17 Eukaryotic DNA Kinases

Eukaryotic DNA Kinases STEVEN B . ZIMMERMAN BARBARA H . PHEIFFER

I . Introduction and Perspectives . . . . . . . . . . . . . . . . . . I1 . Purification and Properties . . . . . . . . . . . . . . . . . . . .

A . Purification . . . . . . . . . . . . . . . . . . . . . . . . . B . Physical Properties . . . . . . . . . . . . . . . . . . . . . 111. The Catalytic Reaction . . . . . . . . . . . . . . . . . . . . A . Description of the Reaction . . . . . . . . . . . . . . . . . . B . Assay Procedures . . . . . . . . . . . . . . . . . . . . . . C. Stoichiometry and Identification of Products . . . . . . . . . . D. Requirements for Activity . . . . . . . . . . . . . . . . . . E . Reversal of the Reaction and Labeling by Exchange . . . . . . F. Kinetics and Mechanism . . . . . . . . . . . . . . . . . . . G . Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . IV. Comparison of the DNA Kinases with RNA Kinase and Polynucleotide Kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Biological Role . . . . . . . . . . . . . . . . . . . . . . . . . VI . Research Applications . . . . . . . . . . . . . . . . . . . . . Note Added in Proof . . . . . . . . . . . . . . . . . . . . . .

1

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315 316 316 318 318 318 318 319 320 322 324 324 326 327 329 329

Introduction and Perspectives

DNA kinase activity from a eukaryotic source was first demonstrated by Novogrodsky ef (11 . ( 1 ) . Extracts of rat liver nuclei were shown t o transfer phosphate groups from ATP to 5'-hydroxyl termini in DNA . Since then. enzymes with this activity have been partially purified from 1 . A . Novogrodsky. M . Tal. A . Traub. and J . Hurwitz. JBC 241. 2933 (1966)

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THE ENZYMES Vol XIV Copynght @ 1981 by Academic Press. lnc All rights of reproduction in any form reserved ISBN 0-12-122714-6

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S . B . ZIMMERMAN AND B . H. PHEIFFER

rat liver (2, 3 ) and calf thymus ( 4 ) . The DNA kinase from rat liver has proved to be highly specific for DNA ( 3 ) .A preparation of an enzyme from calf thymus with otherwise quite similar properties shows low activity on RNA chains in addition to its activity on DNA (4).In addition, a relatively specific RNA kinase has been partially purified from HeLa cell nuclei (5 ). The restricted acceptor specificity of these eukaryotic enzymes may be contrasted to the broad specificity of the polynucleotide kinase from T2-, T4-, or T6-infected Escherichicr coli, which has comparable activities on both DNA and RNA as well as on oligonucleotides and even on 3’rnononucleotides (6, 7). In this chapter, we will describe the properties of the eukaryotic DNA kinases and contrast them with those of the RNA kinase and of the polynucleotide kinase. The polynucleotide kinase is reviewed by Richardson in this volume (7); both DNA kinase and polynucleotide kinase have been reviewed by Kleppe and Lillehaug (6).

II. Purification and Properties

A. PURIFICATION The most thoroughly characterized DNA kinase is the activity from nuclei of rat liver (1-3,8). This enzyme has been partially purified by two independent procedures. Both preparations start by isolating nuclei. The enzyme is extracted from the nuclei with 0.15-0.2 M NaCl. Subsequent steps in the procedure of Teraoka et ul. ( 2 ) include removal of inactive materials by precipitation at pH 5, gradient elution from a phosphocellulose column, and finally gel filtration on Sephadex G-150. In the procedure of Levin and Zimmerman (-?I the nuclear extract is subjected to gradient elution from a phosphocellulose column and stepwise elution from a sulfopropyl Sephadex column. The specific activities of the final fractions from either procedure are similar and correspond to approximately a 1000-fold purification relative to a crude extract of rat liver. Two useful aspects of the procedure of Levin and Zimmerman (-3) may be 2. 3. 4. 5. 6. 7. 8.

H. Teraoka, K. Mizuta, F. Sato. M . Shimoyachi, and K. Tsukada, EJB 58,297 (1975). C. J. Levin and S. B. Zimmerman, JBC 251, 1767 (1976). G . E. Austin, D. Sirakoff, B . Roop, and G . H. Moyer, BBA 522, 412 (1978). S. Shuman and J. Hurwitz, JBC 254, 10396 (1979). K. Kleppe and J. R . Lillehaug, Adiwn. En:ymo/. 48, 245 ( 1979). C. C. Richardson, this volume, chap. 16. M . Ichimura and K. TsukadaJ. Biochern. (Tokyo)69, 823 (1971).

17. EUKARYOTIC DNA KINASES

3 17

mentioned. First, the phosphocellulose fraction has proved to be very stable, losing little or no activity for periods of 4-6 months at 4" (9); this fraction has been used extensively to characterize the activity. Second, a highly purified DNA ligase is also obtained in separate fractions of the phosphocellulose chromatography (10 ). Although preparations from both procedures are heterogeneous based upon their gel electrophoresis patterns ( I I ) , they are relatively free from interfering activities, as implied by their use for labeling the 5'-hydroxyl termini at single-strand interruptions (nicks) within duplex DNA (8, 10). More direct assays have indicated a general lack of contamination with nuclease, phosphodiesterase, or DNA ligase activities (2, 3 ) ; no phosphatase activity was detected on p-nitrophenyl phosphate (2) or 5 ' phosphate groups in DNA ( 3 ) . A very low level of nuclease activity on denatured DNA can be demonstrated with concentrated samples of the phosphocellulose fraction (/I); this activity, which is apparently the enzyme described by Cordis et d.(/.?), can be abolished with little loss of kinase activity by substituting Ca*+(0.01 M ) for Mg2+in the kinase incubation mixture (/I). A DNA kinase activity has been partially purified from homogenates of calf thymus ( 4 ) . The procedure involved protamine sulfate precipitation of inactive materials, ammonium sulfate fractionation, gradient elution from columns of phosphocellulose, hydroxyapatite and sulphopropyl Sephadex , and finally centrifugation through a glycerol gradient. The final fraction was about 1600-fold purified relative to the crude extract. The purified fractions were relatively unstable. DNA ligase, nuclease, and phosphatase (on 5'-phosphate groups in DNA) were not detected in these fractions. DNA kinase has also been partially purified from extracts of Chinese hamster lung cells grown in tissue culture (II). Extracts of washed cells in 0.2-0.4 M NaCl were made by several cycles of freezing and thawing. Diluted extracts were subjected to the same phosphocellulose chromatography that was used for the rat liver enzyme (3).A single peak of activity with the characteristic inhibition by inorganic sulfate (see Section I11 ,G,3) appeared at the same place in the gradient as did the enzymes from liver (2, 3 ) or calf thymus (4). 9. The sulfopropyl Sephadex fraction of this procedure has a half-life of a few weeks U). Both the phosphocellulose and Sephadex fractions of Teraoka et a / . are stated to be stable for at least a week at 0-4" (2). 10. S. B . Zimmerman and C. J. Levin,JBC 250, 149 (1975). 1 1 . S . B . Zimmerman, C. J . Levin, and B . H . Pheiffer, unpublished results. 12. G . A . Cordis, P. J. Goldblatt, and M. P. Deutscher, Biochemisrry 14, 2596 (1975).

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B. PHYSICAL PROPERTIES The molecular weight of the DNA kinase from rat liver has been estimated at 8 x lo4 based upon its gel filtration properties (2, 3 ) . A sedimentation coefficient ( s ~ , , ,=~ 4.4) was determined by Teraoka et al. (2). The enzyme from calf thymus is similar in size. A molecular weight of 7 x lo4 was estimated from a sedimentation coefficient = 4.3) and a Stokes radius from gel filtration of 3.9 nm, using an assumed value for the partial specific volume (4). 111.

The Catalytic Reaction

A. DESCRIPTION OF THE REACTION DNA kinase catalyzes the reversible transfer of a phosphate group between a nucleoside triphosphate and the 5’-hydroxyl moiety at the terminus of a DNA chain ( 2 - 4 ) NTP

+ 5’-hydroxyl terminus in DNA e NDP + 5’-phosphate terminus in DNA

(1)

Although the reaction is customarily assayed in the forward direction with ATP as the phosphate donor, studies of the specificity of the reverse eaction (13) indicate that the enzyme can use a number of other nucleotides. DNA is indicated as the phosphate acceptor in Eq. (1); the kinase from calf thymus may also have limited activity on 5’-hydroxyl termini of RNA chains (4).

B. ASSAYPROCEDURES The routine assay for DNA kinase measures the rate of transfer of the radioactive phosphate group of [y-32P]ATPinto an acid-insoluble form in the presence of a DNA acceptor containing 5’-hydroxyl termini ( 1 - 4 , 8). In preparation for its use as an acceptor, the DNA is either partially digested with pancreatic DNase to form 5’-phosphate termini followed by treatment with a phosphatase to yield 5’-hydroxyl termini, or is partially digested with micrococcal nuclease, which directly yields 5’-hydroxyl groups. Estimates of DNA kinase activity in crude extracts of cells or nuclei should be evaluated cautiously. Other enzymes can transfer the terminal phosphate of ATP to acid-precipitable acceptors that may be present in 13. B. H. Pheiffer and S. B. Zimmerman, Biochemistry 18, 2960 (1sv79).

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3 19

crude extracts. The characteristic inhibition of DNA kinase by relatively low concentrations of inorganic sulfate (see Section III,G,3) may prove useful in such situations. Other assays have been used for special purposes. For example, the rate of phosphorylation of relatively low molecular weight acceptors that are not acid-precipitable may be followed by adsorbing them to charcoal ( I , 3 ) . Also, the reverse reaction may be assayed by the rate of the nucleoside diphosphate-dependent release of radioactivity from [5'-3'P]phosphate termini in DNA into an acid-soluble form (13).

c. 1.

STOICHIOMETRY AND IDENTIFICATION OF

PRODUCTS The Forward Reuctioti

The labeled product of phosphorylation of DNA with [y-32P]ATPby the rat liver DNA kinase was characterized as a 5'-phosphate terminus of a DNA chain by a number of criteria. As expected for such a product, the label was rendered acid-soluble by treatment with E. coli alkaline phosphatase or pancreatic DNase (3, 8) but not by treatment with pronase, pancreatic RNase, or alkali ( 3 ) .Combined treatment with a phosphodiesterase plus 5'-nucleotidase yielded 32Pi, implying the formation of a labeled 5'-phosphate group (2). The product of the kinase and [y-32P]ATP on dephosphorylated nicked DNA was characterized in some detail. When treated with pancreatic DNase and venom phosphodiesterase, the radioactivity was quantitatively recovered in the four isolated 5'deoxynucleoside monophosphates, clearly indicating the formation of 5 ' phosphate groups by the kinase (10). Further, the labeled product was sealed into phosphodiester linkage by DNA ligase from either rat liver (10, 14) or E. coli (11); these enzymes require the presence of a 5'-phosphate group for their action. The other product of the forward reaction was identified as ADP by its cochromatography with an ADP standard on a DEAE-cellulose column. The amount of ADP formed approximately matched the amount of DNA phosphorylated, consistent with the reaction as written in Eq. (1) ( 2 ) . The product formed by phosphorylation of DNA with the calf thymus enzyme was also identified as a phosphate terminus (4), based upon the sensitivity of the product to degradation by pancreatic DNase, micrococcal nuclease, or E. coli alkaline phosphatase. The radioactivity was released in a form not adsorbed by charcoal after combined treatment with 14. K . Tsukada and M . Ichimura, BBRC 42, 1156 (1971).

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venom phosphodiesterase and 5’-nucleotidase, as expected for a 5 ’ nucleotide product. The acid-precipitable product of the calf thymus enzyme on RNA was identified by its solubilization by pancreatic RNase or NaOH, but not by pancreatic DNase or pronase. 2 . The Reverse Recictioti The reverse reaction catalyzed by rat liver DNA kinase releases radioactivity from DNA bearing [5’-32P]phosphatetermini in the presence of a suitable nucleoside diphosphate ( I S ) . The acid-soluble product of the reverse reaction with ADP and P2P]DNA was identified as PPIATP by its cochromatography with authentic ATP on a DEAE-cellulose column. The 32P released from the DNA was quantitatively accounted for by the amount of [32P]ATPformed. No significant radioactivity was associated with Pi, AMP, ADP, or adenosine tetraphosphate. FOR ACTIVITY D. REQUIREMENTS

1. p H

The rates of the forward (2, 3 ) and reverse reactions (I.?) of the DNA kinase from rat liver have a similar sharp optimum at about pH 5.5, with diminished but significant activity up to at least pH 8 (Fig. 1). The rate of the forward reaction of the calf thymus enzyme has a similar pH dependence ( 4 ) . 2 . Divu I en t Cations The DNA kinase from both rat liver and calf thymus requires a divalent cation for significant activity. For the rat liver enzyme ( 2 , 3 ) ,a number of metals support kinase activity (Mg2+,Mn’+, Co2+,Zn2+,Ni2+,and Ca2+), whereas Cu2+is inhibitory. With the calf thymus enzyme (4),Mg2+, Mn2+, and Zn2+were shown to be active. 3 . Specijicity .for Nucleoside Triphosphrites ATP is the only nucleoside triphosphate that has been shown to be active in the forward reaction of the DNA kinase ( K , = 2 4 y M ) (/-4). However, the wide range of nucleoside diphosphates that are active in the reverse reaction ( I 3 1 suggests that all of the common ribo- and deoxyribonucleoside triphosphates can participate in the forward reaction. The forward reaction with labeled ATP is inhibited by the presence of these unlabeled ribo- and deoxyribonucleoside triphosphates in a manner consistent with such a broad specificity for the phosphate donor ( 3 , 4 ) .

32 1

17. EUKARYOTIC DNA KINASES

PH

FIG. 1. Effect of pH on rat liver DNA kinase. Open and closed circles indicate Trismaleate and sodium succinate buffers, respectively. From Levin and Zimmerman ( 2 ) , reproduced with permission.

4. Specificity .for the Phosphate Acceptor

The first descriptions of DNA kinase activity in crude extracts did not define its ability to phosphorylate other than DNA acceptors (I, 8). Subsequent detailed studies indicated that the DNA kinase purified from rat liver is specific for DNA (and long oligodeoxynucleotides) and has little or no activity on RNA (3, 15). In these studies, all of the potential substrates tested were also assayed with the polynucleotide kinase from T4-infected cells (7) to ensure that negative results with the rat liver enzyme were a function of the specificity of that enzyme, and not due to a defect in the substrate. a. DNA versus R N A . The routine substrate for the DNA kinase from rat liver is DNA that has been enriched in 5’-hydroxyl termini (see Section 15. Teraoka et r d . ( 2 ) cite preliminary results with the purified enzyme from rat liver which indicated to them that RNA can act as a phosphate acceptor. In the absence of their experimental results and in view of the experimental evidence cited here in opposition to their conclusion, it is our opinion that the rat liver DNA kinase is indeed highly specific for DNA relative to RNA.

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111,B). In contrast to its activity on such DNA preparations, the DNA kinase was inactive on similarly treated samples of RNA or of poly(rA) (Table I) (3, 15). The lack of activity on these materials was not due to contaminating nucleases that destroyed either the phosphate acceptors or the phosphorylated products. Such possibilities were ruled out by the results of sequential incubations of the rat liver and T4 kinases. The lack of activity of the rat liver enzyme on RNA was also maintained in the presence of divalent cations other than Mg2+, as well as under the T4 kinase assay conditions (3). The calf thymus preparation differed from that of rat liver in that the thymus preparation phosphorylated RNA containing 5'-hydroxyl termini at about 10% of the rate with which it acted upon DNA (4). This apparent difference in acceptor specificity is further discussed in Section IV. The rat liver kinase phosphorylates 5'-hydroxyl termini in DNA that are joined to any of the four usual bases (see Section III,C, 1). Further, any of these four bases can also be present on the 3'-hydroxyl side of a nick that is being phosphorylated (10). Both native and denatured DNA were generally phosphorylated at similar rates (Table I) and to similar extents, except that the extent of phosphorylation of micrococcal nuclease-treated DNA increased greatly upon denaturation, suggesting an inhibition by 3'-phosphate groups adjacent to a site of phosphorylation (3). b . Nucleotides and Oligonucleotides as Phosphate Acceptors. Several deoxydinucleoside monophosphates and deoxynucleoside 3'-mOnOphosphates (as well as ribonucleoside 3'-monophosphates) were not substrates for the rat liver enzyme (Table I), although they were all readily phosphorylated by the T4 kinase ( 3 ) . The dependence of the kinase activity on the chain length of oligodeoxynucleotides was tested with a series of partial DNase I digests of calf thymus DNA (3). After dephosphorylation, the digests were incubated with DNA kinase and [y-32P]ATP.Digests with average chain length of -6-9 residues were relatively inactive, whereas digests with average chain length of 13 or more residues were acted upon at rates similar to the rate on denatured DNA. These results suggested that a chain length of more than 10-12 residues is required for rapid phosphorylation. The size distribution of labeled oligodeoxynucleotides in a partial digest was consistent with this conclusion. E.

REVERSALOF THE REACTIONAND LABELING BY

EXCHANGE

Reversal of the kinase reaction could be readily demonstrated (131, although the rate was several orders of magnitude slower than the rate of

323

17. EUKARYOTIC DNA KINASES

TABLE I

PHOSPHATE ACCEPTORSPECIFICITY OF RAT LIVERDNA KINASE~ Relative rate of kinase activity Experiment

Substrate

(%)

A

DNA, micrococcal nuclease-treated DNA, micrococcal nuclease-treated, heat 5 min at loo", quench poly(rA), micrococcal nuclease-treatedb RNA, micrococcal nuclease-treated" RNA, micrococcal nuclease-treated, heat 5 min at loo", quench DNA, micrococcal nuclease-treated Ado 3'-P, Guo 3'-P, Cyt 3'-P, or Urd 3'-P dCyt 3'-P or dThd 3'-P dT-dC or dC-dT

100

B

83 <2 <2

Adapted from Levin and Zimmerman (3).

'' The lack of activity of the kinase on these substrates was observed on samples treated

with levels of micrococcal nuclease producing from 6 to 50% acid-soluble materials. With the T4 kinase, these samples gave rates of activity within twofold of the rate of that enzyme upon micrococcal nuclease-treated DNA.

the forward reaction. The reverse reaction required the presence of a divalent cation and any one of a variety of nucleoside diphosphates; ADP, GDP, CDP, UDP, dADP, dGDP, dCDP, and dTDP all supported the reverse reaction, whereas ATP, AMP, PPi, and Pi were not active. (Identification of products is summarized in Section III,C,2.) The pH dependence of the reverse reaction showed a sharp optimum at -pH 5.5, which was indistinguishable from that of the forward reaction. There was also no obvious differential effect of incubation temperature on the forward and reverse reactions, both rates increasing about fourfold between 20"and 37" and more than eightfold between 0 and 20". The reverse reaction with heat-denatured DNA occurred at a rate 5 times less than that with native DNA. It is pJssible to introduce radioactivity at nicks without prior dephosphorylation by using the DNA kinase in an exchange reaction (13) similar to that previously demonstrated for polynucleotide kinase (7). DNA bearing unlabeled 5'-phosphate termini was incubated with a mixture of ADP and [y-32P]ATPin the presence of the enzyme. As expected for an exchange reaction, the rate of incorporation of label into DNA is increased by the presence of the ADP, as opposed to labeling of dephosphorylated nicks by the forward reaction where ADP acts as an inhibitor.

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F. KINETICSAND MECHANISM The reaction catalyzed by the rat liver DNA kinase apparently proceeds by a sequential mechanism (i.e., without involvement of a covalently linked enzyme-substrate complex) (13). Double-reciprocal plots of the initial velocity as a function of ATP concentration at different DNA levels or of DNA concentration at several ATP levels both gave series of nonparallel lines, indicative of a sequential mechanism. Also, attempts to demonstrate an enzyme-dependent exchange reaction between nucleoside di- and triphosphates that would be expected to occur with a covalent enzyme-substrate complex were unsuccessful. Either DNA alone or ATP alone protected the kinase from thermal inactivation under assay conditions ( 1 3 ) . Hence, the enzyme can apparently interact with either substrate in the absence of the other, suggesting a random order of interaction with the substrates. There is no information on the order of release of products. G. INHIBITORS 1. Ionic Strength

The kinases from rat liver (3)and calf thymus ( 4 )are subject to a similar nonspecific inhibition by monovalent salts, with 50% inhibition at about 0.2 M salt. The rat liver enzyme shows a small stimulation at lower ionic strengths (3 ).

2. Sirlfi ydryl Conipouiitis The enzymes from both liver (2, 3 ) and thymus (4) appear to have essential sulfhydryl groups. A mercaptan such as dithiothreitol or P-mercaptoethanol was generally added to enzyme preparations and their assay mixtures. Although omission of the mercaptan from these media usually had only slight effects, dialysis of enzyme in its absence caused significant losses of activity. The rat liver kinase was inhibited by p-chloromercuribenzoate (2, 3 ) ; both the rat liver and thymus enzymes were inhibited by iodoacetate or AgN03 (3, 4). 3. Inorganic Sulfnte and Other Inorganic Anioris The forward reaction of the rat liver kinase is inhibited by remarkably low levels of inorganic sulfate (-50% inhibition by 0.3 m M N%S04under routine assay conditions) and related compounds, such as N+Se04 or Na2W04( 2 , 3 )(Fig. 2). The inhibition by Na,S04 is competitive with ATP ( K i = 0.2 mM) and noncompetitive with DNA (13). Inhibition of the

SALT (rnM)

FIG.2. Effect of various anions on rat liver DNA kinase. From Levin and Zrnmerman (3). reproduced with permission.

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S. B. ZIMMERMAN AND B . H. PHEIFFER

reverse reaction requires much higher levels of sulfate (50% inhibition at 4 mM N%,S04)(13).The kinase activity is inhibited by relatively low levels of PPi (50% inhibition at 0.2 mM), but not by NaF (> 10% inhibition at 10 mM) (2, 3 ) . The calf thymus enzyme appears to be less sensitive to inorganic sulfate [60% inhibition at 0.01 M (NH4)2S04]and is also inhibited by PPi (4). 4. Organic Sufur. Compounds

The rat liver DNA kinase was inhibited by low levels of certain sulfurcontaining polymers (3, 13). For example, dextran sulfate or heparin gave 50% inhibition at -20 or 60 ng/ml, respectively, under assay conditions, whereas samples of agar or chondroitin sulfates A or C (or dextran) did not inhibit significantly even at 15 pg/ml. Dextran sulfate inhibition was competitive with respect to both DNA and ATP, and inhibited the forward and reverse reactions to similar extents. The calf thymus enzyme was inhibited 75% by a sample of heparin at 1 mg/ml and was not inhibited by agar at three times that level (4). 5. Miscellaneous Compounds

A number of compounds that either influence the rate of the microbial polynucleotide kinase or that are of intrinsic interest were tested with the rat liver kinase (spermine, spermidine, adenosine 3', 5'-monophosphate, adenosine 5'-phosphosulfate); all were without strong effect (3, 13). Actinomycin D did not inhibit the activity of the enzyme in crude extracts of liver nuclei (1, 8).

IV.

Comparison of the DNA Kinases with RNA Kinase and Polynucleotide Kinase

This review has been primarily concerned with the isolation and properties of the DNA kinases from rat liver and calf thymus. These enzymes are notable for the much greater rate at which they phosphorylate DNA termini than RNA termini, although the calf thymus preparations show some activity on RNA termini. In this connection, the report by Shuman and Hurwitz (5) of a eukaryotic RNA kinase is particularly interesting. They found that extracts of nuclei from HeLa cells contained two physically separable kinase activities, namely, a DNA kinase activity that is probably similar to those previously described, and in addition an RNA kinase with distinctly different properties. The RNA kinase has been partially purified and characterized in some detail. Although it is relatively

17. EUKARYOTIC DNA KINASES

327

specific for RNA termini, it also shows a low rate of phosphorylation on DNA termini. The demonstration of the RNA kinase emphasizes a need for caution in evaluating the low levels of activity on RNA that were shown by partially purified preparations of DNA kinase from calf thymus. As suggested by Austin et ril. ( 4 ) ,it is possible that the activities they observed on DNA termini and RNA termini were actually a result of the presence of two separate enzymes. In addition to these eukaryotic enzymes that phosphorylate 5’-hydroxyl termini of DNA or RNA, there is also the polynucleotide kinase from T2-, T4-, or T6-infected E . coli, which phosphorylates both species of termini, as well as other acceptors (6, 7). A comparison of some distinctive properties of all of these kinases is made in Table 11. V.

Biological Role

The biological function of the DNA kinase is not known. It seems reasonable to ascribe to the enzyme a role in the repair of damage to DNA (2-41, particularly in view of its activity at single-strand breaks in duplex DNA. DNA kinase can convert 5’-hydroxyl termini to 5’-phosphate groups at such nicked locations, thus providing the proper substrate for subsequent joining to apposed 3’-hydroxyl groups by the DNA ligase (19, 20). The distribution of the DNA kinase is consistent with a role in DNA repair processes. The activity is apparently confined to the cell nucleus in rat liver (2, 3 ) . The enzyme is found in cells of several species: rat ( I ,3 ) , hamster ( I I ) , and probably also in human (5, 11, 11, 21) and frog (13). There is no evidence as yet for a control function of the DNA kinase. Its activity was not changed after partial hepatectomy in the rat (2),nor was it significantly different in extracts of Chinese hamster lung cells from the exponential or stationary phases of growth ( I I ). The apparent polynu16. C . C. Richardson, PNAS 54, 158 (1965). 17. R. Wu, BBRC 43, 927 (1971). 18. J . R. Lillehaug and K. Kleppe, B i o c h e r n i s ~ y14, 1225 (1975). 19. I . R. Lehman, Scietice 186, 790 (1974). 20. S. Soderhall and T. Lindahl, FEES (Fed. O r r . Riocliem. S o c . ) Lett. 67, 1 (1976). 21. G . C. F. Pedrali Noy, L. Dalpra’, A. M. Pedrini, G . Ciarrocchi, E. Giulotto, F. Nuzzo, and A. Falaschi, Nircleic Acids Res. I , 1183 (1974). 22. A. M. Pedrini, L. Dalpra’, G. Ciarrocchi, G. C. F. Pedrali Noy, S. Spadari, F. NUZZO, and A. Falaschi, Nrrcleic Acids Res. 1, 193 (1974). 23. H. Saiga and T. Higashinakagawa, Nircleic Acids Res. 6, 1929 (1979).

TABLE I1

KINASES,RNA KINASEAND T4 POLYNUCLEOTIDE KINASE

COMPARISONS OF DNA

DNA kinase Rat liver Characteristic pH optimum Apparent K , for ATP ( p m ) Phosphate acceptors Divalent cations

Teraoka et al. ( 2 )

Levin and Zimmerman ( 3)

5.5

5.5

2

DNA Mg2+, Mn2+, Cap+ 0.5

5.5 4

2

RNA kinase ( 5 )

DNA, RNA, oligonucleotides, nucleoside 3'-phosphates (6. 7) Mg2+, Mn'+ , Znp+, Co2+ , Nil+ , Ca2+ (Cu2+inhibitory) ( / )

1

(Cu2+ inhibitory) 0.3

8 x 10'

8 x 104

T4 Polynucleotide kinase

7.9 to 8.9 500

DNA, oligodeoxynucleotides with n > 10-12 Mg2+, Mn2+, Zn2'. COW Ni2+ Ca2+ 7

[SOZ-] for 50% inhibition (mM) Molecular weight

Calf thymus Austin et a / . (4)

b

7 x 104

-

14 x lo4 ( 6 )

10 mM (NH,),SO, produced 60% inhibition ( 4 ) .

* Inorganic sulfate is inhibitory to the T4 polynucleotide kinase (K, = 600 &ml)

actually stimulate activity in assays conducted at low ionic strength (18).

(/7), although sulfate at high concentrations has been reported to

17. EUKARYOTIC DNA KINASES

329

cleotide kinase activity did not undergo large changes in lymphocytes as a function of time after phytohemaglutinin stimulation (2f,24), nor was it greatly different in tissue culture cells from several patients with xeroderma pigmentosum as compared to control cells (2,24). It is not known whether the DNA kinase has an intrinsic 3’-phosphatase activity in addition to its phosphorylating capacity. If the eukaryotic enzyme turns out to be similar to polynucleotide kinase (6, 7) in having both activities, this will certainly strengthen the case for the involvement of the DNA kinase in DNA repair. Finally, it should be noted that preparations of both the DNA kinase from calf thymus (4) and the RNA kinase from HeLa cells (5) have appreciable activity on both RNA and DNA termini, albeit with quite different relative rates, pH optima, etc. If the relatively low activity on RNA of the calf thymus DNA kinase preparations turns out to be due to the DNA kinase per se, the latter enzyme would be a second agent (along with the RNA kinase) capable of phosphorylating RNA termini. As noted by Shusuch phosphorylation may well man and Hurwitz (5) and by Winicov (3), be involved in the sequence of reactions for “capping” and “splicing” during RNA processing. In a related way, it may be noted that the RNA kinase activity on DNA termini provides a second source of phosphorylation of DNA termini. VI.

Research Applications

The DNA kinase of rat liver has been used for some years to introduce labeled 5’-phosphate groups at single-strand breaks in duplex DNA (14). Such labeled DNA may be conveniently used to assay DNA ligase activity (14, 2 ) . Also, we note that although such application has not yet been made, the specificity of the various eukaryotic kinases discussed in this review suggests their possible use to quantitate RNA and DNA termini in heterogeneous samples. Note Added in Proof

Since this paper was submitted, a further purification of the bovine DNA kinase has been described [S. Tamura, H. Teraoka, and K. Tsukada, EJB 1 15, 449 (1981)l. In contrast to earlier results ( 4 ) , the more highly purified enzyme is inactive on RNA; hence both the bovine and rat DNA kinases appear to be specific for DNA. 24. The kinase assays in references (21)and (22)were done under conditions optimal for the T4 polynucleotide kinase and may not be a measure of the DNA kinase in these cells. 25. 1. Winicov, Biorhemistry 16, 4233 (1977).