Eur. J. Biochem. 77, 521 527 (1977) ~
A DNA Polymerase from Ustilago maydis Evidence of Proof-Reading by the Associated 3' +. 5' Deoxyribonuclease Activity Geoffrey T. YARRANTON and Geoffrey R. BANKS National Institute for Medical Research, London (Received February 7,1977)
The 3' + 5' deoxyribonuclease activity associated with an Ustilago maydis DNA polymerase hydrolysed non-complementary 3'-primer termini about 12 times more rapidly than complementary termini. An analysis of its substrate specificity suggested that, although it was unable to hydrolyse fully single-stranded polynucleotides, it could hydrolyse such regions less than about four nucleotides in length covalently bound to a primer molecule which was base-paired to a complementary template strand. Template-primer combinations containing complementary or non-complementary primer termini both supported polynucleotide synthesis, but whereas the former were conserved, the latter were hydrolysed during the reaction thus allowing synthesis to occur. No addition of nucleotides onto a conserved non-complementary 3'-primer terminus was detected. The deoxyribonuclease activity therefore fulfilled a proof-reading function during DNA synthesis in vitro. Biochemical and genetical studies in prokaryotes have elegantly demonstrated the central role played by DNA polymerases in determining the fidelity of DNA replication [l - 131. Those studies provide evidence that the 3' 3 5' deoxyribonuclease activity associated with these DNA polymerases is able to excise a 3'-terminal primer deoxyribonucleotide which is not complementary to the template polydeoxyribonucleotide strand and also probably those misincorporated into newly synthesized DNA by the polymerase activity in vitro. Such reactions occurring in vivo would provide a means of controlling and maintaining the fidelity of DNA replication. Purified eukaryotic DNA polymerases do not, in general, possess this associated deoxyribonuclease activity [14,15], but, as reported previously [16,17], a polymerase purified from the eukaryotic organism Ustilago maydis does so. The evidence presented in this paper strongly suggests that the associated deoxyriboAbbreviations. [5'-"P](dT)m - [3H](dN)a denotes poly(thymidylic acid) containing an average of 320 dTMP nucleotides, labelled at its 5'-terminus with a ["P]phosphate group and at its 3'-terminus with an average of x residues of another [3H]deoxyribonucleotide dNMP. This and other abbreviations follow CBN rules, see Eur. J . Biochem. 15,203-208 (1970). Enzymes. DNA polymerase or deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase (EC 2.7.7.7); terminal transferase or deoxynucleosidetriphosphate :oligodeoxynucleotide deoxynucleotidyltransferase (EC 2.7.7.31); bovine spleen phosphodiesterase or orhtophosphoric diester phosphohydrolase (EC 3.1.4.1); micrococcal nuclease or nucleate 3'-oligonucleotidohydrolase(EC 3.1.4.7).
nuclease activity, can perform a proof-reading function in vitro. MATERIALS AND METHODS Materials Deoxyribonucleoside triphosphates, polydeoxyribonucleotides and oligodeoxyribonucleotides were from PL Biochemicals Inc., radiochemicals from the Radiochemical Centre (Amersham), Sephadex G-100 from Pharmacia Fine Chemicals and poly(ethy1eneimine)-cellulose thin-layer chromatography sheets (Macherey-Nagel Polygram CEL 300 PEI 20 x 20 cm) from CamLab. Bovine spleen phosphodiesterase and micrococcal nuclease were from the Sigma (London) Chemical Co. and terminal transferase was a gift from Dr I. R. Johnson. U. maydis DNA polymerase (fraction V) was prepared by the method of Banks et al. [16]. Labelled Polydeoxyribonucleotides Synthesis. [5'-32P](dT), was prepared by the method of Kelly et al. 1181 except that [5'-32P](dT)~o was used as the primer (161. The reaction mixture 2.5 mM dTTP, contained: 0.08 mM [S'-32P](dT)~~, 0.2 M potassium cacodylate pH 7.0, 1 mM 2-mercaptoethanol, 1 mM cobalt chloride, 0.5 mg/ml bovine serum albumin and 20 pg/ml terminal transferase. It was incubated at 37 "C for 7 h and terminated by incubation in a boiling water bath for 2 min. The
522
Proof-Reading by a U . maydis DNA Polymerase
product was purified by Sephadex G-100 chromatography. Deoxyribonucleotides were added to the 3’terminus of [5‘-32P](dT)m by terminal transferase with [3H]deoxyribonucleoside triphosphates [4]. Polymers synthesised were : E ~ ’ - ~ ~ PI ([ ~ 3 ~T1 )( d~ ~ ) m , [ 5 ‘ - 3 2 ~ i ( d ~ )m[ 3 ~ i ( d ~ ) 3 m ,
Table 1 . Distribution of the 3‘-terminal nucleotides on synthetic polvdeoxyribonucleotides The theoretical distributions were calculated by assuming that addition of nucleotides onto primers by the terminal transferase follows a Poisson distribution [19]. The observed values were determined from the deoxyribonuclease degradation products as described in Materials and Methods Pol ynucleotide
theoretical
[5’-32P](dT)m - [3H](dC),B$and [5’-32P](dT),
-
observed
[3H](dC)n .
Composition. The addition of nucleotides to oligodeoxyribonucleotide primers by terminal transferase follows a Poisson distribution [19]. The above labelled polydeoxyribonucleotides were degraded by the sequential action of micrococcal nuclease and bovine spleen phosphodiesterase [20,21] and the products separated by thin-layer chromatography on poly(ethy1eneimine)-cellulose sheets eluted with water. Internal chain deoxyribonucleotides were converted to their deoxyribonucleoside 3‘-monophosphates and the 3‘-terminal ones to their deoxyribonucleosides. Table 1 shows the experimentally determined compositions, which are in good agreement with those predicted by the Poisson distribution. Chain Length. The number-average chain length of the [5’-32P](dT)3, was determined from the specific activity of the 5’-phosphate label and the deoxyribonucleotide concentration measured spectrophotometrically. Enzyme Assays
3H label at 3’-terminus
nucleotides and inorganic pyrophosphate. Reaction mixtures were incubated at 37 “C for 30 rnin and terminated in a boiling water bath for 2 min. Aliquots (0.05 ml) of the mixture were applied to the origin of Whatman 3MM chromatography paper which was eluted overnight with isobutyric acid/concentrated NH40H/water (66/1/33). After drying, the chromatogram was cut longitudinally into 1-cm strips and radioactivity determined in each. Nearest-Neighbour Base Analysis DNA polymerase reactions were carried out as described above with [ce3’P]dTTP as the labelled substrate. After incubation at 37 “C for 30 min, the product was precipitated with 10 % trichloroacetic acid and pelleted by centrifugation at 7000 rev./min for 15 min. The supernatant solution was removed and the pellet washed three times with ether. After redissolving the DNA, it was digested by micrococcal nuclease and then bovine spleen phosphodiesterase [20,21]. The resulting mixture, along with appropriate markers was resolved by chromatography on Whatman 3MM paper, eluting overnight with isobutyric acid/concentrated NH40H/O. 1 M EDTA/water (100/2.3/1.6/50), which separated all four 3‘deoxyribonucleoside monophosphates. Radioactivity on the chromatogram was determined as above.
The rates of hydrolysis of the 3’4abelled polydeoxyribonucleotides were measured essentially as described by Brutlag and Kornberg [4]. Reaction mixtures (0.15 ml total volume) contained 1.5 nmol [5’32P](dT)m - [3H](dN),, 1.5 nmol poly(dA) where relevant, 50 mM Tris-HC1 pH 7.5, 10 mM MgCL, 17 mM dithiothreitol and 60 mM KCl. Aliquots (0.02 ml) were removed at specific time intervals of incubation and absorbed onto DEAE-cellulose chromatography paper cut into strips 1.5-cm wide and marked longitudinally into 1.5-cm segments. They were washed three times by gentle agitation in cold 0.3 M ammonium formate and once in cold 95% RESULTS ethanol. After air drying, the strips were cut into 1.5 x 1.5-cm squares and radioactivity determined in Substrate Specificity toluene-based scintillant. Polymerisation reactions were monitored in a similar manner using [ C I - ~ ~ P I - It was reported previously that the 3’ -, 5’ deoxyribonuclease activity associated with the U. maydis deoxyribonucleoside triphosphate substrates. DNA polymerase digested single-stranded DNA about Pyrophosphorolysis was measured using the above 50% as efficiently as double-stranded DNA [17]. reaction conditions, except that 3.3 mM tetrasodium This result was obtained with 3’-terminally labelled pyrophosphate was also present, and determined the native and heat-denatured activated calf thymus formation of labelled deoxyribonucleoside triphosDNA substrates, both of which contain doublephates from the labelled primer terminal deoxyribo-
523
G. T. Yarranton and G. R. Banks
0
5
10 Time
A
A
I
15 (min)
20
25
-0
4
8
0
4
8
Time (min)
Fig. 1. Substrute specijicity of the 3’ + 5‘ deoxyribonuclease activity. Standard reaction mixtures contained [5‘-3zP](dT)m - [’H](dT), (0--0) or poly(dA) . [5’-3’P](dT), - rH](dT)= (---O) and 1G units of DNA polymerase
Fig. 2. Time course of complementary und non-complementary primer terminus hydrolysis. Standard reaction mixtures contained 1G units of DNA polymerase and (a) poly(dA) . [5’-32P](dT)jx- [3H](dT),, (O--O), poly(dA) . [5’-3zP](dT)m - [3H](dA)m (0-0) or poly(dA) . [5‘-3’P](dT)32,-- [3H](dC)m ( L O ) incubated at 37 “C and (b) poly(dA) . [5’-3ZP](dT)32T - [3H](dT),i (0-- -0) or poly(dA) . [5’-32P](dT)3T6- [3H](dA),,j (0--0) incubated at 25 “C
stranded regions, thereby making an interpretation of the DNA specificity questionable. The homopolymer [5’-3zP](dT)E, - [3H](dT)il-i, alone and in combination with poly(dA), was therefore used to determine this specificity more rigorously. No degradation of the single-stranded homopolymer was detected after incubation at 37 “C for 40 min (Fig. 1). In the double-stranded structure poly(dA) . [5’-32P](dT), - [3H](dT),, however, only 2- 3 of the terminal [3H]dTMP remained in polymeric form after a 10-min incubation. This result strongly suggests that the deoxyribonuclease activity is specific for double-stranded DNA.
such as poly(dA) . (dT),, are unstable at temperatures around 37 “C, resulting in partial denaturation or fraying of the two strands at their termini. Complete hydrogen bonding can be approached, however, at lower temperatures [22]. The rates of terminal [3H]dTMP and [3H]dAMP hydrolyses were therefore, determined at 25 “C and 37 “C (Fig.2A, B). The rate for the complementary [3H]dTMP was reduced 11fold from 0.22 at 37 “C to 0.02 pmol/min at 25 “C, whilst that for [3H]dAMP was reduced only 3-fold from 0.78-0.24 pmol/min. This suggests that the deoxyribonuclease activity recognises and hydrolyses the non-base-paired primer termini much more efficiently.
Hydrolysis of Complementary and Non-Complementary Primer Termini
Using the experimentally determined values for the average number of labelled deoxyribonucleotides at the 3’-terminus of each primer chain (Table l), a comparison of their rates of hydrolysis by the 3‘ + 5’ deoxyribonuclease was made. The non-complementary, to the poly(dA) template strand, [3H]dAMP in poly(dA) . [5’-32P](dTh- [3H](dA)m was hydrolyzed about 4-fold more rapidly than the complementary [3H]dTMP termini in poly(dA) . [5’-32P](dT)m - [3H](dT),. This increased rate did not reflect a specificity for a purine rather than a pyrimidine nucleotide because the [3H]dCMP termini in poly(dA) . [5’-32P](dT)m - C3H](dC>,, were hydrolysed at almost the same rate as the [3H]dAMP (Fig.2A). The effect of primer terminus secondary structure on their hydrolysis rates was studied in more detail by varying the reaction temperature. Homopolymer pairs
Polynucleo tide Synthesis and Primer Termini Hydrolysis
As previously reported, DNA synthesis protected the primer from hydrolysis by the 3’ .+ 5‘ deoxyribonuclease activity [17].When poly(dA) . [5’-32P](dT), - [3H](dT), was incubated with the DNA polymerase in the presence of [ U - ~ ~ P I ~ T[32P]poly(dT) TP, was synthesised accompanied by an initial hydrolysis of about 10% of the [3H]dTMP in primer termini, but no further hydrolysis (Fig. 3 A). Protection of these termini complementary to the template strand required polydeoxyribonucleotide synthesis rather than nonspecific triphosphate binding to the enzyme, because only dTTP and dUTP afforded protection (Table 2). dATP, dGTP or dCTP, which are not complementary to the poly(dA) template and could not be polymerised [16], did not protect the base-paired [3H]dTMP termini from hydrolysis.
524
Proof-Reading by a U. maydis DNA Polymerase
Table 2. The influence of deoxyrihonuclease triphosphates on primer terminus hydrolysis Standard reaction mixtures contained a single dNTP as indicated to a final concentration of 13 3 WM+ indicates enzyme-catalysed conversion of 3H label in the polydeoxyribonucleotides to dNMP - Indicates no such conversion detected Polynucleotide
Poly(dA) [S’-32P](dT),
dNTP present -
[3H](dT)ii
Poly(dA) . [S’-32P](dT)m - [3H](dA)T,-i
none dTTP ordUTP dATP, dGTP ordCTP none dTTP ordUTP dATP, dGTP ordCTP
3‘-Terminal hydrolysis
+ -
+
+ +
+
0
0
5
15
10
20
Time (min)
Fig. 4. Polynucleotide synthesis and primer terminus hydrolysis. Standard reaction mixtures were as in the legend to Fig.3 but with the template-primer poly(dA) . [5’-3’P](dTh - [3H](dC),,. (--O) [32P]Poly(dT)synthesis; (-0) [3H]dCMP hydrolysis. Hydrolysis in the absence of dTTP was identical to that shown in its presence
. [5’-32P](dT)m - [3H](dA)m (Fig.4). Also only about 10 % of the non-complementary [3H]dCMP nucleotides were susceptible to hydrolysis in the absence of dTTP. These observations suggest that the [3H]dCMP non-complementary terminal nucleotides of only a small fraction of primer molecules were susceptible to hydrolysis. Assuming that terminal transferase added deoxyribonucleotides during the preparation of these primer molecules in agreement with a Poisson distribution [19], Eqn (1) gives an estimation of that fraction of the total population of primer molecules which possess any given number of added [3HIdCMP molecules. Time (min)
Fig. 3 Polynucleotide synthesis and primer terminus hydrolysis. Standard reaction mixtures contained 10 units of DNA polymerase. 13 3 pM [e3’P]dTTP (320 counts min-’ pmol-’) and (a) poly(dA) . [5’-32P](dT)m - [3H](dT)m or (b) poly(dA) [S‘-32P](dT)5[3H](dA)m (w) [32P]poly(dT) synthesis, (-0) [3H]dTMP (a) or [3H]dAMP (b) hydrolysis
Incontrast, when poly(dA). [5’-32P](dT)m- [3H](dA)m was incubated with the DNA polymerase and [u-~’P]~TTP, complete removal of the non-basepaired [3H]dAMP was observed as polymerisation proceeded (Fig. 3B and Table 2), suggesting that prior removal of non-complementary termini was required before polynucleotide synthesis onto the primer could occur. Similar observations were made with poly(dA) . [5’-32P](dTh- [3H](dC)m (results not shown). The rate and extent of [32P]poly(dT)synthesiswhen - [’H](dC), was incubatpoly(dA) . [5’-32P](dT)-jZ, ed with the enzyme and [u-~’P]~TTP was only 10% of that observed with the template-primer poly(dA)
X !
where A = mean number of added deoxyribonucleotides, x = number of deoxyribonucleotides added per chain under consideration and P = mole fraction of total population with x deoxynucleotides added. Using Eqn (l), it can be calculated that about 10 “4 of the total [3H]dCMP in poly(dA) . [5’-32PJ(dT), - [’H](dC),, is contained in primers with 1-3 [3H]dCMP nucleotides and that this class represents about 10% of the total primer population. We suggest that it is also this class which is susceptibleto the 3‘ .+ 5’ deoxyribonuclease activity and that after removal of these [3H]dCMP non-complementary deoxyribonucleotides, primed poly(dT) synthesis can occur. Nearest-Neighbour Base Analysis of DNA Synthesis Products
To verify that polydeoxyribonucleotide synthesis required a base-paired 3’-primer terminus and cannot
525
G. T. Yarranton and G. R. Banks Table 3. Nearest-neighbour base analyses of D N A polymerase reaction products Standard reaction mixtures contained the template-primers indicated, 10 units of U. rnaydis DNA polymerase and [CG’~P]~TTP (13.3 pM, 2 x lo3 counts min-’ pmol-’). The products were subjected to nearest-neighbour base analyses as described in Materials and Methods Template-primer
Analysis product
Amount of product detected
Table 4. Primer terminus specificity of the pyrophosphorolysis reaction The reaction mixture and conditions of assay are described in Materials and Methods. + and - denote the presence and absence respectively of 3.3 mM tetrasodium pyrophosphate in the reaction mixture Template-primer
PyroProduct as phosphate [’HIdNMP [’HJdNTP
% total label present
pmol Poly(dA) . [5’-32P](dT), - [’H](dT)n
Poly(dA) . [5’-32P](dT)m- [’H](dA)W
3’-dTMP 131 0.07 3‘-dAMP 0.06 3‘-dCMP
Poly(dA) . [5’-32P](dT), - [3~1(d~),
3’-dTMP 130 3‘-dAMP 0.08 0.05 3’-dCMP
Poly(dA) . [5‘-’’P](dT)m - [3~1(d~),,
Poly(dA) . [5‘-’’Pl(dT)m - [’H](dC)m
3’-dTMP 126 0.06 3’-dAMP 0.05 3’-dCMP
Poly(dA) . [5‘-32P](dT),
3’-dTMP 3’-dAMP 3’-dCMP
-
[3H](dC)n
20 0.G5 0.04
occur from a non-paired one, the products of polymerisation reactions directed by the template-primers were subjected to nearest-neighbour base analysis (Table 3). The analysis was sensitive enought to detect single deoxyribonucleotide additions to 1% of the primer termini. No addition onto any non-basepaired 3’-primer termini was detected. These results also again suggest that hydrolysis of these terminal deoxyribonucleotides must occur before poly(dT) synthesis can occur.
The 3 ‘-Primer Terminus Specijkity of Pyrophosphorolysis Pyrophosphorolysis is the direct reversal of the polymerisation reaction involving the formation of deoxyribonucleoside triphosphate from DNA and inorganic pyrophosphate. Its primer-template specificities should, therefore follow those of the polymerisation reaction [4]. When poly(dA) . [5’-32P](dT)m - [3H](dT)i-i- was incubated with the DNA polymerase at 37 “C for 30 min the only detectable 3H label was in dTMP (Table 4). When inorganic pyrophosphate was also present in the reaction mixture, 53% and 47% of the 3H label was in dTMP and dTTP - [3H]respectively. With poly(dA) . [5’-”2P](dT), (dA),, all the 3H label was in dAMP both in the absence and presence of pyrophosphate. Hence pyrophosphorolysis activity of the DNA polymerase resembled the polymerisation reaction in its specificity for a base-paired 3‘-primer terminus.
-
+ -
+
100
< 0.01
41
53
99.5 100
0.5 < 0.01
DISCUSSION The experiments described above suggest that the 3‘ + 5‘ deoxyribonuclease activity associated with the U. maydis DNA polymerase is able to proof-read in vitro. It hydrolysed noncomplementary terminal primer nucleotides faster than complementary ones, but the specificity decreased when the latter were destabilized by heat (Fig. 2). Whereas primers with 3’-terminal nucleotides complementary or non-complementary to the template directed polynucleotide synthesis by the DNA polymerase, only the former termini were conserved (Fig. 3). We conclude that the enzyme is unable to elongate from a non-complementary primer terminus prior to its removal and restoration of base-pairing, an interpretation in accord with the product nearest-neighbour base analyses (Table 3) and pyrophosphorolysis specificity (Table 4). The specificity of the deoxyribonuclease activity for a non-base-paired 3‘-terminus is apparently in conflict with its inability to hydrolyse a single-stranded homopolymer (Fig. 1). This contradiction may be explained by the experiments reported with poly(dA) . [5’-32P](dT)m - [3H](dC),, which comprised primer molecules with 0 to 10 non-complementary nucleotides (Fig.4). Only about 10% of the total [3H]dCMP was hydrolysed, a figure which agrees well with that predicted for primer chains having 1 - 3 [’HIdCMP nucleotides per primer molecule. The rate of poly(dT) synthesis directed by this template-primer combination was also only 10% of that observed with poly(dA) . [5’-32P](dT)& - [3H](dA)m, in which over- 90 % of the [3H]dAMP nucleotides were hydrolysed. Since the enzyme was in molar excess over primer termini, the rate of poly(dT) synthesis was proportional to the number of available primer sites. This suggests therefore, that only 10% of these primer molecules was
526
available for elongation, probably those with 1 - 3 [3H]dCMP nucleotides which first could be removed by the deoxyribonuclease activity. We suggest that the U . maydis DNA polymerase is able to bind to doublestranded DNA (probably at a nick or gap in one strand) but not to fully single-stranded DNA. When this binding region includes less than about 4 non-base-paired nucleotides at a 3’-terminus in one strand, the 3‘nucleotide is stereochemically in alignment with the active deoxyribonuclease site and exonucleolytic hydrolysis of the unpaired nucleotides can occur. If, however, this region is longer than 3 nucleotides, the terminal one is not in such a position and hydrolysis cannot occur. The properties of the U. maydis DNA polymerase described in this paper are, with the exception of its DNA secondary structure specificity, similar to those of the bacteriophage T4 DNA polymerase and the DNA polymerases I and I1 of Escherichiu coli [4]. Our inability to detect addition of deoxyribonucleotides directly onto a non-base-paired primer terminus distinguishes it from the DNA polymerase p of mammalian cells and the avian myeloblastosis virus enzyme, both of which lack a deoxyribonuclease activity and catalase such an addition [14,15,23]. Its ability to hydrolyse non-base-paired 3’-primer terminal nucleotides also distinguishes it from the DNA polymerase a of mammalian cells which again possesses no such activity but does catalyse synthesis from such a primer [23]. Although the above viral and DNA polymerase p enzymes synthesise DNA in vitro with greater fidelity than predicted from the stability of Watson/Crick base-pairing in DNA, they are less accurate than prokaryotic polymerases or DNA polymerase a [23- 261. Thus although the deoxyribonuclease activity might be important in increasing the fidelity of DNA synthesis, it is possible that DNA polymerase a which lacks this activity but is accurate, has evolved to initially select nucleotides for incorporation into DNA with enhanced fidelity. That such an increased selectivity can occur is illustrated by the studies of Gillin and Nossal with the DNA polymerase from the T4 CB120 antimutator DNA polymerase mutant [13]. Clearly the slow rate at which the U. maydis DNA polymerase synthesises DNA in vitro must enhance its proof-reading efficiency as found also for the latter T4 mutant enzyme, which possesses reduced strand displacement and polymerisation rates compared to the wild-type enzyme and also an increased proof-reading efficiency [12]. It may be significant that when the rate of DNA synthesis directed by heat-denatured DNA was increased by the U. maydis DNA unwinding protein [27,28], a significant reduction in the rate of nucleotide turnover (nucleotide incorporation into DNA followed by hydrolysis) by the associated deoxyribonuclease activity was observed [17,29].
Proof-Reading by a U. maydis DNA Polymerase
Although the association of deoxyribonuclease activities with prokaryotic DNA polymerases has been known for some time, it is only recently that a probable association with eukaryotic polymerases has been reported [17,30-321. Most of these reports have involved enzymes from lower eukaryotes, but recently a new enzyme (DNA polymerase S) has been isolated from mammalian cells and shown to posses deoxyribonuclease activity [33]. It seems possible, therefore, that such proof-reading activities are ubiquitous. So far, only one DNA polymerase has been positively identified in U. maydis. There is some evidence that it is required for chromosome replication [34,35], but a role in genetic repair is not excluded [28]. It is possible to isolate mutator strains of U. maydis and one antimutator is also known [34] (and R. Holliday, personal communication). If the change in mutation rate can be directly related to a change in the proofreading ability of the DNA polymerase, this will not only show that the enzyme does replicate chromosomes, but also that the mutation frequency in a eukaryote is in part controlled by the exonuclease function of the enzyme. We wish to thank Dr R. Holliday, F.R.S. for valuable discussion and Dr I. R. Johnston for a generous gift of terminal transferase.
REFERENCES 1. Speyer, J. F., Karam, J. D. & Lenny, A. B. (1966) Cold Spring Harbor Symp. Quant. Biol. 31, 693 - 697. 2. Drake, J. W. & Allen, E. F. (1968) Cold Spring Harbor Symp. Quant. Biol. 33, 339 - 344. 3. Hall, Z. W. & Lehman, I. R. (1968) J . Mol. Bid. 36, 321 - 333. 4. Brutlag, D. & Kornberg, A. (1972) J . Biol. Chem. 247, 241248. 5. Hershfield, M. S. & Nossal, N. G. (1972) J . Biol. Chem. 247, 3393 - 3404. 6 . Muzyczka, N., Poland, R. L. & Bessman, M. J. (1972) J . Biol. Chem. 247,7116-7122. 7. Hershfield, M. S. (1973) J . Biol. Chem. 248, 1417- 1423. 8. Bessman, M. J., Muzyczka, N., Goodman, M. F. & Schnaar, R. L. (1974) J . Mol. Biol. 88, 409-422. 9. Goodman, M. F., Gore, W. C., Muzyczka, N. & Bessman, M. J. (1974) J. Mol. Biol. 88, 423-435. 10. Lo, K.-J. & Bessman, M. J. (1976) J . Biol. Chem. 251, 24752479. 11. Lo, K.-J. & Bessman, M. J. (1976) J . Biol. Chem. 251, 24802486. 12. Gillen, F. D. & Nossal, N. G. (1976) J . Bid. Chem. 251, 52195224. 13. Gillen, F. D. & Nossal, N. G. (1976) J . Bid. Chem. 251, 52255232. 14. Chang, L. M. S. & Bollum, F. J. (1973) J . Bid. Chem. 248, 3398 - 3404. 15. Battula, N. & Loeb, L. A. (1976) J . Biol. Chem. 251, 982-986. 16. Banks, G. R., Holloman, W. K., Kairis, M. V., Spanos, A. & Yarranton, G. T. (1976) Eur. J. Biochem. 62, 131- 142. 17. Banks, G. R. & Yarranton, G. T. (1976) Eur. J . Biochem. 62, 143-150. 18. Kelly, R. B., Cozareili, N. R., Deutscher, M. R., Lehman, I. R. & Kornberg, A. (1973) J . Bid. Chem. 245, 39-45.
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G. T. Yarranton and G . R. Banks 19. Hayes, F. M., Mitchell, V. E., Ratliff, R. L. & Williams, D. L. (1967) Biochemistry, 6,2488 - 2492. 20. Josse, J., Kaiser, A. D. & Kornberg, A. (1961) J. Biol. Chem. 236, 864 - 890. 21. Wu, R. & Kaiser, A. D. (1968) J . Mol. Biol. 35, 523-537. 22. Cassani, G. R. & Bollum, F. J. (1969) Biochemistry, 8, 39283936. 23. Chang, L. M. S. (1973) J . Biol. Chem. 248, 6983-6992. 24. Springgate, C. F. & Loeb, L. A. (1973) Proc. Natl Acad. Sci. U.S.A. 70,245-249. 25. Battula, N. & Loeb, L. A. (1974) J. Biol. Chem. 249, 40864093. 26. Battula, N. & Loeb, L. A. (1975) J . Biol. Chem. 250, 44054409.
27. Banks, G. R. & Spanos, A. (1975) J . Mol. Biol. 93, 63-77. 28. Yarranton, G. T., Moore, P. D. & Spanos, A. (1976) Mol. Gen. Genet. 145, 215-218. 29. Yarranton, G . T. (1976) Ph.D. Thesis, C.N.A.A., London. 30. Helfman, W. B. (1973) Eur. J. Biochem. 32, 42-50. 31. Crerar, M. & Pearlman, R. E. (1974) J . Biol. Chem. 249, 3123-3131. 32. McLennan, A. G. & Keir, H. M. (1975) Biochem. J . 151,239247. 33. Byrnes, J. J., Downey, K. M., Black, V. L. & So, A. G. (1976) Biochemistry, 15,2817-2823. 34. Jeggo, P. A. & Banks, G. R. (1975) Mol. Gen. Genet. 142, 209 - 224. 35. Unrau, P . (1977) Mol. Gen. Genet. 150, 13-19.
G. T. Yarranton, Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts, U.S.A. 02139 G. R. Banks, M.R.C. National Institute for Medical Research, The Ridgeway, Mill Hill, London, Great Britain, NW7 IAA
A DNA Polymerase from Ustilago maydis Evidence of Proof-Reading by the Associated 3' +. 5' Deoxyribonuclease Activity Geoffrey T. YARRANTON and Geoffrey R. BANKS National Institute for Medical Research, London (Received February 7,1977)
The 3' + 5' deoxyribonuclease activity associated with an Ustilago maydis DNA polymerase hydrolysed non-complementary 3'-primer termini about 12 times more rapidly than complementary termini. An analysis of its substrate specificity suggested that, although it was unable to hydrolyse fully single-stranded polynucleotides, it could hydrolyse such regions less than about four nucleotides in length covalently bound to a primer molecule which was base-paired to a complementary template strand. Template-primer combinations containing complementary or non-complementary primer termini both supported polynucleotide synthesis, but whereas the former were conserved, the latter were hydrolysed during the reaction thus allowing synthesis to occur. No addition of nucleotides onto a conserved non-complementary 3'-primer terminus was detected. The deoxyribonuclease activity therefore fulfilled a proof-reading function during DNA synthesis in vitro. Biochemical and genetical studies in prokaryotes have elegantly demonstrated the central role played by DNA polymerases in determining the fidelity of DNA replication [l - 131. Those studies provide evidence that the 3' 3 5' deoxyribonuclease activity associated with these DNA polymerases is able to excise a 3'-terminal primer deoxyribonucleotide which is not complementary to the template polydeoxyribonucleotide strand and also probably those misincorporated into newly synthesized DNA by the polymerase activity in vitro. Such reactions occurring in vivo would provide a means of controlling and maintaining the fidelity of DNA replication. Purified eukaryotic DNA polymerases do not, in general, possess this associated deoxyribonuclease activity [14,15], but, as reported previously [16,17], a polymerase purified from the eukaryotic organism Ustilago maydis does so. The evidence presented in this paper strongly suggests that the associated deoxyriboAbbreviations. [5'-"P](dT)m - [3H](dN)a denotes poly(thymidylic acid) containing an average of 320 dTMP nucleotides, labelled at its 5'-terminus with a ["P]phosphate group and at its 3'-terminus with an average of x residues of another [3H]deoxyribonucleotide dNMP. This and other abbreviations follow CBN rules, see Eur. J . Biochem. 15,203-208 (1970). Enzymes. DNA polymerase or deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase (EC 2.7.7.7); terminal transferase or deoxynucleosidetriphosphate :oligodeoxynucleotide deoxynucleotidyltransferase (EC 2.7.7.31); bovine spleen phosphodiesterase or orhtophosphoric diester phosphohydrolase (EC 3.1.4.1); micrococcal nuclease or nucleate 3'-oligonucleotidohydrolase(EC 3.1.4.7).
nuclease activity, can perform a proof-reading function in vitro. MATERIALS AND METHODS Materials Deoxyribonucleoside triphosphates, polydeoxyribonucleotides and oligodeoxyribonucleotides were from PL Biochemicals Inc., radiochemicals from the Radiochemical Centre (Amersham), Sephadex G-100 from Pharmacia Fine Chemicals and poly(ethy1eneimine)-cellulose thin-layer chromatography sheets (Macherey-Nagel Polygram CEL 300 PEI 20 x 20 cm) from CamLab. Bovine spleen phosphodiesterase and micrococcal nuclease were from the Sigma (London) Chemical Co. and terminal transferase was a gift from Dr I. R. Johnson. U. maydis DNA polymerase (fraction V) was prepared by the method of Banks et al. [16]. Labelled Polydeoxyribonucleotides Synthesis. [5'-32P](dT), was prepared by the method of Kelly et al. 1181 except that [5'-32P](dT)~o was used as the primer (161. The reaction mixture 2.5 mM dTTP, contained: 0.08 mM [S'-32P](dT)~~, 0.2 M potassium cacodylate pH 7.0, 1 mM 2-mercaptoethanol, 1 mM cobalt chloride, 0.5 mg/ml bovine serum albumin and 20 pg/ml terminal transferase. It was incubated at 37 "C for 7 h and terminated by incubation in a boiling water bath for 2 min. The
522
Proof-Reading by a U . maydis DNA Polymerase
product was purified by Sephadex G-100 chromatography. Deoxyribonucleotides were added to the 3’terminus of [5‘-32P](dT)m by terminal transferase with [3H]deoxyribonucleoside triphosphates [4]. Polymers synthesised were : E ~ ’ - ~ ~ PI ([ ~ 3 ~T1 )( d~ ~ ) m , [ 5 ‘ - 3 2 ~ i ( d ~ )m[ 3 ~ i ( d ~ ) 3 m ,
Table 1 . Distribution of the 3‘-terminal nucleotides on synthetic polvdeoxyribonucleotides The theoretical distributions were calculated by assuming that addition of nucleotides onto primers by the terminal transferase follows a Poisson distribution [19]. The observed values were determined from the deoxyribonuclease degradation products as described in Materials and Methods Pol ynucleotide
theoretical
[5’-32P](dT)m - [3H](dC),B$and [5’-32P](dT),
-
observed
[3H](dC)n .
Composition. The addition of nucleotides to oligodeoxyribonucleotide primers by terminal transferase follows a Poisson distribution [19]. The above labelled polydeoxyribonucleotides were degraded by the sequential action of micrococcal nuclease and bovine spleen phosphodiesterase [20,21] and the products separated by thin-layer chromatography on poly(ethy1eneimine)-cellulose sheets eluted with water. Internal chain deoxyribonucleotides were converted to their deoxyribonucleoside 3‘-monophosphates and the 3‘-terminal ones to their deoxyribonucleosides. Table 1 shows the experimentally determined compositions, which are in good agreement with those predicted by the Poisson distribution. Chain Length. The number-average chain length of the [5’-32P](dT)3, was determined from the specific activity of the 5’-phosphate label and the deoxyribonucleotide concentration measured spectrophotometrically. Enzyme Assays
3H label at 3’-terminus
nucleotides and inorganic pyrophosphate. Reaction mixtures were incubated at 37 “C for 30 rnin and terminated in a boiling water bath for 2 min. Aliquots (0.05 ml) of the mixture were applied to the origin of Whatman 3MM chromatography paper which was eluted overnight with isobutyric acid/concentrated NH40H/water (66/1/33). After drying, the chromatogram was cut longitudinally into 1-cm strips and radioactivity determined in each. Nearest-Neighbour Base Analysis DNA polymerase reactions were carried out as described above with [ce3’P]dTTP as the labelled substrate. After incubation at 37 “C for 30 min, the product was precipitated with 10 % trichloroacetic acid and pelleted by centrifugation at 7000 rev./min for 15 min. The supernatant solution was removed and the pellet washed three times with ether. After redissolving the DNA, it was digested by micrococcal nuclease and then bovine spleen phosphodiesterase [20,21]. The resulting mixture, along with appropriate markers was resolved by chromatography on Whatman 3MM paper, eluting overnight with isobutyric acid/concentrated NH40H/O. 1 M EDTA/water (100/2.3/1.6/50), which separated all four 3‘deoxyribonucleoside monophosphates. Radioactivity on the chromatogram was determined as above.
The rates of hydrolysis of the 3’4abelled polydeoxyribonucleotides were measured essentially as described by Brutlag and Kornberg [4]. Reaction mixtures (0.15 ml total volume) contained 1.5 nmol [5’32P](dT)m - [3H](dN),, 1.5 nmol poly(dA) where relevant, 50 mM Tris-HC1 pH 7.5, 10 mM MgCL, 17 mM dithiothreitol and 60 mM KCl. Aliquots (0.02 ml) were removed at specific time intervals of incubation and absorbed onto DEAE-cellulose chromatography paper cut into strips 1.5-cm wide and marked longitudinally into 1.5-cm segments. They were washed three times by gentle agitation in cold 0.3 M ammonium formate and once in cold 95% RESULTS ethanol. After air drying, the strips were cut into 1.5 x 1.5-cm squares and radioactivity determined in Substrate Specificity toluene-based scintillant. Polymerisation reactions were monitored in a similar manner using [ C I - ~ ~ P I - It was reported previously that the 3’ -, 5’ deoxyribonuclease activity associated with the U. maydis deoxyribonucleoside triphosphate substrates. DNA polymerase digested single-stranded DNA about Pyrophosphorolysis was measured using the above 50% as efficiently as double-stranded DNA [17]. reaction conditions, except that 3.3 mM tetrasodium This result was obtained with 3’-terminally labelled pyrophosphate was also present, and determined the native and heat-denatured activated calf thymus formation of labelled deoxyribonucleoside triphosDNA substrates, both of which contain doublephates from the labelled primer terminal deoxyribo-
523
G. T. Yarranton and G. R. Banks
0
5
10 Time
A
A
I
15 (min)
20
25
-0
4
8
0
4
8
Time (min)
Fig. 1. Substrute specijicity of the 3’ + 5‘ deoxyribonuclease activity. Standard reaction mixtures contained [5‘-3zP](dT)m - [’H](dT), (0--0) or poly(dA) . [5’-3’P](dT), - rH](dT)= (---O) and 1G units of DNA polymerase
Fig. 2. Time course of complementary und non-complementary primer terminus hydrolysis. Standard reaction mixtures contained 1G units of DNA polymerase and (a) poly(dA) . [5’-32P](dT)jx- [3H](dT),, (O--O), poly(dA) . [5’-3zP](dT)m - [3H](dA)m (0-0) or poly(dA) . [5‘-3’P](dT)32,-- [3H](dC)m ( L O ) incubated at 37 “C and (b) poly(dA) . [5’-3ZP](dT)32T - [3H](dT),i (0-- -0) or poly(dA) . [5’-32P](dT)3T6- [3H](dA),,j (0--0) incubated at 25 “C
stranded regions, thereby making an interpretation of the DNA specificity questionable. The homopolymer [5’-3zP](dT)E, - [3H](dT)il-i, alone and in combination with poly(dA), was therefore used to determine this specificity more rigorously. No degradation of the single-stranded homopolymer was detected after incubation at 37 “C for 40 min (Fig. 1). In the double-stranded structure poly(dA) . [5’-32P](dT), - [3H](dT),, however, only 2- 3 of the terminal [3H]dTMP remained in polymeric form after a 10-min incubation. This result strongly suggests that the deoxyribonuclease activity is specific for double-stranded DNA.
such as poly(dA) . (dT),, are unstable at temperatures around 37 “C, resulting in partial denaturation or fraying of the two strands at their termini. Complete hydrogen bonding can be approached, however, at lower temperatures [22]. The rates of terminal [3H]dTMP and [3H]dAMP hydrolyses were therefore, determined at 25 “C and 37 “C (Fig.2A, B). The rate for the complementary [3H]dTMP was reduced 11fold from 0.22 at 37 “C to 0.02 pmol/min at 25 “C, whilst that for [3H]dAMP was reduced only 3-fold from 0.78-0.24 pmol/min. This suggests that the deoxyribonuclease activity recognises and hydrolyses the non-base-paired primer termini much more efficiently.
Hydrolysis of Complementary and Non-Complementary Primer Termini
Using the experimentally determined values for the average number of labelled deoxyribonucleotides at the 3’-terminus of each primer chain (Table l), a comparison of their rates of hydrolysis by the 3‘ + 5’ deoxyribonuclease was made. The non-complementary, to the poly(dA) template strand, [3H]dAMP in poly(dA) . [5’-32P](dTh- [3H](dA)m was hydrolyzed about 4-fold more rapidly than the complementary [3H]dTMP termini in poly(dA) . [5’-32P](dT)m - [3H](dT),. This increased rate did not reflect a specificity for a purine rather than a pyrimidine nucleotide because the [3H]dCMP termini in poly(dA) . [5’-32P](dT)m - C3H](dC>,, were hydrolysed at almost the same rate as the [3H]dAMP (Fig.2A). The effect of primer terminus secondary structure on their hydrolysis rates was studied in more detail by varying the reaction temperature. Homopolymer pairs
Polynucleo tide Synthesis and Primer Termini Hydrolysis
As previously reported, DNA synthesis protected the primer from hydrolysis by the 3’ .+ 5‘ deoxyribonuclease activity [17].When poly(dA) . [5’-32P](dT), - [3H](dT), was incubated with the DNA polymerase in the presence of [ U - ~ ~ P I ~ T[32P]poly(dT) TP, was synthesised accompanied by an initial hydrolysis of about 10% of the [3H]dTMP in primer termini, but no further hydrolysis (Fig. 3 A). Protection of these termini complementary to the template strand required polydeoxyribonucleotide synthesis rather than nonspecific triphosphate binding to the enzyme, because only dTTP and dUTP afforded protection (Table 2). dATP, dGTP or dCTP, which are not complementary to the poly(dA) template and could not be polymerised [16], did not protect the base-paired [3H]dTMP termini from hydrolysis.
524
Proof-Reading by a U. maydis DNA Polymerase
Table 2. The influence of deoxyrihonuclease triphosphates on primer terminus hydrolysis Standard reaction mixtures contained a single dNTP as indicated to a final concentration of 13 3 WM+ indicates enzyme-catalysed conversion of 3H label in the polydeoxyribonucleotides to dNMP - Indicates no such conversion detected Polynucleotide
Poly(dA) [S’-32P](dT),
dNTP present -
[3H](dT)ii
Poly(dA) . [S’-32P](dT)m - [3H](dA)T,-i
none dTTP ordUTP dATP, dGTP ordCTP none dTTP ordUTP dATP, dGTP ordCTP
3‘-Terminal hydrolysis
+ -
+
+ +
+
0
0
5
15
10
20
Time (min)
Fig. 4. Polynucleotide synthesis and primer terminus hydrolysis. Standard reaction mixtures were as in the legend to Fig.3 but with the template-primer poly(dA) . [5’-3’P](dTh - [3H](dC),,. (--O) [32P]Poly(dT)synthesis; (-0) [3H]dCMP hydrolysis. Hydrolysis in the absence of dTTP was identical to that shown in its presence
. [5’-32P](dT)m - [3H](dA)m (Fig.4). Also only about 10 % of the non-complementary [3H]dCMP nucleotides were susceptible to hydrolysis in the absence of dTTP. These observations suggest that the [3H]dCMP non-complementary terminal nucleotides of only a small fraction of primer molecules were susceptible to hydrolysis. Assuming that terminal transferase added deoxyribonucleotides during the preparation of these primer molecules in agreement with a Poisson distribution [19], Eqn (1) gives an estimation of that fraction of the total population of primer molecules which possess any given number of added [3HIdCMP molecules. Time (min)
Fig. 3 Polynucleotide synthesis and primer terminus hydrolysis. Standard reaction mixtures contained 10 units of DNA polymerase. 13 3 pM [e3’P]dTTP (320 counts min-’ pmol-’) and (a) poly(dA) . [5’-32P](dT)m - [3H](dT)m or (b) poly(dA) [S‘-32P](dT)5[3H](dA)m (w) [32P]poly(dT) synthesis, (-0) [3H]dTMP (a) or [3H]dAMP (b) hydrolysis
Incontrast, when poly(dA). [5’-32P](dT)m- [3H](dA)m was incubated with the DNA polymerase and [u-~’P]~TTP, complete removal of the non-basepaired [3H]dAMP was observed as polymerisation proceeded (Fig. 3B and Table 2), suggesting that prior removal of non-complementary termini was required before polynucleotide synthesis onto the primer could occur. Similar observations were made with poly(dA) . [5’-32P](dTh- [3H](dC)m (results not shown). The rate and extent of [32P]poly(dT)synthesiswhen - [’H](dC), was incubatpoly(dA) . [5’-32P](dT)-jZ, ed with the enzyme and [u-~’P]~TTP was only 10% of that observed with the template-primer poly(dA)
X !
where A = mean number of added deoxyribonucleotides, x = number of deoxyribonucleotides added per chain under consideration and P = mole fraction of total population with x deoxynucleotides added. Using Eqn (l), it can be calculated that about 10 “4 of the total [3H]dCMP in poly(dA) . [5’-32PJ(dT), - [’H](dC),, is contained in primers with 1-3 [3H]dCMP nucleotides and that this class represents about 10% of the total primer population. We suggest that it is also this class which is susceptibleto the 3‘ .+ 5’ deoxyribonuclease activity and that after removal of these [3H]dCMP non-complementary deoxyribonucleotides, primed poly(dT) synthesis can occur. Nearest-Neighbour Base Analysis of DNA Synthesis Products
To verify that polydeoxyribonucleotide synthesis required a base-paired 3’-primer terminus and cannot
525
G. T. Yarranton and G. R. Banks Table 3. Nearest-neighbour base analyses of D N A polymerase reaction products Standard reaction mixtures contained the template-primers indicated, 10 units of U. rnaydis DNA polymerase and [CG’~P]~TTP (13.3 pM, 2 x lo3 counts min-’ pmol-’). The products were subjected to nearest-neighbour base analyses as described in Materials and Methods Template-primer
Analysis product
Amount of product detected
Table 4. Primer terminus specificity of the pyrophosphorolysis reaction The reaction mixture and conditions of assay are described in Materials and Methods. + and - denote the presence and absence respectively of 3.3 mM tetrasodium pyrophosphate in the reaction mixture Template-primer
PyroProduct as phosphate [’HIdNMP [’HJdNTP
% total label present
pmol Poly(dA) . [5’-32P](dT), - [’H](dT)n
Poly(dA) . [5’-32P](dT)m- [’H](dA)W
3’-dTMP 131 0.07 3‘-dAMP 0.06 3‘-dCMP
Poly(dA) . [5’-32P](dT), - [3~1(d~),
3’-dTMP 130 3‘-dAMP 0.08 0.05 3’-dCMP
Poly(dA) . [5‘-’’P](dT)m - [3~1(d~),,
Poly(dA) . [5‘-’’Pl(dT)m - [’H](dC)m
3’-dTMP 126 0.06 3’-dAMP 0.05 3’-dCMP
Poly(dA) . [5‘-32P](dT),
3’-dTMP 3’-dAMP 3’-dCMP
-
[3H](dC)n
20 0.G5 0.04
occur from a non-paired one, the products of polymerisation reactions directed by the template-primers were subjected to nearest-neighbour base analysis (Table 3). The analysis was sensitive enought to detect single deoxyribonucleotide additions to 1% of the primer termini. No addition onto any non-basepaired 3’-primer termini was detected. These results also again suggest that hydrolysis of these terminal deoxyribonucleotides must occur before poly(dT) synthesis can occur.
The 3 ‘-Primer Terminus Specijkity of Pyrophosphorolysis Pyrophosphorolysis is the direct reversal of the polymerisation reaction involving the formation of deoxyribonucleoside triphosphate from DNA and inorganic pyrophosphate. Its primer-template specificities should, therefore follow those of the polymerisation reaction [4]. When poly(dA) . [5’-32P](dT)m - [3H](dT)i-i- was incubated with the DNA polymerase at 37 “C for 30 min the only detectable 3H label was in dTMP (Table 4). When inorganic pyrophosphate was also present in the reaction mixture, 53% and 47% of the 3H label was in dTMP and dTTP - [3H]respectively. With poly(dA) . [5’-”2P](dT), (dA),, all the 3H label was in dAMP both in the absence and presence of pyrophosphate. Hence pyrophosphorolysis activity of the DNA polymerase resembled the polymerisation reaction in its specificity for a base-paired 3‘-primer terminus.
-
+ -
+
100
< 0.01
41
53
99.5 100
0.5 < 0.01
DISCUSSION The experiments described above suggest that the 3‘ + 5‘ deoxyribonuclease activity associated with the U. maydis DNA polymerase is able to proof-read in vitro. It hydrolysed noncomplementary terminal primer nucleotides faster than complementary ones, but the specificity decreased when the latter were destabilized by heat (Fig. 2). Whereas primers with 3’-terminal nucleotides complementary or non-complementary to the template directed polynucleotide synthesis by the DNA polymerase, only the former termini were conserved (Fig. 3). We conclude that the enzyme is unable to elongate from a non-complementary primer terminus prior to its removal and restoration of base-pairing, an interpretation in accord with the product nearest-neighbour base analyses (Table 3) and pyrophosphorolysis specificity (Table 4). The specificity of the deoxyribonuclease activity for a non-base-paired 3‘-terminus is apparently in conflict with its inability to hydrolyse a single-stranded homopolymer (Fig. 1). This contradiction may be explained by the experiments reported with poly(dA) . [5’-32P](dT)m - [3H](dC),, which comprised primer molecules with 0 to 10 non-complementary nucleotides (Fig.4). Only about 10% of the total [3H]dCMP was hydrolysed, a figure which agrees well with that predicted for primer chains having 1 - 3 [’HIdCMP nucleotides per primer molecule. The rate of poly(dT) synthesis directed by this template-primer combination was also only 10% of that observed with poly(dA) . [5’-32P](dT)& - [3H](dA)m, in which over- 90 % of the [3H]dAMP nucleotides were hydrolysed. Since the enzyme was in molar excess over primer termini, the rate of poly(dT) synthesis was proportional to the number of available primer sites. This suggests therefore, that only 10% of these primer molecules was
526
available for elongation, probably those with 1 - 3 [3H]dCMP nucleotides which first could be removed by the deoxyribonuclease activity. We suggest that the U . maydis DNA polymerase is able to bind to doublestranded DNA (probably at a nick or gap in one strand) but not to fully single-stranded DNA. When this binding region includes less than about 4 non-base-paired nucleotides at a 3’-terminus in one strand, the 3‘nucleotide is stereochemically in alignment with the active deoxyribonuclease site and exonucleolytic hydrolysis of the unpaired nucleotides can occur. If, however, this region is longer than 3 nucleotides, the terminal one is not in such a position and hydrolysis cannot occur. The properties of the U. maydis DNA polymerase described in this paper are, with the exception of its DNA secondary structure specificity, similar to those of the bacteriophage T4 DNA polymerase and the DNA polymerases I and I1 of Escherichiu coli [4]. Our inability to detect addition of deoxyribonucleotides directly onto a non-base-paired primer terminus distinguishes it from the DNA polymerase p of mammalian cells and the avian myeloblastosis virus enzyme, both of which lack a deoxyribonuclease activity and catalase such an addition [14,15,23]. Its ability to hydrolyse non-base-paired 3’-primer terminal nucleotides also distinguishes it from the DNA polymerase a of mammalian cells which again possesses no such activity but does catalyse synthesis from such a primer [23]. Although the above viral and DNA polymerase p enzymes synthesise DNA in vitro with greater fidelity than predicted from the stability of Watson/Crick base-pairing in DNA, they are less accurate than prokaryotic polymerases or DNA polymerase a [23- 261. Thus although the deoxyribonuclease activity might be important in increasing the fidelity of DNA synthesis, it is possible that DNA polymerase a which lacks this activity but is accurate, has evolved to initially select nucleotides for incorporation into DNA with enhanced fidelity. That such an increased selectivity can occur is illustrated by the studies of Gillin and Nossal with the DNA polymerase from the T4 CB120 antimutator DNA polymerase mutant [13]. Clearly the slow rate at which the U. maydis DNA polymerase synthesises DNA in vitro must enhance its proof-reading efficiency as found also for the latter T4 mutant enzyme, which possesses reduced strand displacement and polymerisation rates compared to the wild-type enzyme and also an increased proof-reading efficiency [12]. It may be significant that when the rate of DNA synthesis directed by heat-denatured DNA was increased by the U. maydis DNA unwinding protein [27,28], a significant reduction in the rate of nucleotide turnover (nucleotide incorporation into DNA followed by hydrolysis) by the associated deoxyribonuclease activity was observed [17,29].
Proof-Reading by a U. maydis DNA Polymerase
Although the association of deoxyribonuclease activities with prokaryotic DNA polymerases has been known for some time, it is only recently that a probable association with eukaryotic polymerases has been reported [17,30-321. Most of these reports have involved enzymes from lower eukaryotes, but recently a new enzyme (DNA polymerase S) has been isolated from mammalian cells and shown to posses deoxyribonuclease activity [33]. It seems possible, therefore, that such proof-reading activities are ubiquitous. So far, only one DNA polymerase has been positively identified in U. maydis. There is some evidence that it is required for chromosome replication [34,35], but a role in genetic repair is not excluded [28]. It is possible to isolate mutator strains of U. maydis and one antimutator is also known [34] (and R. Holliday, personal communication). If the change in mutation rate can be directly related to a change in the proofreading ability of the DNA polymerase, this will not only show that the enzyme does replicate chromosomes, but also that the mutation frequency in a eukaryote is in part controlled by the exonuclease function of the enzyme. We wish to thank Dr R. Holliday, F.R.S. for valuable discussion and Dr I. R. Johnston for a generous gift of terminal transferase.
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G. T. Yarranton and G . R. Banks 19. Hayes, F. M., Mitchell, V. E., Ratliff, R. L. & Williams, D. L. (1967) Biochemistry, 6,2488 - 2492. 20. Josse, J., Kaiser, A. D. & Kornberg, A. (1961) J. Biol. Chem. 236, 864 - 890. 21. Wu, R. & Kaiser, A. D. (1968) J . Mol. Biol. 35, 523-537. 22. Cassani, G. R. & Bollum, F. J. (1969) Biochemistry, 8, 39283936. 23. Chang, L. M. S. (1973) J . Biol. Chem. 248, 6983-6992. 24. Springgate, C. F. & Loeb, L. A. (1973) Proc. Natl Acad. Sci. U.S.A. 70,245-249. 25. Battula, N. & Loeb, L. A. (1974) J. Biol. Chem. 249, 40864093. 26. Battula, N. & Loeb, L. A. (1975) J . Biol. Chem. 250, 44054409.
27. Banks, G. R. & Spanos, A. (1975) J . Mol. Biol. 93, 63-77. 28. Yarranton, G. T., Moore, P. D. & Spanos, A. (1976) Mol. Gen. Genet. 145, 215-218. 29. Yarranton, G . T. (1976) Ph.D. Thesis, C.N.A.A., London. 30. Helfman, W. B. (1973) Eur. J. Biochem. 32, 42-50. 31. Crerar, M. & Pearlman, R. E. (1974) J . Biol. Chem. 249, 3123-3131. 32. McLennan, A. G. & Keir, H. M. (1975) Biochem. J . 151,239247. 33. Byrnes, J. J., Downey, K. M., Black, V. L. & So, A. G. (1976) Biochemistry, 15,2817-2823. 34. Jeggo, P. A. & Banks, G. R. (1975) Mol. Gen. Genet. 142, 209 - 224. 35. Unrau, P . (1977) Mol. Gen. Genet. 150, 13-19.
G. T. Yarranton, Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts, U.S.A. 02139 G. R. Banks, M.R.C. National Institute for Medical Research, The Ridgeway, Mill Hill, London, Great Britain, NW7 IAA
