]poly(A) by the dependence of the reaction on added poly(A).

to an alkaline phosphatase resistant form in the presence of 3’-OH, 5’-OH poly(dA) was a consequence of poly(A) joining. The phosphatase resistant 32P remained susceptible to alkali and the reaction occurred independent of poly(dA). Varying the ratio of [32P]poly(A) to poly(dA) from I:3 to 1: 1 to 3: 1 or the incubation temperature from 30” to 25” or 37” did not alter the inability of T4 DNA ligase to join [5’-32P]poly(A) to poly(dA).

Joining of Poly(dT) and Poly( U) in Presence of Poly(dA)

E. coli DNA Ligase-E. coli DNA ligase joins poly(dT) but does not link poly(U) (23) in the presence of poly(dA). No detectable covalent joining of poly(dT) to poly(U) occurred (Table III). All [5’-32P]poly(dT) converted to an alkaline phosphatase resistant form in the presence of 3’-OH, 5’-OH poly(U) was stable to alkaline hydrolysis. This indicates that joining of poly(dT) occurs without participation of poly(U) (Experiment III, Table III).

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TABLE III TABLE IV

Joining of poly(dT) and poly(U) by Escherichia coli DNA ligase

Joining of poly(dT) and poly(U) by T4 DNA ligase

The reaction conditions are as described in the legend to Table I. The polynucleotide concentrations were: Experiment I, 300 pmoles of [32P]poly(dT) + 340 pmoles of poly(dA); Experiment II, 300 pmoles of [32P]poly(U) plus 340 pmoles of poly(dA); Ex- periment III, 300 pmoles of [32P]poly(dT) plus 350 pmoles of poly(U) plus 1280 pmoles of poly(dA); Experiment IV, 300 pmoles of [32P]po1y(U) plus 320 pmoles of poly(dT) plus 1280 pmoles of

The experimental conditions were as reported in the legend to Table II. The polynucleotide concentrations used were: Experi- ment I, 300 pmoles of [32P]poly(dT) plus 310 pmoles of poly(dA); Experiment II, 300 pmoles of [32P]poly(U) plus 310 pmoles of poly(dA); Experiment III, 160 pmoles of [32P]po1y(dT) plus 170 pmoIes of poly(U) plus 660 pmoles of poly(dA); Experiment IV, 150 pmoles of [32P]poly(U) plus 140 pmoles of poly(dT) plus 660 pmoles of poly(dA).

poly(dA).

Experi merit Additions Alkaline phospha-

mse-resistant zzPa -.

i n

, D i

% nput ==P

I A B C

II A B

III

Poly(dT) (3.38 pmoles of 5’-32P) 1.0 unit E. coli DNA ligase 0.2 unit E. coli DNA ligase As in B; followed by NaOH treatment Poly(U) (2.43 pmoles of 5’-32P) 1.0 unit E. coli DNA ligase 0.2 unit E. coli DNA ligase Poly(dT) (3.52 pmoles of 5’-32P) + 3’-

1.06 32 1.15 34 1.43 40

0.00 0.00

<l <l

A B C D E

IV

OH, 5’-OH poly(U) 0.2 unit E. coli DNA ligase As in A; omit poly(U) As in A; omit NAD As in A; omit poly(dA) As in A; followed by NaOH treatment Poly(U) (0.50 pmole of 5’-32P) + 3’.

0.76 22 0.80 23 0.02 <l 0.00 <l 0.72 21

A OH, 5’-OH poly(dT)

0.2 unit E. coli DNA ligase 0.00 <1

a A: Ikz dine phosphatase-resistant 32P detected in the absence of DNA ligase in Experiments I to IV was 92, 110, 64, and 57 cpm, respectively; the input radioactivity was 13,376, 9,628, 13,926, and 1,982 cpm, respectively.

T-4 DNA Ligase-Under the conditions described in Table IV,

this enzyme efficiently joined poly(dT) residues but did not link

poly(U) residues in the presence of poly(dA). In contrast to the E’. coli enzyme, the T4 DNA ligase formed covalent struc-

tures between [5’-32P]poly(dT) and 3’.OH, 5’.OH poly(U) (Table IV, Experiment III). However, covalent structures with

[5’-32P]poly(U) and 3’.OH, 5’-OH poly(dT) were not formed. The yield of covalent hybrid (Experiment III, Table IV) repre- sented nearly 40% of the input 32P; this reaction was slightly increased (to 50%) by decreasing the [5’-32P]poly(dT) concen- tration from 160 to 90 pmoles in the presence of poly(U) and poly(dA) as described in Table IV.

The rate of joining of [5’J2P]poly(dT) to poly(U), judged by the formation of alkali-labile alkaline phosphatase-resistant 32P (inset, Fig. 3) was similar to that of poly(dT) alone. Both re- actions were complete in 20 min and the final yields were not increased by the addition of more enzyme.

Further proof of the formation of covalent structures between [5’-32P]poly(dT) and poly(U) was obtained as follows. Poly(U) and [32P]poly(dT) joined products sedimented faster than poly- (dT) and poly(U) in formaldehyde sucrose gradient. After alkaline hydrolysis, 40% of the 32P from this covalent hybrid product was isolated as 2’(3’)-UMP. The latter identification was based on migration in high voltage paper electrophoresis and by the resistance of the mononucleotide to 5’-nucleotidase action.

Experi- ment

I A B C

II A B C

III

A B C

IV

A B

Additions

Poly(dT) (3.48 pmoles of 5’-32P) 0.02 unit T4 DNA ligase 0.05 unit T4 DNA ligase 1.5 units T4 DNA ligase Poly(U) (2.63 pmoles of 5’-32P) 0.02 unit T4 DNA ligase 0.05 unit T4 DNA ligase 1.5 units T4 DNA ligase Poly(dT) (2.01 pmoles of 5’-32P) + 3’-

OH, 5’-OH poly(U) 0.05 unit T4 DNA ligase As in A; omit poly(U) As in A; followed by NaOH treatment Poly CU) (0.30 pmole of 5’.3*P) + 3’.OH,

5’.OH poly(dA) 0.05 unit T4 DNA ligase As in A; omit poly(dT)

Alkaline phospha- tase” resistant 12P-

pmo1es/30 I:

% nprt rap

2.28 66 3.08 89 3.30 95

0.00 0.00 0.00

1.70 1.49 0.95

0.00 0.00

<l <l <l

85 74 47

<l <l

a The amount of alkaline phosphatase-resistant 32P detected in the absence of DNA ligase in Experiments I to IV was 68, 81, 55, and 30 cpm, respectively, while the input radioactivity was 12,461, 9,423, 7,109, and 1,037 cpm, respectively.

Formation of [5’-32P]Poly[d(A-T)] [3’-3H]U~\fP Covalent Circular Structures

E. coli DNA ligase converted 25 to 35% of the input [5’-32P]- poly[d(A-T)] to an alkaline phosphatase (and alkali) resist,ant form (Experiment I, Table V). As demonstrated by Modrich and Lehman (27) all of this product consists of covalently closed circles. .4 similar joining reaction was observed when at least 50% of the [5’-““P]poly[d(A-T)] was substituted with UMP at the 3’-OH terminus (Experiment II, Table V). In the presence

of 1 unit of ligase (Experiment IIE), 1.33 pmoles of [5’-32P]poly-

[d(A-T)] containing UMP at 3’-OH ends were converted to an alkaline phosphatase-resistant form. When this product was

treated with alkali, 0.88 pmole of the 32P became susceptible to alkaline phosphatase (viz. 1.33 - 0.45 pmole). As summarized in Scheme 2, this represents the joining of 5’-32P of poly[d(A-T1]

to the 3’-OH UMP residue (8, 22, and 26% in C, D, and E of Experiment II, respectively), while the alkali-resistant 32P repre-

sents poly[d(A-T)] circles devoid of UMP (7, 10, and 14% in C, D, and E of Experiment II, respectively).

When [5’-32P]poly[d(A-T)] containing [3H]UMP at the 3’.OH terminus was incubated with E. coli DNA ligase, 45% of the input 32P was converted into an alkaline phosphatase as well as

exonuclease III-resistant form (Fig. 4A). Simultaneously, 4Ooj, of the input 3H became resistant to the action of exonuclease

III, indicating the inclusion of UMP in the circular forms.3 In

3 The conclusion that products formed from poly[d(A-T)] were circular is based solely on its resistance to exonuclease III action as first described by Modrich and Lehman (27).

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[= 1 P poly CdT, + ply t”) (alkali treated1

.,-----,------- 4 ------ ---------- LL --_--_-__-_--_- ~

I I I I I I I5 30 45 60 75 90

TIME (mln)

FIG. 3. Rate of joining [5’-32P]poly(dT) t,o 3’-OIi poly(U) by T4 DNA ligase. lteaction mixtures, as described in Table II, were increased to 0.81 ml in experiments with [32P]poly(dT) plus poly(U) and to 0.21 ml in experiments with poly(U). The amount of polynucleotides added in the two casts were 0.68 nmole of [32P]- poly(dT) plus 1.73 nmoles to 3’.OH, 5’.OH poly(U) plus 5.24 nmoles of poly(dA) and 0.18 nmole of [32P]poly(dT) plus 1.31 nmoles of poly(dA), respectively. Preincubation was carried out at 37” for 15 min followed by 120 min at 30”. Reactions mere initiated with 2 mM MgCle and 0.51 unit of T4 DNA ligase per ml. Portions were removed at indicated intervals and subjected to the action of alkaline phosphatase as well as to alkaline hydrolysis as described in Fig. 2. At, 30 min, indicated by the arrow, an aliquot was incubated in the presence of an additional amount, of T4 DNA ligase (0.38 unit per ml). The symbols are as de- scribed in Fig. 2. The inset represents the alkali-labile radio- activity due to the transfer of 32P from the 5’ terminus of poly(dT) to the 3’ terminus of poly(U).

accord with this conclusion, when this exonuclease III-resistant 32P product (constituting 45y0 of input 3*P) was further treated with alkali, a substantial amount of the 3*P became sensitive to the action of alkaline phosphatase and exonuclcase III (Fig. 4B). Simultaneously, all of the 3H became susceptible to the action of exonuclease III (Fig. 4B). Identical results were obtained when T4 DNA ligase was used in place of E. coli ligase.

The rate of conversion of 5’ 9 termini of poly[d(A-‘I’)] or poly[d(A-T)] substituted with [“H]UMP to internal phospho- diester bonds was identical (Fig. 5). In both cases the joining reaction plateaued after 60% of the input 321’ became resistant to the action of alkaline phosphatase. In reactions containing poly[d(A-T)] substituted with [3H]UMP, 35% of the 3H remained acid-insoluble after exonuclease III treatment (Fig. 5B). This value was low due to the presence of an exonuclease in DNA ligase preparations which resulted in the loss of [3H]Uh’II’ during the incubation (Fig. 5B). The nature of this activity is un- known, but it had no effect on [3H]UMI’ once it was covalently linked in a circular form.

Joining of (A) 3(dA) 3oo on Template Poly(dT)

With the substrates described above (Tables I to IV), no de- tectable joining reaction between 5’-phosphate termini of poly- ribonucleotidcs and 3’-OH termini of polydeoxyribonucleotides was detected with either DNA ligase preparations. Further studies utilizing [5’-32P]ApApApdA---pdA as substrate, however, indicated that T4 DNA ligase could catalvze such reactions.

SCHEME 2. Formation of [5’-32P]poly[d(A-T)].[3’-3H]UMP cir- cles by DNA ligase.

3685

TAIILE V Joining of [5’-33P]p01y[d(A-T)] and [5’-““P]polll[rl(A-~)I. [.!I’-3H]-

UMP by Escherichia coli DNA ligase

The conditions were as described in Table I with the following modifications. Labeled poly[d(A-T)] in Tris.IICl, pII 8.1 (80. to 90.~1 volume) was heated at 100” for 2 min followed by rapid chilling in ice water for 5 min. It was then supplemented with dithiothreitol, bovine serum albumin, and NAD and preincu- bated at 37” for 25 min. The reaction was initiated at 30” and terminated after 30 min as previously described. Alkaline phos- phatase treatment was carried out by incubating the mixture with 1 unit of bacterial alkaline phosphatase at 70” for 30 min. Subsequent treatment with NaOH consisted of incubation with 0.5 N NaOII at 100” for 10 min. Such alkali-treated samples were again treated with alkaline phosphatase after neutralization with HCl and the addition of 100 mM Tris.HCl, pH 8.0. Acid-insolu- ble radioactivity was determined as before. The amount of poly- [d(A-T)] used was 3.4 pmoles of 32P termini (containing 2.3 X 10’ cpm) in lcxperiment I and 3.3 pmoles of 32P termini (2.21 X lo4 cpm) containing 3.9 pmoles of [“H]UMP (3.40 X lo4 cpm) at 3’ termini in Experiment II.

I A

B

II C

D

E

Additions Ii kid-insoluble a2P

pmo1es % [5’-32P]Poly(d (A-T)] 0.2 unit of E. coli DNA ligase

(a) Alkaline phosphatasc (b) As in (a); followed by NaOII,

then alkaline phosphatasc 0.05 unit of E. coli DNA ligase

(c) As in (a) (d) As in (b)

0.85 25

1.00 30

1.24 37 1.16 34

[5’-32P]Poly[d (A-T)]. [3’-3H]UMP 0.1 unit of E. coli DNA ligase

(a) Alkaline phosphatase (b) As in (a), followed by NaOH (c) As in (b), then alkaline phos-

phatase

0.51 0.50

16 15

0.24 7 0.5 unit of E. coli DNA ligase

(d) As in (a) (e) As in (c)

1.0 unit of E. coli DNA ligase

(f) As in (a) (8) As in (b) (h) As in (c)

1.07 32 0.36 10

pdApdT------dApdTOH bdApdT------dApdTpuo”

dApdT-pdApdT

Alkllli

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3686

20 40 60 SO

TIME (min) TIMECmlnl

FIG. 4. Formation of covalently closed circles of [5’-32P]poly- [d(A-T)].[3H]UMP by Escherichia coli DNA ligase. A, reaction conditions described in Table V were used and included 19.9 pmoles of 32P (5.55 X 10” cpm) and 27 pmoles of [3H]UMP (2.38 X IO5 cnm) containing nolvfd(A-T)l and 3 units of E. coli DNA ligase. _ The reaction-was “carried out in a total volume of 0.3 ml for 30 min at 30”. Portions (4 ~1) were removed and alka- line phosphatase or exonuclease III-resistant, acid-insoluble ra- dioactivity determined. Incubation with I unit of alkaline phosphatase was carried out at 70”, while incubat,ion with 1.8 units of exonuclease III was carried out at 37” in the presence of 10 mM 2-mercantoethanol. B. another nortion of the DNA liease

FIG. 5. Rate of formation of covalently closed circles of [5’-32Pl- poly[d(A-T)] and [5’-3zP]poly[d(A-T)]. [+I]UMP catalyzed by Escherichia coli DNA ligase. Reaction mixtures described in Table V (0.3 ml) included 2 units of E. coli DNA ligase and either 16.4 pmoles of 32P (6.8 X IO4 cpm) poly[d(A-T)] or 13.3 pmoles of 32P (5.5 X lo4 cpm) and 18 pmoles of 3H (1.54 X lo5 cpm) poly[d(A-T)]UMP. Portions (25 ~1) were removed at indicated intervals and added to 2 ml of 20 mM Tris.HCl, pH 8.0 and heated for 90 s at 100”. Portions were incubated with 0.2 unit of alkaline phosphatase (X--X) or with 0.6 unit of exonuclease III (0=-O) as described in Fig. 4 and acid-insoluble radioactivity determined. A and B indicate the fate of 32P and 3H. resnec-

I I I 0 20 40 60

TlME(m,nl

m= 60 L y ~- 2

c-mot ; 40 ..-..-.. i>.. ---- 4 * ,, _______ -..-.,*

$ 20 1.. ‘.., mrphotorP

I :.&..I ,....!.wFa 0 20 40 60

TIME (mln)

reaction mixture (0.18 ml) was first treated with 24 units of exo- nuclease III at 37” for 30 min followed by incubation with 0.3 N NaOH at 37” for 18 hours and finally dialyzed against 25 mM Tris. HCl, pH 8.0. Portions of this alkali-treated product were again subjected to alkaline phosphatase and exonuclease III action and acid-insoluble radioactivity determined. The control values represent the amount of input radioactivity remaining acid- insoluble after the initial exonuclease III treatment.

This reaction occurred to a limited extent (Table VI). No join- ing reaction could be detected when E. coli DNA ligase was used (Experiment IA). The joining of the [5’J2P]AMP terminus to the 3’-OH terminus of the (dA) polymer by T4 DNA ligase (Experiment IB and Experiment II) was dependent on ATP and poly(dT). The reaction resulted in the conversion of alkali- sensitive 32P to an alkali-resistant material (Scheme I). The alkali resistant 32P was located in a phosphodiester bond (Scheme 3c) and inaccessible to alkaline phosphatase action (Experiment IIA).

Further confirmation of the [5’-32P]AMP linkage to the 3’.OH terminus of poly(dA) was provided by the exonuclease activity of DNA polymerase I on alkali-treated DNA ligase products as summarized in Scheme 3. DNA polymerase I catalyzes exo- nucleolytic cleavage from 3’45 direction (poorly from 5’43’ direction) on polynucleotides carrying only 3’-OH termini and not 3’-phosphate termini (28, 29). Treatment of the T4 DNA ligase product with alkali generated (dA),o,, [32P]Ap; this 32P was resistant to the exonuclease II attack. Removal of the 3’- phosphate residue with alkaline phosphatase, on the other hand, resulted in the acid-solubilization of the 32P by DNA polymerase I preparations.

DISCUsSION

Since T4 DNA ligase joins DNA to DNA (7-9) as well as RNA to RNA (10, II), it was not surprising that this enzyme would join RNA to DNA in either direction. The linkage of

tively. _

5’.phosphate termini of poly(dA) and poly(dT) to 3’.OH termini of poly(A) and poly(U), respectively (Tables II, IV, and Fig. 3), as well as the formation of poly[d(A-T)] circles with UMP sub-

stituted at the 3’-OH end of the polymer occurred readily. The inverse reaction, namely the joining of 5’.phosphate termini of polyribonucleotides to the 3’-OH termini of polydeoxyribonucleo- tide, was difficult to observe. It did not occur with poly(A) or poly(U) as substrates but was observed with a poly(dA) sub- strate containing AMP residues at the 5’ termini of the polymer. Even then, the joining reaction occurred poorly and the yield of the reaction was only 5% (Table VI). Attempts to increase this yield either by altering the substrate to template ratio or the incubation conditions failed. Since T4 DNA ligase catalyzes the linkage of poly(A) to poly(A) in the presence of poly(dT), the enzyme is capable of transferring AMP to the 5’-phosphate terminus of a polyribonucleotide and can subsequently form phosphodiester bonds from such structures. Thus, the finding that poly(dA) is not linked to [5’-32P]poly(A) and that [5’-32P]- (A3) (dA)300 is only poorly joined together by T4 DNA ligase must reflect some structural difficulty with poly(dA) derivatives. Indeed, the joining of poly(dA) occurs to a much more limited extent than the joining of poly(dT). It is also possible that T4 DNA ligase does not favor the joining reaction between [5’-32P]- ribo and 3’-deoxyribopolynucleotides. Using rephcative form II of c$X DNA, presumably containing RNA covalently attached to the 5’ terminus of DNA, Westergaard et al. (32) observed that T4 DNA ligase in conjunction with T4 DNA polymerase yielded a covalently closed replicative form (RF I), which was alkali sensitive. E. coli DNA ligase did not catalyze such a closure (32).

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3687

TABLE VI

Joining of [5’-32P](A~) (dA)3~~ in presence of (dT)sooo by T4 DNA Ligase

In Experiments I and II the input 32P was 9.09 X lo3 cpm. The amount of alkaline phosphatase-resistant or NaOH-resistant 32P detected in the absence of enzyme was 0.03 and 0.05 pmole in Experiments I and II, respectively. These values have been substracted from those presented above. The reaction conditions were as described in Table I for E. coli DNA ligase and Table II for T4 DNA ligase. In Experiment I, 1.11 nmoles of (dT)s,,oo template and 1.01 nmoles of [3zP](A3)(dA)soo (2.9 pmoles of 32P) were used and incubation was carried out at 30” for 60 min. Each reaction mixture was then divided in three equal portions in order to determine the susceptibility of 32P to alkaline phospha- tase or NaOH; the third portion was used as control. In Experi- ment II, reaction mixtures containing 0.68 nmole of poly(dT)8000 primer and 0.4 nmole of [32P](A3)(dA)s0,, (1.2 pmoles of 32P) were used and the entire mixture was treated with alkaline phospha- tase or NaOH as indicated.

Experiment

I A

B

II A

B

C

Additions

2.5 units of E. coli DNA Ligase (a) Alkaline phosphatase (b) NaOH

0.25 unit of T4 DNA Ligase (c) As in (a) (d) As in (b)

0.1 unit of T4 DNA Ligase (a) Alkaline phosphatase (b) NaOH (c) As in (a); followed by NaOH (d) As in (b); then alkaline phos-

phatase As in A, omit ATP

(e) As in (a) As in A, omit poly(dT)

(j) As in (a)

Acid-insoluble p*P

pvde %

0.01 0.00

<1 <1

0.05 0.04

5 4

0.07 6 0.06 5 0.05 4

0.05

0.00

0.00

4

<l

<1

al pApApApdA-------dApdAol, Exe II ssnsltive =P (29)

T4 DNA ligose

followed by

alkaline phosphotasr 1

b) OHApA----dApdA--pApA--dApdAon

I NaOH

Delayed Exo II

scmaltive 32P

Cl OHApdA----pdA--pAp Em II resistant “P

I

(29,29)

alkaline phosphotase

action.

d) OHdApdA----pdA--pAOn Exo II ssnsltive “P

SCHEME 3. Joining of [5’-32P](A)3(dA)300 substrate by coliphage T4 DNA ligase and susceptibility of products to exonuclease II

4 Hirose et al. (37) have reported that RNA is not located at the 3’ end of DNA fragments since the DNA is degraded by 3’+5’ exonuclease action of T4 DNA polymerase. We have observed . . that exonuclease activity of T4 DNA polymerase preparations is equally effective on poly[d(A-T)] and poly[d(A-T)] substituted with UMP or AMP at the 3’ termini.

In our experiments with E. coli DNA ligase, we could not de- tect the joining of the 5’ terminus of poly(A) and poly(U) with the 3’ terminus of poly(dA) and poly(dT), respectively (Tables I and III). Olivera and T -hman (23) were also unable to join 5’-32P-polyribonucleotides with the 3’.OH end of polydeoxyribo- nucleotides. The polymer [5’-32P](A3)(dA)300 which served as a poor substrate for T4 DNA ligase was inactive with E. coli DNA

ligase (Table VI). In contrast., joining of the 5’ terminus of poly(dA) or poly[d(A-T)] with the 3’-OH terminus of poly(A) or UMP occurred readily. The failure to link poly(dT) to the 3’-OH terminus of poly(U) by E. coli DNA ligase (Table III) is in contrast to the reaction catalyzed by T4 DNA ligase. This could be due to the formation of multiple-stranded structures of poly(U) with poly(dA), which could also explain the inability of T4 DNA ligase to link poly(U) residues (Table IV, (11)).

The ability of E. coli DNA ligase to join 5’-3*P-deoxyribopoly- mers to 3’-OH ends of RNA chains increases the list of reactions yielding covalently linked RNA and DNA. Elongation of DNA chains with ribonucleotides by RNA polymerase (25, 33\, or elongation of RNA chains with deoxyribonucleotides by DNA polymerases (34, 35) involves the synthesis of new RNA and DNA in the generation of covalent hybrid structures. DNA ligase, on the other hand, covalently links preformed RNA and DNA. It is obvious then that the finding of covalently linked RNA.DNA molecules does not necessarily mean that DNA synthesis has been initiated from RNA chains. Newly synthe- sized DNA fragments in E. coli (Okazaki fragments) have been shown to contain RNA chains at 5’ termini of DNA fragments (36, 37). These RNA chains have been shown to contain 5’- triphosphate termini as expected of initiation events catalyzed by RNA polymerase. However, the existence of ribonucleo- tides at the 3’-OH termini of DNA fragments has not as yet been ruled out.4

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Kamalendu Nath and Jerard HurwitzCatalyzed by Deoxyribonucleic Acid Ligase

Covalent Attachment of Polyribonucleotides to Polydeoxyribonucleotides

1974, 249:3680-3688.J. Biol. Chem. 

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