Deoxyribonucleoside Triphosphate Stimulation of Exonucleolytic Activity of the Micrococcus Zuteus Deoxyribonucleic Acid Polymerase*

SUMMARY The rate of 5’ to 3’ exonucleolytic degradation by the Micrococcus lufeus DNA polymerase is stimulated by deoxyribonucleoside triphosphates. A single deoxynucleoside triphosphate can stimulate 6-fold the rate of degradation of an alternating sequence DNA polymer. It is neither necessary nor sufficient that the deoxyribonucleoside triphosphate be complementary to the bases of the DNA polymer in order to stimulate the rate of exonucleolytic degradation of that polymer. The deoxyribonucleoside triphosphate is not chemically altered due to its role as a stimulator. The Micrococcus Zuteus DNA polymerase has a low level of exonucleolytic activity which gives a high percentage of dinu-aleotides as products (1). This activity is part of the polymerase protein of certain nucleoside triphosphates the rate of exonucleolytic

to the bases of the DNA polymer in order to stimulate the rate of exonucleolytic degradation of that polymer.
The deoxyribonucleoside triphosphate is not chemically altered due to its role as a stimulator.
The Micrococcus Zuteus DNA polymerase has a low level of exonucleolytic activity which gives a high percentage of dinualeotides as products (1). This activity is part of the polymerase protein (2, 3). The presence of certain nucleoside triphosphates markedly stimulates the rate of exonucleolytic degradation (1,2,4,5). A study of this behavior was performed in order to better understand the mechanism of DNA synthesis.
Comparative properties of the &I. Zuteus DNA polymerase and the Escherichia coli DNA polymerase are discussed.
EXPERIMENTAL PROCEDURE GeneraGMany of the materials and methods are described in the preceding paper (3). They include the following: nucleotides, nucleic acids, M. Zuteus DNA polymerase, nuclease reactions, determinations of the products of nucleolytic degradation, and polyacrylamide gel electrophoresis. dADP was purchased from Sigma and was purified by column chromatography with DEAE-cellulose in the carbonate form (gift of. R. W. Sweet).
Each slice was placed in a test tube containing 0.05 ml of the following reaction mixture: 50 mM Tris-HCl, pH 7.8; 5 mM MgCL, 0.5 ItXM 2-mercaptoethanol, and 30 PM [r4C]dATP (specific activity 4.5 X 104 cpm per nmole). The gel slices, immersed in this reaction mixture, were incubated for 18 hours at 37". Following incubation, 0.02-ml aliquots were withdrawn from the reaction mixture and added to 0.015 ml of marker solution (5 PM dATP, 5 PM dADP, and 0.10 M EDTA).
This mixture was spotted on Whatman No. 1 chromatography paper and the products were resolved by descending chromatography in Solvent A (3). The entire length of all chromatograms was scanned with a Packard radiochromatogram scanner.

Stimulation of Rate of ExonucZeoZytic Degradation by Single Deoxyribonucleotide Triphosphate
The rate of exonucleolytic degradation of certain doublestranded DNA polymers is increased several fold by the addition of one deoxyribonucleoside triphosphate. PoZy(dA-dT) .poly(dA-dT)-Stimulation of the rate of exonucleolytic degradation of poly(dA-dT) .poly(dA-dT) by the addition of various deoxyribonucleoside triphosphates is shown in Fig. 1. In the absence of triphosphates, degradation proceeds at a slow linear rate. Addition of dATP alone results in a 6-fold increase in the initial rate of degradation.
Addition of dTTP alone increases the rate of degradation 2-fold. The addition of both dATP and dTTP results in a rate of degradation slightly less than the rate observed for dATP added alone. Under these conditions, however, the deoxyribonucleoside triphosphates serve as substrates for polymerization, resulting in a decreasing concentration of triphosphate stimulators and an increasing concentration of unlabeled poly(dA-dT) . poly(dA-dT).
Hence the B-fold increase in the rate of degradation observed with the addition of dATP alone is obscured by the synthetic reaction when dATP and dTTP are added simultaneously. Fig. 1 also shows that the addition of dCTP, a nucleotide that is not complementary to either of the bases of the DNA substrate, stimulates the rate of nucleolytic degradation 5-fold. nucleotides. Each of the two polymers described above has self complementary and identical sequences in both strands. Fig. 3 shows the rate of exonucleolytic degradation of the poly-(dC-dA) strand of poly(dT-dG) .poly(dC-dA). Addition of dATP stimulates the rate of exonucleolytic degradation a-fold above the rate observed in the absence of deoxyribonucleoside triphosphates.
Addition of dTTP stimulates the rate approximately 2.5-fold.
Addition of dCTP has essentially no effect on the rate. The simultaneous addition of dCTP and dATP shows a slower rate of degradation than the rate observed for dATP alone. However, a synthetic reaction can occur in this case. Also, the simultaneous addition of all four complementary deoxyribonucleoside triphosphates results in an inhibition of the degradation rate. The results obtained with poly(dA-dT) .poly(dA-dT), poly-(dI-dC) .poly(dI-dC), and poly(dT-dG (e) A pyrimidine nucleoside triphosphate stimulates degradation most effectively when it is not contained in the DP\TA polymer strand which is degraded.
(f) Conditions permitting DNA synthesis decrease the rate of nucleolytic degradation observed in the presence of the purine nucleoside triphosphate alone.
Poly(dG-dC) .poly(dG-&)-The effect of deoxyribonucleoside triphorphates on the rate of poly(dG-dC) .poly(dG-dC) degradation is shown in Fig. 4. When tested individually, dATP, dGTP, and dCTP have no effect on the rate of degradation. Simultaneous addition of both dGTP and dCTP results in a decreased rate of degradation as expected for conditions permitting synthesis.
Addition of dITP alone resulted in a small increase in the rate of nucleolytic degradation. However, the simultaneous addition of both dITP and dCTP resulted in a 4-fold increase in the rate of nucleolytic degradation. These conditions permit the rapid synthesis of poly(dI-dC) (7). Poly-(dI-dC) .poly(dI-dC) is degraded 10 times more rapidly than poly(dG-dC) .poly(dG-dC) (compare Figs. 2 and 4). It would be expected that the hybrid polymer poly(dG-dC) .poly(dI-dC) would be degraded at a rate intermediate to these two cases. Hence the stimulation of poly(dG-dC) .poly(dG-dC) degradat*ion by addition of dITP and dCTP may be a result of the formation of poly(dG-dC) .poly(dI-dC) . With this unique exception, the stimulation observed with the poly(dG-dC) . poly (dG-dC) conforms with the other alternating base sequence polymers. In general, however, poly (dG-dC) .poly(dG-dC) is much less susceptible to nucleoside triphosphate stimulation and its degradation can be stimulated only by dITP.

Concentration of Deoxyribonucleoside
Triphosphate for

Stimulation of Exonucleolytic Degradation
The effect of the concentration of dATP on the amount of stimulation of nucleolytic activity is shown in Fig. 5a. The relative increase in the rate of nucleolytic degradation of both poly(dA-dT) .poly(dA-dT) and poly(dI-dC) .poly(dI-dC) was measured.
The "fold" stimulation is calculated by dividing the nanomoles of DNA substrate degraded in the presence of dATP by the nanomoles of DNA substrate degraded in the absence of dATP.
For both poly(dA-dT) .poly(dA-dT) and poly(dI-dC) . poly(dI-dC), the amount of stimulation observed increases with an increasing concentration of dATP. Maximal stimulation of the nucleolytic degradation rate is achieved at 200 JAM dATP and concentrations higher than 200 PM have essentially no further effect on the amount of stimuliition.
A slightly different pattern was observed for several other deoxynucleoside triphosphates.
In Fig. 5b, the amount of stimulation of poly(dA-dT) .poly(dA-dT) degradation by dGTP or by dCTP, when tested individually, is plotted as a function of their concentration.
For these nucleoside triphosphates, the increase in the amount of stimulation continues through the concentration range of 200 to 500 pM.  Fig. 6, the rate of nucleolytic degradation of poly (dI-dC) . poly-(dI-dC) is stimulated 3.5-fold by dTTP when tested alone, and 4.0.fold by dATP when tested alone. Simultaneous addititin of both dATP and dTTP results in the same rate as that observed for dATP alone. The concentration of dATP is saturating under these conditions.
Hence, the effects of dATP and dTTP are not additive.
The same result was obtained with poly(dT-dG) . poly (dC-dA) for these two nucleoside triphosphates (results not shown).
The effect of simultaneous addition of dGTP and dATP on the rate of poly(dA-dT) epoly(dA-dT) degradation is shown in Fig. 7. Although there is a small increment in the rate of degradation above that observed for dATP alone, the effects of dATP and dGTP are not strictly additive.
Thus, the various nucleoside triphosphates act at the same site in stimulating nucleolytic degradation.
Presuming that the triphosphates are bound only to the "triphosphate binding site," this result is consistent with the finding (8) that the E. coli DNA polymerase I has only a single binding site for triphosphates.

Stability of Deoxyribonucleoside Triphosphate
Stimulator-To determine whether deoxyribonucleoside triphosphate was altered chemically as a result of its stimulatory function, [l%]dATP was incubated with the enzyme in the presence and absence of poly-(dA-dT) poly(dA-dT) No breakdown was observed which could be correlated with nuclease reaction.
However, the dATP was converted to dADP by the enzyme preparation at a slow linear rate (6 nmoles per hour) and no other products were formed.
This putative modification of the stimulator is not coupled with nucleolytic degradation since: (a) the same rate is observed in the presence and absence of DNA; (b) the rate of conversion of dATP to dADP is only 200/, of the rate of nucleolytic degradation under similar conditions; (c) gel electrophoresis of the enzyme preparation demonstrated that the enzyme which converts dATP to dADP and the polymerase (nuclease) are different proteins (Fig. 8) 0.12 (3) in this gel electrophoresis system. Assay of t,he gels for nuclease activity in the presence and absence of dATP by the gel slice method showed that the nucleolytic act.ivity was stimulated by dATP when resolved from the dATPase activity (results not shown).
Thus it is concluded that t'he dhTPase activity is a trace contaminating enzyme and that the stimulation of nucleolytic degradation does not require the breakdown of the nucleoside triphosphat,e to the diphosphate form.
It should be noted that the nucleoside diphosphokinase activity which is associated with DNA polymerase (9) does not affect this assay for dATPase activity.
The dATPase assay involves only one nucleoside triphosphate which, in the nucleoside diphosphokinase reaction, would not yield a net increase of triphosphate.
In the cases where a deoxyribonucleoside triphosphate stimulator is complementary to the base sequence of the DNA substrate, it is possible that a limited end addition reaction could occur at the 3'-hydroxyl end of the DNA substrate. During the dATPase assay in which [I%]dATP was incubated with the M. Zuteus DNA polymerase in the presence of poly(dA-dT) . poly (dA-dT) no detectable DNA synthesis occurred ( < 1 nmole per ml).
A very limited DNA synthesis reaction, however, does occur under these conditions but is detected only when radioactive dL4TP of very high specific activity is used> When [a(-32P]dATP (specific activity 1 X 10' cpm per nmole) was incubated with poly(dA-dT) .poly(dA-dT) and the n/r. Zuteus DNA polymerase, a small amount of [32P]dAMP was rapidly incorporated into DNA. The reaction ceased when the amount of dAMP incorporated was equal to the calculated number of 3' termini present in the poly(dA-dT) .poly(dA-dT) sample. This limited 3' end-addition reaction of a single nucleotide is expected for an alternating base sequence polymer when a single complementary nucleoside triphosphate is provided. A limited end-addition reaction has been reported for the E. coli DNA polymerase I with native DNA (10) and is being utilized for DNA sequencing with T-4 DNA polymerase (11).
The role of deoxyribonucleoside triphosphate stimulation is not strictly a result of its role in the limited end-addition reaction since the concentration of deoxynucleoside triphosphate required for maximal stimulation of the exonucleolytic rate is 200 pM (or greater in some cases). This concentration is at least IO-fold higher than the concentration required to saturate the polymerase for the end-addition reaction. Furt,hermore, certain purine deoxyribonucleoside triphosphates, which are not complementary to the DNA substrate, stimulate exonucleolytic degradation.
However, the end-addition reaction might be a clue to determining why a complementary pyrimidine nucleoside triphosphate, which may be incorporated at the 3' terminus, is unable to stimulate nucleolytic degradation. This would require that the nucleotide at the 3' end of a DNA polymer have a significant effect on the 5' to 3' exonuclease activity.

Product Distribution in Presence and Absence of dATP
To determine whether the stimulation of nucleolytic degradation by nucleoside triphosphates affected the proportion of mononucleotides, dinucleotides, and trinucleotides produced, [3H]poly-(dA-dT) .poly (dA-dT) was degraded in the presence and absence of dATP.
Issue of bZay 10, 1972 L. K. Miller and R. D. Wells 2679 Table I shows that the same relative distribution of label is observed in both the absence and presence of dATP. This is true although only 287; of the poly(dA-dT) .poly(dA-dT) substrate was degraded in the absence of dATP, whereas 98% degradation was observed in its presence in the same time period.
Hence, the stimulation of nucleolytic degradation increase5 the rate of formation of all the various types of products.
In the preceding paper (3) it was shown that the majority of exo- Two nuclease reaction mixtures (see "Methods") were incubated in the presence or absence of 200 PM dATP.
The products of degradation were analyzed after 2 hours of incubation at 37" by paper chromatography as described in "Methods." The nanomoles per ml of each product was calculated from the ratio of nonorigin counts per min to origin cpm. nucleolytic activity observed on double-stranded DNA was a result of 5' to 3' exonucleolytic degradation which produced mononucleotides, dinucleotides and trinucleotides. Thus, it is apparent that deoxyribonucleoside triphosphates stimulate the rat'e of 5' to 3' exonucleolytic degradation.

Effect of Deoxyribonucleoside
Triphosphates on PoZy(dA) .poly(dT) Degradation The stimulation of degradation of the poly(dT) strand of poly(dA) .poly(dT) by dATP, dTTP, and a combination of these two deoxynucleoside triphosphates is shown in Fig. 9. dATP alone stimulates the rate of nucleolytic degradation 2-fold. dTTP alone initially stimulated the degradation 3.5-fold but at later times, is inhibitory.
A combination of both dATP and dTTP stimulates the initial rate of degradation 6-fold. Degradation of the DNA substrate is essentially complete within 30 min in this reaction.
A rigorous interpretation of these data is made complex by the fact that polymer synthesis occurs in all these reactions. Hence, although dATP stimulates the rate of nucleolytic degradation 2-fold, it cannot be concluded that this stimulation is similar to that observed with poly(dA-dT) . poly(dA-dT). The dATP may merely be used for the synthesis of poly(dA).
Continual synthesis of poly(dA) would ensure that the poly(dT) strand is in a double stranded form which, as noted previously, is a better substrate for the 5' to 3' exonuclease than single st'randed poly (dT) . Nuclease reactions bated at 37" and at intervals, aliquots were removed and assayed for acid-soluble radioactivity. contained 30 PM poly(dA) annealed to 30 pM 3H-labeled poly (dT) No triphosphate addition ( l ) ; 500 ,uM dATP (0) ; 500 rclM dTTP (A); and 500 pM of both dATP (specific activity 1800 cpm per nmole), triphosphates as indicated, and dTTP (a). and 30 units per ml of M. Zuteus DNA polymerase.
The mixtures were incubated at 37" and at intervals, aliquots were withdrawn The rate of degradation of the poly(dA) strand of poly(dA) . poly (dT) in the presence and absence of deoxynucleoside triphosphates is shown in Fig. 10. dATP, dTTP, and a combination of these two triphosphates do not show a marked stimulation of the initial rate of degradation and inhibit the rate at later times. Again, a rigorous interpretation of these results is difficult since DNA synthesis occurs in all cases.

E$ect of Deoxyribonucleoside
Triphosphates on Rate of M. luteus DNA Degradation The rate of exonucleolytic degradation of native M. luteus DNA and the effect of addition of various deoxyribonucleoside triphosphates is shown in Fig. 11. The addition of dATP alone has no effect. Addition of a mixture of dTTP, dCTP, and dGTP also has no effect. triphosphates. This is in direct contrast to the stimulation observed with poly(dT) annealed to poly(dA) (Fig. 9).
It was shown (3)  For example, dCTP stimulates the rate of poly(dA-dT) .poly(dA-dT) degradation 4-fold but has no effect on the degradation of poly(dI-dC) .poly(dT-dC) or the poly(dC-dA) strand of poly (dT-dG) . poly(dC-dA). However, dTTP stimulates the degradation of the latter two polymers but has essentially no effect on the rate of poly(dA-dT) .poly(dA-dT) degrada-tion. Since a limited end-addition reaction is possible when the deoxyribonucleoside triphosphate is complementary to a base of an alternating base sequence DNA, it would appear that when the 3'-OH terminus of a DNA polymer is a pyrimidine nucleotide and the stimulatory nucleoside triphosphate is also a pyrimidine, no stimulation is observed. However, if the 3'.OH terminal nucleotide is a purine, either pyrimidine or purine nucleoside triphosphates stimulate degradation. The nucleotide stimulator is not chemically altered as a function of its stimulatory role. Substrate concentrations, not catalytic concentrations, of nucleos de triphosphates are necessary for maximum stimulation; the presence of a stimulator does not alter the product distribution.
These facts strongly suggest that the microenvironment at the 3'-OH DNA terminus binding site or the occupation of the triphosphate site (or both) influence the activity of the 5' to 3' exonuclease.
The enzyme may undergo a conformational change on binding triphosphates; other studies (12) suggested that DNA polymerase is subject to conformational changes.
Although our data do not rule out the possibility that DNA synthesis can stimulate 5' to 3' exonucleolytic degradation as suggested for the E. coli DNA polymerase (13), our studies with alternating sequence polymers indicate that DNA synthesis is not required for the stimulation of the rate of 5' to 3' exonucleolytic degradation.
The M. Zuteus DNA polymerase is similar to the E. coli DNA polymerase I in several respects.
Although the exonuclease of the M. luteus DNA polymerase is less than 1 yc of the polymerase activity (I), whereas the exonuclease of the E. coli DNA polymerase is 10% of the polymerase activity (14), both enzymes possess 5' to 3' exonuclease activities which specifically degrade double-stranded DNA. Both enzymes also possess 3' to 5' exonuclease activities which degrade single stranded DNA's to mononucleotides.
The 5' to 3' exonuclease of E. coli DNA polymerase I degrades the poly(dT) strand of poly(dA) .poly(dT) to mononucleotides, dinucleotides, and small oligonucleotides (13). Kadohama and McCarter (15) have reported that the exonucleolytic activity of the E. coli DNA polymerase also degrades poly (dA-dT) .poly(dA-dT) to yield a large proportion of dinucleotides as initial degradation products. These results are similar to the M. luteus enzyme with one exception.
The 3' to 5' exonucleolytic activity of the E. coli DNA polymerase rapidly degrades dinucleotides to mononucleotides (15-17); dinucleotides and short oligonucleotides are resistant to further degradation by the exonuclease of the M. luteus DNA polymerase.
The 5' to 3' exonucleases of both the M. luteus DNA polymerase and the E. coli DNA polymerase are stimulated by deoxyribonucleoside triphosphates.
The rate of degradation of naturally occurring DNA by the E. coli DNA polymerase I is stimulated several fold by the addition of four deoxyribonucleoside triphosphates (13). This is also observed with the M. Zuteus enzyme. Thus, for a natural template perhaps DNA synthesis is required for the triphosphate stimulation. When the 5' to 3' exonuclease is cleaved from E. coli DNA polymerase I by proteolytic treatment (18) and separated from the polymerase fragment, no stimulation is observed unless the polymerase fragment is again added to the exonuclease fragment (19). Our observation that the stimulation of 5' to 3' exonuclease activity by deoxynucleoside triphosphates apparently involves a change in the 3'-OH primer terminus binding site or the triphosphate binding site, explains why the exonuclease