Mismatch Extension Ability of Yeast and Human DNA Polymerase eta *

M. Todd Washington, Robert E. Johnson, Satya Prakash, and Louise PrakashDagger

From the Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1061

Received for publication, October 3, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

DNA polymerase eta  (Poleta ) functions in error-free replication of UV-damaged DNA, and in vitro it efficiently bypasses a cis-syn T-T dimer by incorporating two adenines opposite the lesion. Steady state kinetic studies have shown that both yeast and human Poleta are low-fidelity enzymes, and they misincorporate nucleotides with a frequency of 10-2-10-3 on both undamaged and T-T dimer-containing DNA templates. To better understand the role of Poleta in error-free translesion DNA synthesis, here we examine the ability of Poleta to extend from base mismatches. We find that both yeast and human Poleta extend from mismatched base pairs with a frequency of ~10-3 relative to matched base pairs. In the absence of efficient extension of mismatched primer termini, the ensuing dissociation of Poleta from DNA may favor the excision of mismatched nucleotides by a proofreading exonuclease. Thus, we expect DNA synthesis by Poleta to be more accurate than that predicted from the fidelity of nucleotide incorporation alone.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

DNA polymerase eta  (Poleta )1 functions in error-free replication of UV-damaged DNA, and mutations in the gene encoding this enzyme result in increased UV mutability in both yeast and humans (1). In humans, inactivation of Poleta causes the variant form of the cancer prone syndrome xeroderma pigmentosum (2, 3). Poleta is unique among eukaryotic DNA polymerases in its ability to efficiently replicate DNA containing a cis-syn T-T dimer, and it does so by incorporating two adenines across from the two thymines of the dimer (3-6).

The high fidelity of replicative DNA polymerases arises, in part, because their active sites are intolerant of the distorted geometry resulting from mispairs between the template residue and the incoming nucleotide (7). Steady state kinetic studies of yeast and human Poleta have indicated that it is a low-fidelity enzyme, misincorporating nucleotides with a frequency of 10-2-10-3 on undamaged DNA (5, 8). Remarkably, however, Poleta synthesizes DNA opposite a T-T dimer with the same efficiency and accuracy as opposite undamaged T residues (5, 6). The low fidelity of Poleta may reflect an unusual tolerance of its active site for deviant geometry arising from distorting template lesions such as a T-T dimer.

The accuracy of synthesis by DNA polymerases depends on the frequency of incorporation of incorrect nucleotides into DNA and on the frequency of extension of the mismatched primer termini. Extension of mismatched primers is a critical step in mutation fixation, because in the absence of efficient extension, the mismatched nucleotide can be excised by a proofreading exonuclease, or if the mismatch is not excised, cell death may ensue as a result of incomplete DNA synthesis. Thus, for a mutation to be expressed, extension from the misincorporated nucleotide must occur. To better understand how Poleta , with a low nucleotide insertion fidelity, can function in an error-free pathway of translesion DNA synthesis in vivo, here we examine the ability of Poleta to extend from base mispairs. We find that yeast and human Poleta extend from mismatched primer-templates with a frequency of ~10-3 relative to matched primer-templates. This implies that Poleta , which has a low processivity, will have a greater likelihood of dissociating from the DNA template after the incorporation of an incorrect nucleotide than a correct one. That would lower the error rate of DNA synthesis in vivo, because the mismatched primer terminus could then be subjected to the proofreading 3'right-arrow5' exonuclease activity of other protein factors.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

DNA Substrates-- DNA substrates containing all possible correct base pairs or mispairs at the 3' primer terminus were generated using four different oligodeoxynucleotide primers and four oligodeoxynucleotide templates. The four 45-nucleotide primers have the following sequence: 5'-GTTTT CCCAG TCACG ACGAT GCTCC GGTAC TCCAG TGTAG GCATN, where N is G, A, T, or C. The four 52-nucleotide templates have the following sequence: 5'-TTCGT ATNAT GCCTA CACTG GAGTA CCGGA GCATC GTCGT GACTG GGAAA AC, where N is G, A, T, or C. The various combinations of primers and templates were annealed by mixing 1 µM 32P-end labeled primer with 1.5 µM template in 50 mM Tris-HCl, pH 7.5, and 100 mM NaCl and heating to 90 °C for 2 min before slowly cooling to room temperature over several hours.

Steady state Kinetics Assays-- Yeast and human Poleta were expressed in and purified from yeast strain BJ5464 as described (4, 5). The steady state kinetics of single nucleotide incorporation were measured by incubating 1 nM yeast or human Poleta with 20 nM DNA substrate in 25 mM sodium phosphate, pH 7.0, buffer containing 5 mM magnesium chloride, 5 mM dithiothreitol, 10 µg/ml bovine serum albumin, and 10% glycerol for 10 min at 25 °C. For nucleotide incorporation following a correctly base paired or mispaired primer terminus, the concentration of dATP was varied from 0 to 5 µM or from 0 to 2000 µM, respectively. Reactions were quenched after 10 min by adding 10 volume of loading buffer (95% formamide, 0.03% bromphenol blue, and 0.3% cyanol blue). Samples were then run on 10% polyacrylamide sequencing gels to separate the unextended and extended DNA primers. Gel band intensities were quantified using a PhosphorImager and ImageQuant software (Molecular Dynamics). The observed rate of nucleotide incorporation was calculated by dividing the amount of reaction product formed by the 10-min incubation time. The observed rate of nucleotide incorporation was then plotted as a function of nucleotide concentration, and the apparent Km and Vmax parameters were obtained from the best fit to the Michaelis-Menten equation using nonlinear regression (Sigma Plot 4.0). The intrinsic efficiency of mismatch extension, fexto, which is a constant that represents the efficiency of extending mismatched termini in competition with matched termini at equal DNA concentrations, was calculated as described (7, 9, 10) using the following equation: fexto = (Vmax/Km)mismatch/(Vmax/Km)matched.


    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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We examined the steady state kinetics of nucleotide incorporation by Poleta following the correctly base paired and mispaired termini in primer-template substrates (7, 9, 10). For example, the rate of incorporation of an A residue by yeast Poleta opposite a template T residue following a G·C base pair or an A·C, T·C, or C·C mispair was measured over a broad range of dATP concentrations (Fig. 1A). Gel band intensities were evaluated, and the rate of nucleotide incorporation was plotted as a function of nucleotide concentration. As shown in Fig. 1B, these plots yield curves typical of Michaelis-Menten kinetics. The apparent values of Vmax and Km for extension of each primer terminus were obtained from the best fit to the Michaelis-Menten equation using nonlinear regression. The frequency of mispair extension (fexto), which is the ratio of the apparent Vmax/Km of extension from the mispair to the apparent Vmax/Km of extension from a correct base pair, was then calculated (7, 9, 10).



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Fig. 1.   Mismatch extension by yeast Pol eta . A, dAMP incorporation by yeast Poleta opposite a template T following a G·C base pair or A·C, T·C, and C·C mispairs. Yeast Poleta (1 nM) was incubated with DNA substrate (20 nM) and varying concentrations of dATP at 25 °C for 10 min. B, observed rate of nucleotide incorporation by yeast Poleta following a G·C base pair or A·C, T·C, and C·C mispairs graphed as a function of dATP concentration. The obtained Vmax and Km parameters are listed in Table I.

As shown in Table I, for the incorporation of an A residue following a G·C base pair, the apparent Km for yeast Poleta is 0.20 µM, and the Vmax is 0.28 nM/min, whereas for the incorporation of an A following an A·C mispair, the apparent Km is 21 µM, and the Vmax is 0.25 nM/min, respectively. Thus, for the A·C mispair, fexto is 8.5 × 10-3, and similarly, the fexto values for the T·C and C·C mispairs are 1.5 × 10-3 and 1.1 × 10-3, respectively. The fexto values were determined for all the possible mispairs, and overall, yeast Poleta extends from mispairs with an average frequency of 3.1 × 10-3 (Table I).


                              
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Table I
Frequencies of extension from matched and mismatched primer-template termini by yeast Poleta on undamaged DNA
Extension was examined in the presence of dATP, the next correct nucleotide for template T.

For most DNA polymerases, the frequency of extension from a given mispair (fexto) is approximately the same as the frequency of incorporating that same mispair (finc; Refs. 7, 10). Fig. 2A compares the fexto values with the previously reported finc values (8) for yeast Poleta for each possible mispair. Points lying above the dashed line represent mispairs with a higher efficiency of extension than insertion, whereas those below the line indicate mispairs with a lower efficiency of extension than insertion. Because most of the points lie near or below the dashed line, yeast Poleta is somewhat less efficient at extending from mispairs than at forming mispairs.



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Fig. 2.   Comparison of mispair extension and mispair insertion efficiencies by yeast Poleta (A) and human Poleta (B). The finc value for inserting a wrong nucleotide opposite a template base is plotted against the corresponding fexto value for extending from that mispair. In the base mispairs shown, the first base is in the primer, and the second base is in the template. The dashed line corresponds to fexto = finc.

We also examined human Poleta for its ability to extend from base mispairs. As was observed for yeast Poleta , the fexto values for human Poleta range from 10-2 to 10-3, with an average of 2.5 × 10-3 (Table II), and a comparison of fexto values with the previously published finc values (5) indicates that human Poleta is also somewhat less efficient at extending from mispairs than at inserting mispaired bases (Fig. 2B).


                              
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Table II
Frequencies of extension from matched and mismatched primer-template termini by human Poleta on undamaged DNA
Extension was examined in the presence of dATP, the next correct nucleotide for template T.

Poleta replicates through a cis-syn T-T dimer with the same efficiency and fidelity as through undamaged T nucleotides (5, 6). Furthermore, our steady state kinetic analyses of base mispair extension across from the T-T dimer indicate that these mispairs are also inefficiently extended and with the same frequency as mispairs in undamaged DNA.2

When compared with other DNA polymerases, the mispair extension ability of Poleta is greater than that of the high-fidelity DNA polymerase alpha , the fexto of which ranges from 10-3 to 10-6 (10). However, its mispair extension ability is considerably lower than that of the most promiscuous extender of mispairs known, yeast Polzeta , which extends from mispaired template primer termini with a frequency of 10-1 to 10-2 (11). Polzeta plays an essential role in mutagenic bypass of DNA lesions, and it specifically functions in damage bypass by extending from nucleotides placed opposite DNA lesions by another DNA polymerase (11).

Poleta has low processivity (5, 8), and thus it has a modest probability (0.2-0.3) of dissociating from the DNA template after each nucleotide incorporation. Our observation that both yeast and human Poleta extend from mismatched primer termini with a frequency of ~10-3 relative to a matched primer terminus implies that Poleta has a substantially higher probability of dissociating from the primer terminus after the incorporation of an incorrect nucleotide than a correct nucleotide. Dissociation of Poleta would prevent mutation fixation, because any mispairs left in DNA would then be subject to removal by the proofreading exonucleolytic activity of Poldelta or other proofreading exonucleases. Thus, DNA synthesis by Poleta would be more accurate than is indicated from the fidelity of nucleotide incorporation (finc) values. Because Poleta extends from mismatched bases opposite a T-T dimer with the same efficiency as from undamaged DNA, we predict that the error frequency during T-T dimer bypass will also be lower than that suggested from the finc values for the incorporation of wrong nucleotides opposite the two T nucleotides of the T-T dimer (5, 6).

We expect the activity of Poleta to be restricted to DNA synthesis during damage bypass. The Rad6-Rad18 complex, which is essential for damage bypass and which contains ubiquitin conjugating and DNA binding activities (12), may be crucial for modulating the specific targeting of Poleta to sites where replication has stalled at a DNA lesion and for ensuring the dissociation of Poleta from DNA once the lesion has been bypassed. Furthermore, association with other protein factors may increase the fidelity of nucleotide incorporation by Poleta . Thus, in vivo, damage bypass by Poleta would be much more accurate than 10-2-10-3, the frequency of nucleotide misincorporation.


    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM19261.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Sealy Center for Molecular Science, University of Texas Medical Branch, 6.104 Medical Research Bldg., 11th and Mechanic Sts., Galveston, TX 77555-1061. Tel.: 409-747-8601; Fax: 409-747-8608; E-mail: lprakash@scms.utmb.edu.

Published, JBC Papers in Press, October 27, 2000, DOI 10.1074/jbc.M009049200

2 M. T. Washington, R. E. Johnson, S. Prakash, and L. Prakash, unpublished observations.


    ABBREVIATIONS

The abbreviation used is: Poleta , polymerase eta .


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES


1. Johnson, R. E., Washington, M. T., Prakash, S., and Prakash, L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 12224-12226[Free Full Text]
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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.