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J. Biol. Chem., Vol. 282, Issue 34, 24689-24696, August 24, 2007

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Increased Catalytic Activity and Altered Fidelity of Human DNA Polymerase in the Presence of Manganese^*

From the Laboratory of Genomic Integrity, NICHD, National Institutes of Health, Bethesda, Maryland 20892-2725

Received for publication, March 13, 2007 , and in revised form, July 2, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
All DNA polymerases require a divalent cation for catalytic activity. It is generally assumed that Mg²⁺ is the physiological cofactor for replicative DNA polymerases in vivo. However, recent studies suggest that certain repair polymerases, such as pol , may preferentially utilize Mn²⁺ in vitro. Here we report on the effects of Mn²⁺ and Mg²⁺ on the enzymatic properties of human DNA polymerase (pol ). pol exhibited the greatest activity in the presence of low levels of Mn²⁺ (0.05–0.25 mM). Peak activity in the presence of Mg²⁺ was observed in the range of 0.1–0.5 mM and was significantly reduced at concentrations >2 mM. Steady-state kinetic analyses revealed that Mn²⁺ increases the catalytic activity of pol by 30–60,000-fold through a dramatic decrease in the K_m value for nucleotide incorporation. Interestingly, whereas pol preferentially misinserts G opposite T by a factor of 1.4–2.5-fold over the correct base A in the presence of 0.25 and 5 mM Mg²⁺, respectively, the correct insertion of A is actually favored 2-fold over the misincorporation of G in the presence of 0.075 mM Mn²⁺. Low levels of Mn²⁺ also dramatically increased the ability of pol to traverse a variety of DNA lesions in vitro. Titration experiments revealed a strong preference of pol for Mn²⁺ even when Mg²⁺ is present in a >10-fold excess. Our observations therefore raise the intriguing possibility that the cation utilized by pol in vivo may actually be Mn²⁺ rather than Mg²⁺, as tacitly assumed.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
It has been known for several decades that DNA polymerases (pol)² require a divalent cation as an activator for phosphotidyl transfer (1–3). Two metal ions are usually coordinated by three acidic amino acids within the active site of the polymerase, so as to form a metal bridge between the enzyme and the terminal phosphoryl group of the substrate (4, 5), thereby facilitating the departure of the pyrophosphate moiety (6). Based upon its cellular abundance, it is generally believed that Mg²⁺ is the activating cofactor in vivo. However, Mn²⁺, Co²⁺, Ni²⁺, and Zn²⁺ have the capacity to substitute for Mg²⁺ under certain conditions in vitro (2, 3, 7), but usually at the consequence of reduced fidelity and in some cases decreased processivity. The effect of substituting Mg²⁺ with Mn²⁺ on the activity of A- and B-family polymerases has been widely studied (8–12). In addition to reducing fidelity, Mn²⁺ also helps facilitate translesion synthesis by certain replicative polymerases, including Escherichia coli pol I (13) and herpes simplex virus-1 UL30 protein (14) but not others, such as T4 DNA polymerase, or bovine pol (12, 14).

The effect of Mn²⁺ on X-family polymerases has also been studied. In the case of pol , Mn²⁺ decreases the K_m value of nucleotide incorporation by 30-fold (15), such that there is little regard for the instructions provided by the templating base (16). Recent data also suggest that the phylogenetically related pol may have actually evolved to utilize Mn²⁺ under physiological conditions, because the enzyme is active over a wide range of Mn²⁺ concentrations and is inhibited by high levels of Mg²⁺ (17, 18).

To date, there have been few studies on the effects of Mn²⁺ on the Y-family of DNA polymerases. In the single published report, Sulfolobus solfataricus Dpo4 behaved in a similar manner to the replicative polymerases, in that 5 mM Mn²⁺ increased the efficiency of lesion bypass, but with a concomitant 3–4-fold decrease in the overall fidelity of the enzyme (19). The effect of Mn²⁺ on other Y-family polymerases, including human DNA polymerases , , and , is largely unknown. We were therefore very interested in determining the effects of various metal ions on the activity of the human Y-family polymerases and in particular pol , because its reported biochemical properties are quite unusual. Indeed, pol exhibits a remarkable template-dependent fidelity in vitro. When replicating template T in the presence of Mg²⁺, pol preferentially misinserts G over the correct base A by a factor of 3–10-fold (20–24). In contrast, when replicating template A, the enzyme is relatively accurate with misincorporations occurring in the range of 10^–3–10^–4 (20–22). Thus, the fidelity of pol can vary by up to 100,000-fold depending upon the template sequence being replicated. Here we report on the enzymatic properties of pol in vitro in the presence of various divalent cations, in particular Mg²⁺ and Mn²⁺.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Enzymes—N-terminal glutathione S-transferase-tagged human pol was expressed and purified from baculovirus-infected insect Sf9 cells as described (20). C-terminal His-tagged human pol (25) was also expressed in baculovirus-infected Sf21 cells and purified by nickel-agarose, Q-Sepharose, and Bio-Gel HT hydroxyapatite chromatography. N-terminal His-tagged human pol (26) was purchased from Enzymax (Lexington, KY).

Primers and Templates—All primers, undamaged templates, and the abasic site-containing template, TXT30, were synthesized by Lofstrand Laboratories (Gaithersburg, MD) and gelpurified prior to use. The cyclobutane pyrimidine dimer containing template, TTA48, was synthesized and purified by Phoenix Biotechnologies, Inc. (Huntsville, AL). The pyrimidine-pyrimidone (6-4)-containing template, 6-4TT30, was a kind gift from Shigenori Iwai (Osaka University). The benzo-[a]pyrene diol epoxide-containing template, BPDE29, was a kind gift from Don Jerina (NIDDK, National Institutes of Health). For the experiments presented in Fig. 1, the template was T50, 5'-CGT CTA GAC GAA TTC ACG GCT CAA GCT TGC TTG CGC ATG CTC TGC AGG CG-3', and the primer was a 22-mer that was complementary to the template nucleotides underlined. The template used for the experiments presented in Figs. 2 and 5 and Table 1, parts B and C, was T50M13, 5'-CGG TAA TGA TTC CTA CGA TGA AAA TAA AAA CGG CTT GCT TGT TCT CGA TG-3', which corresponds to M13mp18 (nucleotides 3486–3437) (27). The primer was a 20-mer complementary to the template nucleotides underlined. The experiments described in Fig. 3 and Table 1, Part A utilized 48-mer templates UTTA and TTA48, with the sequence 5'-TCG ATA CTG GTA CTA ATG ATT AAC GAA TTA AGC ACG TCC GTA CCA TCG-3'. The primer was a 16-mer that was complementary to the template nucleotides underlined. The adjacent Ts were covalently linked to form a cyclobutane pyrimidine dimer (CPD) in template TTA48. The experiments described in Fig. 4 utilized three lesion-containing templates. TXT30 was a 30-mer with the sequence 5'-CTC GTC AGC ATC TXC ATC ATA CAG TCA GTG-3', where the X was a synthetic tetrahydrofuran moiety (abasic site). The primer was a 16-mer that was complementary to the template nucleotides underlined. The second template, BPDE29, was a 29-mer with the sequence 5'-GCT CGT CAG CAG ATT TAG AGT CTG CAG TG-3', where the A has a benzo[a]pyrene adduct at its N⁶ position (28). The primer was a 16-mer that was complementary to the template nucleotides underlined. The third template, 6-4TT30, was a 30-mer with the sequence 5'-CTC GTC AGC ATC TTC ATC ATA CAG TCA GTG-3', where the two Ts indicate a 6-4 pyrimidine-pyrimidone dimer (29). The primer was a 16-mer that was complementary to the template nucleotides underlined. All primers were 5'-labeled with [-³²P]ATP (5000 Ci/mmol; 1 Ci = 37 gBq) (GE Healthcare).

View this table: [in this window] [in a new window]   TABLE 1 Kinetics of pol (mis)insertion in the presence of Mg2+ and Mn2+

Primer Extension Reaction—Standard replication reactions (10 µl) contained 40 mM Tris·HCl (pH 8.0), 2.5% glycerol, 0.1 mg/ml bovine serum albumin, 10 mM dithiothreitol, 10 nM of 5'-³²P-labeled primer-template DNA, 2 nM glutathione S-transferase-pol , 5 nM His-pol , or His-pol . Reaction times, divalent metal ions, in the form of –Cl₂ salt, and dNTPs concentrations are specified in the figures and legends. After incubation at 37 °C, reactions were terminated by addition of 10 µl of 95% formamide, 10 mM EDTA, and samples were heated to 100 °C for 5 min and then immediately transferred on ice. The reaction products were separated by 15% polyacrylamide, 8 M urea gel electrophoresis and analyzed using a Fuji FLA-5100 PhosphorImager and ImageGauge software.

View larger version (62K): [in this window] [in a new window]   FIGURE 1. Catalytic activity of human DNA polymerases , , and in the presence of various divalent cations. The ability of pol (A), pol (B), or pol (C) to extend a 32P-labeled 22-mer primer annealed to template "T50" was assayed in the presence of 50 µM of all four dNTPs and 0.5 or 5 mM divalent metal ions (as the chloride salt), for 30 min at 37 °C.

View larger version (75K): [in this window] [in a new window]   FIGURE 2. Determination of the optimal concentration of Mn2+ or Mg2+ required for maximal activity of human pol and pol in vitro. The ability of pol (top) and pol (bottom) to extend a 32P-labeled 20-mer primer annealed to template "T50M13" was assayed in the presence of 50 µM of all four dNTPs and various concentrations of MnCl2 or MgCl2 ranging from 0.05 to 8 mM in 20-min reactions at 37 °C. The sequence of the template immediately downstream of the primer (Pr.) is shown on the left-hand side of each gel. The arrows indicate the location of a template T that is located 6 bp from the end of the primer terminus. In the presence of Mg2+, pol frequently misincorporates G opposite template T. The mispair is poorly extended compared with the correct base pair, and as a consequence a strong pause site is observed 6 bp from the primer terminus. Thus, one measurement of the overall catalytic activity of pol is its ability to extend beyond the normal pause site at template T. When products 7 bp and longer are plotted as a function of Mn2+ () or Mg2+ (), it is clear that pol exhibits greatest activity at 0.075 mM Mn2+ and 0.25–0.5 mM Mg2+. Although pol does not pause at template T, we used the same method to determine the optimal conditions for pol activity. As can be seen (lower panels), pol exhibits greatest activity in 0.05–0.1 mM Mn2+ and 0.1–2 mM Mg2+.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Titration of Mn²⁺ or Mg²⁺ in pol and pol Replication Reactions—We next determined the optimum concentration of Mg²⁺ or Mn²⁺ required to promote peak activity of pol or pol by performing primer extension assays in the presence of Mg²⁺ or Mn²⁺ ranging in concentrations from 0.05 to 8 mM. The template for these assays was a 50-mer oligonucleotide that has a run of five template As followed by a T and then four additional As immediately downstream of the primer. This sequence context was chosen, as we have previously shown that in the presence of 5 mM Mg²⁺ pol readily incorporates T opposite the five template As, but then frequently misinserts G opposite template T (29). The efficiency of G (or A) insertion opposite T is much lower than that of the incorporation of T opposite A (20). Further elongation from the mispair is also reduced compared with that of the correct A:T base pair (24), and as a consequence, there is a strong pol -dependent pause 5–6 bases from the starting primer terminus (Fig. 2, top, 0.1–0.5 mM Mg²⁺). The percentage of replication products longer than 6 bp therefore provides a reasonably accurate method of measuring the catalytic activity of pol . By using this approach, it is readily obvious that pol is very active in the presence of low levels of Mn²⁺ (Fig. 2, top, left-hand panel). Presumably, the ability of pol to overcome the kinetic block to replication when the enzyme encounters a template T reflects a change in the specificity of nucleotide (mis)insertion (see below for more detailed analysis), as well as through an increased ability to extend G:T mispairs.³ The peak activity of pol was observed in the range of 0.05–0.2 mM Mn²⁺. Similarly, pol was also much more active in the presence of low levels of Mg²⁺, with peak activity occurring within the range of 0.2–0.5 mM Mg²⁺. Indeed, higher concentrations of Mg²⁺ appear to inhibit pol , as under these assay conditions the limiting amount of pol in the reaction was barely sufficient to elongate the primer by 1–2 bp (Fig. 2, right-hand panel, 8 mM Mg²⁺).

View larger version (82K): [in this window] [in a new window]   FIGURE 3. pol - and pol -dependent bypass of a T-T CPD in the presence of Mn2+ or Mg2+. The ability of pol (A)orpol (B) to extend a 32P-labeled 20-mer primer (Pr.) annealed to the CPD-containing "TTA48" template was assayed in the presence of 100 µM of all four dNTPs and various concentrations of MnCl2 or MgCl2 ranging from 0.05 to 8 mM in 30-min reactions at 37 °C. The location of the T-T CPD in the TTA48 template is indicated (>TT). A graph depicting the percent of lesion bypass (products extended beyond the T-T) in the presence of various concentrations of Mn2+ () or Mg2+ () is shown on the right-hand side of each panel.

In an attempt to further optimize the in vitro reaction conditions for pol synthesis, we also performed replication assays in the presence of low Mn²⁺ or Mg²⁺ in buffers that ranged in pH from 7.5 to 9.0 and in the presence of increasing amounts of NaCl. In general, changing the pH of the assay buffer had little effect on the activity of the enzyme. In contrast, the activity of pol steadily decreased as the concentration of NaCl was increased from 0 to 150 mM.³

Activity of pol and pol on a CPD-containing Template in the Presence of Mg²⁺ and Mn²⁺—We have previously reported that in the presence of 5 mM Mg²⁺, pol is capable of inserting a base opposite the 3'T of a T-T CPD, as well as facilitating low levels of complete translesion synthesis (30, 31). We therefore wanted to reinvestigate the ability of pol to facilitate translesion synthesis over a range of Mg²⁺ and Mn²⁺ concentrations (Fig. 3). In these reactions, the 3' end of the primer was juxtaposed to the CPD and are therefore considered "standing start" reactions. Similar to the observations reported above, maximal activity for pol was observed in the range of 0.075–0.25 mM Mn²⁺ on the damaged template (Fig. 3A). Under these conditions, there was significant extension of primers past the CPD. Indeed, we estimate that approximately 60% of the primer was elongated past the CPD by pol in the presence of 0.2 mM Mn²⁺ (Fig. 3A, right-hand panel). The results with Mg²⁺ were similar to our earlier published observations (30, 31), in that the 3'T of the CPD is a strong kinetic block to pol and elongation past the 3'T was greatest in the range of 0.25–2 mM Mg²⁺. Indeed, we estimate that in the presence of 0.5 mM Mg²⁺, 8% of the primers are elongated past both Ts of the CPD, compared with 2.5% in the presence of 5 mM Mg²⁺, which is consistent with our earlier observations (30, 31).

The ability of pol to bypass a CPD in vitro is unrivalled. It does so with the same or even higher efficiency than it replicates undamaged DNA (32–34). In the presence of Mn²⁺, robust bypass activity was observed from 0.05 to 0.25 mM Mn²⁺, but bypass activity steadily declined as the Mn²⁺ concentration increased (Fig. 3B). In contrast, pol exhibited strong bypass activity in the presence of 0.05–8 mM Mg²⁺.

We conclude from these studies that pol and pol have differing affinities for metal ion activation. Translesion synthesis of a CPD by pol is stimulated 3-fold by low levels of Mg²⁺ and up to 25-fold in the presence of 0.2 mM Mn²⁺ compared with 5 mM Mg²⁺. In contrast, pol appears to exhibit efficient bypass a CPD over a wide range of Mg²⁺ concentrations.

View larger version (73K): [in this window] [in a new window]   FIGURE 4. pol -dependent bypass of an abasic site, BPDE lesion, and 6-4PP lesion in the presence of Mn2+ or Mg2+. The ability of pol to extend 32P-labeled primers (Pr.) annealed to the abasic-containing template, TXT30 (A), the BPDE-containing template, BPDE29 (B), or the 6-4PP containing template, 6-4TT30 (C), was assayed in the presence of 100 µM of all four dNTPs and various concentrations of MnCl2 or MgCl2 ranging from 0.05 to 8 mM in 30-min reactions at 37 °C. The location of the abasic site is indicated by AP, the BPDE lesion by BP, and the 6-4PP indicated by >6-4.A graph depicting the percent of lesion bypass (products extended beyond the abasic site, the BPDE lesion, or the 6-4T-T), in the presence of various concentrations of Mn2+ () or Mg2+ (), is shown on the right-hand side of each panel.

pol Has Higher Affinity for Mn²⁺ than Mg²⁺—As noted earlier, in the presence of Mg²⁺ pol readily misinserts G opposite template T, causing the enzyme to pause. This pausing is much less evident in the presence of Mn²⁺, with the accumulation of a significant proportion of replication products longer than 6 bp (Fig. 2, top). These two distinguishable properties gave us an opportunity to assay which cation pol prefers to utilize when presented with both Mn²⁺ and Mg²⁺. In the experiments depicted in Fig. 5, pol was first preincubated in the presence of various concentrations of Mn²⁺ (Fig. 5A), or Mg²⁺ (Fig. 5B), for 5 min at room temperature. Reactions were started by the addition of various concentrations of either Mg²⁺ or Mn²⁺ and the radio-labeled primer/template, followed by incubation at 37 °C for 20 min. As shown in Fig. 5, it is clear that when preincubated with Mn²⁺ and then challenged with Mg²⁺, the pol -dependent replication products are reminiscent of those observed in the presence of Mn²⁺ alone, with very little pausing at template T (Fig. 5A). This property occurs over a wide range of Mn²⁺/Mg²⁺ concentrations, and is even evident when Mg²⁺ is present in a 40-fold molar excess over Mn²⁺ (see Fig. 5A, 0.05 mM Mn²⁺/2 mM Mg²⁺).

The preference of pol for Mn²⁺ is more evident in the reactions in which pol was preincubated in the presence of Mg²⁺ and then challenged with Mn²⁺ (Fig. 5B). In the absence of added Mn²⁺, the Mg²⁺ reactions exhibited the classic termination pattern, with a strong pause 5–6 bp downstream of the primer. However, in all reactions containing Mn²⁺ this pausing is significantly reduced. Again, Mn²⁺ stimulation occurred over a wide range of Mg²⁺/Mn²⁺ concentrations and was obvious even when Mg²⁺ was present in a 10–40-fold molar excess over Mn²⁺. Based upon these observations, we conclude that pol preferentially utilizes Mn²⁺ as the activator for polymerization, even in reactions containing a vast molar excess of Mg²⁺.

Kinetics of pol -dependent Incorporation in the Presence of Mn²⁺ and/or Mg²⁺—Several groups have previously reported the kinetic parameters for pol -dependent nucleotide incorporation on an undamaged template. In all cases, these values were determined in the presence of 5 or 8 mM Mg²⁺ (20–22). Under these conditions, the two most striking properties of pol are its ability to preferentially misinsert G opposite T and to faithfully and efficiently incorporate A opposite T. However, our present studies indicate that pol is much more catalytically active at much lower concentrations of Mg²⁺ and Mn²⁺. We have therefore determined the kinetic parameters for (mis)insertion opposite template T and A, in the presence of 0.075 mM Mn²⁺, 0.25 mM Mg²⁺, and 5 mM Mg²⁺ either alone (Table 1, parts A and B), or in combination (Table 1, part C). These kinetic studies revealed that the increase in catalytic activity observed in our earlier experiments is largely derived from a dramatic decrease in the K_m value for nucleotide incorporation. Indeed, whereas the V_max was essentially unchanged under all assay conditions, the K_m value varied by as much as 2400-fold at template T and 30,000-fold at template A. For example, the K_m value for the correct incorporation of A opposite T decreased by 245-fold in the presence of 0.25 mM Mg²⁺ compared with 5 mM Mg²⁺, and there was a further 10-fold reduction in the presence of 0.075 mM Mn²⁺. The K_m value for the misincorporation of G opposite T also dropped dramatically but to a slightly lower extent. As a result, there was an overall change in the frequency of misincorporation (f_inc) under the various assay conditions. Although misincorporation of G opposite T was favored by a factor of 2.5-fold in 5 mM Mg²⁺ and by 1.4-fold in 0.25 mM Mg²⁺, in reactions containing 0.075 mM Mn²⁺, the correct incorporation of A was actually favored 2.3-fold more than G. Thus, in contrast to its fidelity-reducing effects on all known polymerases, Mn²⁺ actually increases the fidelity of pol at template T. It should be stressed, however, that even though Mn²⁺ altered the relative ratios of (mis)incorporation opposite template T so as to favor the incorporation of the correct base, pol nevertheless remains extremely error-prone under these conditions.

View larger version (101K): [in this window] [in a new window]   FIGURE 5. Activation of pol catalysis in the presence of Mn2+ and Mg2+. The ability of pol to extend a 32P-labeled 20-mer primer annealed to template "T50M13" was assayed in the presence of 50 µM all four dNTPs and various concentrations of MnCl2 and MgCl2. In the experiments shown in A, pol was preincubated in the presence of Mn2+ for 5 min and reactions were started by the addition of various concentrations of Mg2+ together with the annealed primer template. Reactions were terminated after 20 min at 37 °C. In the experiments shown in B, pol was preincubated in the presence of Mg2+ for 5 min and reactions were started by the addition of various concentrations of Mn2+ together with the annealed primer template and were terminated after 20 min at 37 °C. The sequence of the template immediately downstream of the primer (Pr.) is shown on the left hand side of each panel. The arrows indicate the location of a template T that represents a strong pause site for pol in the presence of Mg2+ alone. As can be clearly seen, pausing at this site is diminished in reactions containing Mn2+.

Overall, the catalytic activity (V_max/K_m)ofpol increased in the presence of low levels of Mg²⁺ or Mn²⁺. The smallest effect was observed for (mis)incorporation opposite A, where the catalytic activity only increased 2–2.5-fold in the presence of 0.25 mM Mg²⁺ compared with 5 mM Mg²⁺. However, much larger effects were observed at template T, where the catalytic activity increased 45–80-fold in 0.25 mM Mg²⁺ compared with 5 mM Mg²⁺. The effects of 0.075 mM Mn²⁺ were much more dramatic, with an additional 11–3300-fold increase in catalytic activity over that observed in the presence of 0.25 mM Mg²⁺.

In the simultaneous presence of 0.075 mM Mn²⁺ and 0.25 mM Mg²⁺, the catalytic activity (V_max/K_m)ofpol for the correct incorporation of T opposite A was intermediate to that observed in the presence of each individual cation. For example, the catalytic activity of pol was 4.5-fold lower than in the presence of 0.075 mM Mn²⁺ alone but 2.6-fold higher than in the presence of 0.25 mM Mg²⁺ alone. With regard to fidelity, Mn²⁺ appears to be dominant, in that pol exhibited 200-fold lower fidelity in the presence of both Mn²⁺ and Mg²⁺ compared with that observed in the presence of 0.25 mM Mg²⁺ alone.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In this study, we have investigated the effects of various divalent cations on the in vitro properties of human DNA polymerases , , and . The most abundant divalent cation is Mg²⁺, with an intracellular concentration of 0.21 to 0.24 mM (36, 37). Given its abundance, Mg²⁺ is therefore generally considered to be the physiologically relevant divalent metal cofactor for most DNA polymerases. In support of this notion, our analyses strongly suggest that the preferred cation for pol and pol is Mg²⁺, as both enzymes were active over a wide range of Mg²⁺ concentrations. In contrast, pol exhibited greatest activity within a much more narrow concentration range of Mg²⁺ (Fig. 2). Peak activity was observed at 0.25 mM Mg²⁺, and synthesis was reduced significantly in reactions containing >2 mM Mg²⁺. As far as we are aware, all of the published reports on the biochemical properties of pol in vitro were performed in the presence of 5 or 8 mM Mg²⁺ and were therefore performed under suboptimal conditions (20–24, 28, 30, 31, 35, 38–42). Indeed, it appears that in the presence of low levels of Mg²⁺, pol may exhibit higher catalytic activity and fidelity than previously thought (Table 1).

Limited synthesis by pol , pol , and pol was observed in the presence of many different divalent cations, but in most cases, the concentration required for replication occurred well outside the physiological concentration of the trace metal, and is therefore probably of little biological significance. The exception was the ability of pol to utilize low levels of Mn²⁺ as an activator for catalysis (Fig. 2). The cellular concentration of Mn²⁺ in mammalian cells is much lower than that of Mg²⁺ and is thought to be in the range of 0.1 to 40 µM (43–45). Our current studies indicate that in vitro, the optimum concentration of Mn²⁺ required for maximal stimulation of pol on an undamaged template occurs around 0.075 mM (75 µM). Although this concentration may lie outside the physiological range of Mn²⁺ available to most cells, pol nevertheless exhibited a strong preference for Mn²⁺ over Mg²⁺, even when Mg²⁺ was in a 10–20-fold excess. Our steady-state kinetic analyses revealed that Mn²⁺ stimulated the catalytic activity of pol through a dramatic decrease in the K_m value for nucleotide incorporation. Interestingly, when both Mn²⁺ and Mg²⁺ were present at optimal concentrations (0.075 and 0.25 mM, respectively), pol exhibited kinetic parameters that more closely resembled those observed in the presence of Mn²⁺ alone (Table 1, part C), which infers that Mn²⁺ is the preferred activator for polymerization.

The structural basis for the enhanced catalytic activity of pol in the presence of Mn²⁺ is presently unknown. However, it is clear that Mn²⁺ has a more relaxed coordination than Mg²⁺, thereby facilitating reactions that are suboptimal or even inhibited in the presence of Mg²⁺ (5). Indeed, the crystal structure of pol reveals that its active site is somewhat distorted and has large side chains protruding into the space normally occupied by the replicating base pair (40). Mn²⁺ may therefore simply allow greater flexibility of the active site of pol , such that it can adopt a conformation that is more favorable for catalysis.

The effects of Mn²⁺ on the fidelity of pol were unique. Although it caused a characteristic reduction in fidelity at template A, Mn²⁺ actually increased fidelity at template T, by 3–5-fold compared with the fidelity of pol in the presence of 0.25 or 5 mM Mg²⁺, respectively. Low levels of Mg²⁺ and Mn²⁺ also had dramatic effects on the ability of pol to traverse a T-T CPD. The efficiency of bypass increased from 2.5% in the presence of 5 mM Mg²⁺ to 8% in the presence of 0.5 mM Mg²⁺ and up to 60% in the presence of 0.2 mM Mn²⁺. Thus, under certain conditions in vitro pol can bypass a T-T CPD relatively efficiently. Presumably conditions favorable for the pol -dependent bypass of CPDs also occur in vivo. Such an assumption is supported by the recent reports that implicate cellular roles for pol in UV-induced mutagenesis and carcinogenesis in mice and humans (46–48). The stimulatory effect of Mn²⁺ on pol -dependent lesion bypass was not limited to the T-T CPD, as bypass of an abasic site increased from 1 to 2% in the presence of Mg²⁺ to 18% in the presence of Mn²⁺. Similarly, bypass of a BPDE lesion also increased from 2.5 to 10% in the presence of Mg²⁺ and Mn²⁺, respectively, thereby raising the possibility that pol may also bypass these lesions in vivo.

In summary, pol is more active in the presence of physiological concentrations of Mg²⁺ than at concentrations previously used in vitro to study the enzymatic properties of the polymerase. We therefore suggest that future in vitro studies on pol be conducted in buffers containing low Mg²⁺ (0.25–2 mM). We also discovered that the enzyme is highly active in low concentrations of Mn²⁺. When asked to select between Mg²⁺ and Mn²⁺, pol clearly prefers to utilize Mn²⁺, even when Mg²⁺ is in large molar excess. Whether a similar situation occurs in vivo remains to be determined, but it nevertheless remains an intriguing possibility certainly worth considering.

	FOOTNOTES

 
^* This work was funded by the Intramural Research Programs of the NICHD, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Bldg. 6, Rm. 1A13, NICHD, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892-2725. Tel.: 301-496-6175; Fax: 301-594-1135; E-mail: woodgate{at}nih.gov.

² The abbreviations used are: pol, DNA polymerase; CPD, cyclobutane pyrimidine dimer; 6-4PP, pyrimidine-pyrimidone (6-4); BPDE, Benzo[a]pyrene diol epoxide.

³ E. G. Frank and R. Woodgate, unpublished observations.

	ACKNOWLEDGMENTS

 
We thank Wei Yang for insights and comments on the possible structural basis for Mn²⁺ stimulation of the catalytic activity of pol .

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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