Induced Polynucleotide Kinase, Polynucleotide Ligase, and Deoxyribonucleic Acid Polymeraset

The mechanism of action of T4-induced polynu-cleotide kinase on short single-stranded deoxyoligonucleotideshas been investigated.It was found that the phosphorylationreaction catalyzed by T, polynucleotide kinase could be re-versed. Thus, a 5 ’-3*P-labeleddeoxyoligonucleotide and ADPon incubation with the enzyme formed [Y-~~PJATP.In addi-tion some radioactive inorganic phosphate was produced.In the presence of ATP the major radioactive product of thereverse reaction was identifiedas adenosine 5’-[6-32P]tetraThe bacteriophage T.i induced polynucleotide kinase catalyzes the transfer of the y-phosphate from ATP to the 5’-hydroxyl terminus of polynucleotides, oligonucleotides, 3’-mononucleotides (Richardson, 1965), and N-protected deoxy-oligonucleotides (van de Smde and Bilsker, 1973). Theenzyme has proved to be an extremely useful tool instructural work on nucleic acids. Thus, labeling of the 5’ endgroups has been used extensively for determining the endgroups and terminal sequences of macromolecular DNAs(Weiss and Richardson, 1967 ;Jacquemin-Sablon and Richard-son, 1970; Wu and Kaiser, 1967) and for structural studyofthc cohesive ends of the bacteriophage X D N A (Wu andKaiser, 1968; Richardson et al., 1908). Similarly, the finger-printing of short oligonucleotides (pyrimidine tracts fromDNAs, nuclease digests of tRNAs) is facilitated by thelabeling of the 5’-end groups (SzCkely and Sanger, 1969:Southern, 1970: Murray, 1973; Simsek er al., 1973).Further, the techniques of introducing label at the terminiof polynucleotide chains facilitate studies of the polynucleo-tide ligase and of D N A enzymology (Weiss et al., 1968b;Gefter et cd.: 1967; Olivera and Lehman, 1967) and 5’-phos-phomonoesterases (Becker and Hurwitz, 1967 ; Weiss et al.? ~~-~~~~~~~ ___~t From the Departments of Biology and Chemistry, MassachusettsInstitute of Technology, Cambridge, Massachusetts 02139. ReceivedJii!l , 2, 1973. This is paper CXXIV in the series Studies on Polynucleo-tides. The preceding paper (CXXIII) in this series is by Panet et n l .(1973). This work has bcen supported by grants from the NationalCancer Institute of the National Institutes of Health, U. S. PublicHealth Service (73675 , CA 051?8), the National Science Foundation(73078, GB-21053X2), and by funds madeavailable to the MassachusettsInstitute of Ttxhiiology by the Sloan Foundation.Present address: Division of Medical Biochemistry, Thz Universityof Calgary, Alberta, Canada.§ Present address: Department of Biochemistry, University of Bergen,Bergen, Norway. 5050 B I O C H E M I S T R Y , V O L . 1 2 , N O . 2 5 , 1 9 7 3phosphate. The revcrse reaction was found to be optimal a tpH 6.5. whereas maximum rate for the forward redction wasobserved at approximately pH 9.0. At pH 7.5 the rate of thereverse reaction was2 of that of the forward reaction. Uti-lizing this reversibility of the T4polynucleotide kinase action,it was shown that deoxyoligonuc!eotides containing unlabeledphosphate at the 5’ end could be quantitatively labeled with?*P at the 5’ end without prior removal of the unlabeled phosphate by phosphatase. 1968a). In this laboratory, it has served as an indispensabletool in the characterization of short synthetic deoxyribopoly-nucleotides andfor monitoring their subsequent joining toform defined bihelical DNAs (Khorana et a!., 1972; Panetet ul., 1973: van de Sande and Bilsker 1973).In view of the usefulness and our continued interest in thepolynucleotide kinase, we have carried out a closer study ofthe reaction catalyzed by this enzyme. A numberof interestingobservations have been made and these form the content ofthis paper. A particularly interesting finding is the revers-ibility of the phosphorylation of the 5’-OH groups in poly-nucleotides. A practical application of this finding is that ifa 5’-phosphate group is already present at the terminus of apolynucleotide chain, it is unnecessary to remove it by a phos-phatase treatment in order to introduce the radioactivelylabeled phosphate group. Materials and Methods Cliernicals. ATP and ADP were purchased from P-L Bio-chemicals Inc. Adenosine 5 ’-tetraphosphate was the productof Sigma Chemical Co. [3HH]ADPwas obtained from NewEngland Nuclear Corporation. [y?P]ATP was prepared ac-cording toa published procedure (Glynn and Chappell,1964).Enzymes. Polynucleotide kinase was isolated from Tq aniN82 phage infected Escherichia coli B62 by a modification(Panet er al., 1973) of the method of Richardson (1965). Theenzyme hada specific activity of 65,000 units,’mg of proteinand showeda single band on sodium dodecyl sulfate gel elec-trophoresis. The enzyme preparation contained no endonu-clease activityas assayed by the method of Weiss et (11.(1969a.b), or exonuclease activity as assayed by method l a ofPanet et al. (1973). Prolonged incubation (12 hr at 37”) of the