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Originally published In Press as doi:10.1074/jbc.M501981200 on March 17, 2005

J. Biol. Chem., Vol. 280, Issue 20, 20051-20058, May 20, 2005
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Biochemical Properties of Saccharomyces cerevisiae DNA Polymerase IV*

Katarzyna Bebenek, Miguel Garcia-Diaz, Steven R. Patishall, and Thomas A. Kunkel{ddagger}

From the Laboratory of Molecular Genetics and Laboratory of Structural Biology, NIEHS, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709

Received for publication, February 22, 2005 , and in revised form, March 14, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Although mammals encode multiple family X DNA polymerases implicated in DNA repair, Saccharomyces cerevisiae has only one, DNA polymerase IV (pol IV). To better understand the repair functions of pol IV, here we characterize its biochemical properties. Like mammalian pol {beta} and pol {lambda}, but not pol µ, pol IV has intrinsic 5'-2-deoxyribose-5-phosphate lyase activity. Pol IV has low processivity and can fill short gaps in DNA. Unlike the case with pol {beta} and pol {lambda}, the gap-filling activity of pol IV is not enhanced by a 5'-phosphate on the downstream primer but is stimulated by a 5'-terminal synthetic abasic site. Pol IV incorporates rNTPs into DNA with an unusually high efficiency relative to dNTPs, a property in common with pol µ but not pol {beta} or pol {lambda}. Finally, pol IV is highly inaccurate, with an unusual error specificity indicating the ability to extend primer termini with limited homology. These properties are consistent with a possible role for pol IV in base excision repair and with its known role in non-homologous end joining of double strand breaks, perhaps including those with damaged ends.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Family X DNA polymerases are evolutionarily conserved, single-subunit enzymes devoid of 3' -> 5' exonuclease activity. The most extensively studied family X polymerase (pol)1 is mammalian DNA pol {beta}, which has a major role in base excision repair (1). Other mammalian family X polymerases also implicated in DNA repair include template-dependent pol µ (2) and pol {lambda} (3–5) and template-independent terminal deoxyribonucleotidyl transferase (TdT) (6). These enzymes share an 8-kDa domain and a polymerase domain comprised of fingers, palm, and thumb subdomains. Nonetheless, they differ in several amino acids likely to be important for function. Pol µ, pol {lambda}, and TdT also have N-terminal BRCT domains not present in pol {beta}. Mammalian family X polymerases also have different biochemical properties. TdT adds nucleotides in a template-independent manner, consistent with its role in increasing the diversity of antigen receptor genes by adding nucleotides to the ends of coding segments during V(D)J recombination (7). Pol {beta} is template-dependent and moderately accurate for both substitution and single base deletion errors; it fills short gaps in DNA in a manner stimulated by a 5'-phosphate on the downstream primer (8) and has an intrinsic dRP lyase activity (9). These properties are all consistent with the well established role of pol {beta} in BER.

Pol {lambda} shares most of the above-mentioned properties with pol {beta}, suggesting that pol {lambda} may also function in BER (10, 11). Pol {lambda} binds dNTPs with high affinity, consistent with polymerization when cellular dNTP concentrations are low, e.g. in cells not in S phase (12). Pol {lambda} generates single base deletions, especially in non-iterated sequence contexts, at a much higher rate than pol {beta}, indicating an ability to use template-primers with minimal base pairing homology. These properties, in combination with a BRCT domain and the ability to physically and functionally interact with several proteins involved in NHEJ of double strand breaks in DNA (13, 14), implicate pol {lambda} in NHEJ.

Pol µ shares many properties in common with pol {lambda}, and it too is implicated in NHEJ. However, pol µ differs from pol {lambda} and pol {beta} in that it lacks dRP lyase activity (10) but relatively efficiently incorporates rNTPs into DNA (15, 16). Recent evidence suggests that pol µ, but not pol {lambda}, can fill short gaps in DNA during NHEJ even when 3' termini lack one or two complementary bases in the template strand.2 Moreover pol µ, but not pol {lambda}, can promote end-joining accuracy during Ig{kappa} recombination in cultured cells, and studies with pol µ knockout mice indicate that it participates in the rearrangement of immunoglobulin light chain genes in vivo. Collectively, these observations indicate that multiple family X members with somewhat different properties are used for the highly specialized DNA repair reactions that occur in mammals.

In contrast to mammals, the only family X DNA polymerase implicated in DNA repair in Saccharomyces cerevisiae is pol IV. Pol IV has highest homology to mammalian pol {lambda} and, like pol {lambda}, pol IV contains a BRCT domain followed by a coding sequence of unknown function that is rich in serine and proline residues. The BRCT domain of pol IV interacts with the Dnl4 subunit of the Dnl4-Lif1 complex (18). This interaction stimulates DNA synthesis by pol IV, which can fill short gaps formed by the alignment of linear duplex DNA molecules with partially complementary ends. These properties, together with genetic studies (19), indicate that pol IV participates in NHEJ. The fact that one yeast polymerase may suffice for NHEJ in yeast whereas three polymerases likely perform specialized NHEJ reactions in mammals, as well as the fact that base excision repair in yeast is not fully understood, led us to further characterize yeast pol IV for biochemical properties that have not been previously examined. The results presented here indicate that yeast pol IV is a hybrid of its mammalian homologs.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—The following oligonucleotides were used to prepare template-primers for polymerase assays: T18A, 5'-ACTGGCCGTCGTTCTAATGTACTCACTGTGATC-3'; P1, 5'-GATCACAGTGAGTAC-3'; G18A, 5'-ACTGGCCGTCGTTCTAAGGTACTCACTGTGATC-3' and 5'-AACGACGGCCAGT-3'. The latter was used with either a hydroxyl (DI), a phosphate (DIP), or a tetrahydrofuran (NL) on the 5' terminus. All oligonucleotides were purchased from Oligos Etc. Materials for the forward mutation assay were from previously described sources (20).

Protein Expression and Purification—His-tagged pol IV was purified to near homogeneity from overexpressing Escherichia coli cells harboring the pET28b-pol IV vector. Cells were induced with 0.5 mM isopropyl 1-thio-{beta}-L-galactopyranoside and incubated at 19 °C overnight. The purification protocol was a modified version of that described by Tseng and Tomkinson (18) and involved incubation with nickel-nitrilotriacetic acid-agarose beads (Qiagen) and elution with 250 mM imidazole followed by HiTrap S column (Amersham Biosciences) chromatography. The final pol IV fraction was tested for 3' -> 5' exonuclease activity by incubation with a 3'-terminal mismatch-containing template-primer.

dRP Lyase Assay—Reaction mixtures (10 µl) contained 50 mM Hepes, pH 7.5, 10 mM MgCl2, 20 mM KCl, 2 mM dithiothreitol, and a 50 nM 32P-labeled substrate prepared as described previously (10). Reactions were initiated by adding 100 nM wild-type or K247A/K248A mutant pol IV or pol {lambda}. After incubation for 10 min at 37 °C, NaBH4 was added to a final concentration of 340 mM, and incubation was continued for 30 min on ice. Products were precipitated with ethanol in the presence of 0.1 µg/ml tRNA, resuspended in 5 µl of deionized water, separated by electrophoresis in a 16% polyacrylamide gel, and visualized by phosphor screen autoradiography.

NaBH4 Trapping Assay—Reaction mixtures (10 µl) contained 50 mM Hepes, pH 7.5, 10 mM MgCl2, 20 mM KCl, 2 mM dithiothreitol, and 100 nM32P-labeled substrate. Reactions were initiated by adding 100 nM wild-type, K247A/K248A pol IV, or 50 nM pol {beta} and 20 mM NaBH4. After 30 min of incubation on ice, reactions were terminated by adding SDS-PAGE loading buffer and resolved in a 10% SDS-polyacrylamide gel. The trapped polymerase-DNA complexes were visualized by phosphor screen autoradiography.

DNA Polymerization on Defined DNA Substrates—A 33-nucleotide template was primed with a 32P-5'-end-labeled 15-mer (P1) as a primer for extension along with a downstream 13-mer with either an OH group (DI), a phosphate group (DIP), or a phosphorylated tetrahydrofuran residue (NL) on the 5' terminus, thus creating a substrate with a five-nucleotide gap. Reaction mixtures (20 µl) contained 50 mM Tris, pH 7.5, 1 mM dithiothreitol, 4% glycerol, 0.1 mg/ml bovine serum albumin, 2.5 mM MgCl2, 100 µM dNTPs, and 200 nM DNA. Reactions were initiated by adding 5 nM pol IV and incubated at 30 °C for 3–9 min. Aliquots were removed and mixed with an equal volume of 99% formamide, 5 mM EDTA, 0.1% xylene cyanol, and 0.1% bromphenol blue. Products were analyzed by electrophoresis in a 16% denaturing polyacrylamide gel and visualized by phosphor screen autoradiography.

Processivity—Processivity was measured by using a 33-nucleotide template (T18A) primed with a 32P-5'-end-labeled 15-mer (T-P) or by using a five-nucleotide-gapped substrate prepared as described above. Reaction mixtures (20 µl) contained the same components used for gap-filling reactions except that the pol IV concentration was 1.5 nM. This results in a DNA to enzyme ratio of 130:1, thereby preventing additional synthesis on previously extended primers as indicated by the fact that termination probabilities remained constant over the time course of the reactions. Reactions were incubated at 30 °C for 3–9 min, and products were analyzed as described above. Termination probability at each site is expressed as a percent, namely the ratio of products at a site to the products at that site plus all greater length products.

Steady State Nucleotide Incorporation—DNA substrates were prepared by hybridizing a 32P-5'-end-labeled 15-nucleotide primer to a 33-nucleotide template, either T18A or G18A. Reaction mixtures (10 µl) contained 50 mM Tris, pH 7.5, 1 mM dithiothreitol, 4% glycerol, 0.1 mg/ml bovine serum albumin, 2.5 mM MgCl2, 200 nM DNA substrate, and pol IV at 2.5 nM or pol {lambda} at 5 nM or 25 nM in reactions with either dATP or rATP, respectively. Reactions were initiated by adding dATP, rATP, dCTP, or rCTP at one of eight concentrations and incubated at 30 °C for 3 or 4 min (with pol IV) or in 37 °C for 4 or 6 min (reactions with pol {lambda}). After adding an equal volume of 99% formamide, 5 mM EDTA, 0.1% xylene cyanol, and 0.1% bromphenol blue, products were resolved on a 16% denaturing polyacrylamide gel and quantified by phosphor screen autoradiography. The data were fit to the Michaelis-Menten equation using nonlinear least squares methods.

Forward Mutation Assay—Reaction mixtures (25 µl) contained 1 nM gel-purified M13mp2-gapped DNA substrate, 50 mM Tris-HCl, pH 8.5, 2.5 mM MgCl2,1 mM dithiothreitol, 2 µg of bovine serum albumin, 4% glycerol, and 10 µM (each) dATP, dGTP, dCTP, and dTTP. Reactions were initiated by adding pol IV at 200 nM, incubated at 37 °C or 30 °C for 1 h, and terminated by adding EDTA to 15 mM. Reaction products were analyzed by agarose gel electrophoresis as described (20). Correct synthesis to fill the 407-nucleotide gap in the otherwise double-stranded substrate produces DNA that yields dark blue phage plaques upon introduction into an E. coli {alpha}-complementation strain and plating on indicator plates. Errors are scored as light blue or colorless mutant phage plaques. DNA from independent mutant clones was sequenced to define the sequence change in each lacZ mutant. As described below, pol IV is highly inaccurate, such that most of the lacZ mutant clones contained multiple changes. The error rates presented here are simply the number of observed mutations divided by the number of nucleotides sequenced.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The coding sequence (Fig. 1a) and inferred domain organization of pol IV (Fig. 1b) are similar to those of mammalian family X polymerases but with some clear differences, e.g. the size and/or location of secondary structural elements and loops and the identity of specific residues known to be important for the functions of the mammalian enzymes. Pol IV has the highest homology to pol {lambda}, with these two enzymes sharing 25% identity in their catalytic cores. These similarities and differences suggest that the biochemical properties of pol IV may be similar but not necessarily identical to those of its mammalian homologs. To test this hypothesis, we expressed full-length recombinant pol IV in E. coli and purified it as described under "Experimental Procedures," The purified polymerase (Fig. 2a) was devoid of detectable 3' -> 5' exonuclease activity and exhibited very low processivity during primer extension with an ungapped template (Fig. 2b, gray bars), properties in common with pol {beta} and pol {lambda}.

Gap-filling DNA Synthesis by Pol IV—Pol {beta} and pol {lambda} can both fill short gaps in DNA that are intended to mimic gapped intermediates in BER and NHEJ. Synthesis to fill short gaps by pol {beta} and pol {lambda} is facilitated by a phosphate group on the 5'-end of gaps that can bind to positively charged regions in their 8-kDa domains (12). In contrast, the activity of pol IV is similar when filling five-nucleotide gaps containing either a 5'-phosphate or a 5'-OH group (Fig. 3). Moreover, primer extension reactions conducted under processive reaction conditions (using a high DNA to pol IV ratio, see "Experimental Procedures") revealed that the processivity of pol IV when filling a five-nucleotide gap with a 5'-phosphate (Fig. 2b, black bars) is not greater than during synthesis on an ungapped template. Surprisingly however, gap-filling synthesis is more robust when a synthetic abasic site, i.e. a tetrahydrofuran residue, is present at the 5'-end of the gap (rightmost lanes in Fig. 3).

dRP Lyase Activity of Pol IV—The sequence alignments in Fig. 1a suggest that the helix-hairpin-helix motif needed for the intrinsic dRP lyase activities of pol {beta} and pol {lambda} is also present in pol IV. Moreover, the major nucleophile in pol {beta} (Lys-72) and pol {lambda} (Lys-312) is conserved in pol IV (Lys-248), but not in pol µ, which lacks dRP lyase activity. Three other residues, proposed to facilitate removal of the dRP group in pol {beta} (His-34, Lys-35, and Tyr-39), are conserved or identical in pol IV (Phe-209, Arg-210 and Tyr-214, respectively). Based on this conservation, we tested pol IV for dRP lyase activity. A duplex oligonucleotide containing a 3'-end-labeled strand with an AP site (Fig. 4a) was incubated with pol IV. Like pol {lambda} (Fig. 4b, lane 4), pol IV (lane 2) excised the 5' dRP group, as indicated by the presence of a 19-mer product.



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  		FIG. 1.
Primary structure of pol IV. a, multiple amino acid alignment of yeast DNA polymerase IV with mammalian members of the X family. The invariant residues are white over a black background, and conserved residues are in bold type. b, modular organization of primary structure of yeast pol IV. Ser-Pro corresponds to the serine-proline rich domain of pol {lambda}, and 8 kDa represents the DNA binding 8-kDa domain.

 
The dRP lyase activities of pol {beta} and pol {lambda} proceed through a {beta} elimination mechanism wherein the catalytic lysine forms a Schiff base intermediate that can be trapped as a covalent enzyme-DNA complex by reduction with sodium borohydride (21). To examine if pol IV uses a similar mechanism, dRP lyase reactions were performed in the presence of sodium borohydride, and reaction products were resolved by SDS-PAGE. Pol IV generated a product band (Fig. 4c, lane 2) at the position expected for a pol IV-DNA complex. Although Lys-248 in pol IV aligns with the major catalytic lysines in pol {lambda} and pol {beta} (Fig. 1a), the adjacent residue in pol IV is also a lysine (Lys-247). On that basis, Lys-247 and Lys-248 were concomitantly changed to alanine, and the mutant protein was purified and tested for dRP lyase activity and the formation of a covalent protein-DNA intermediate. Consistent with the importance of one or both of these residues to dRP lyase activity, the mutant protein was less active and generated substantially less covalent complex (lane 3 in Fig. 4, b and c, respectively). Nonetheless, the pol IV mutant protein retained some dRP lyase activity (Fig. 4b), indicating either that Lys-247 and Lys-248 are not catalytic or that another residue(s) can act as a nucleophile in their absence. The latter possibility is consistent with observations with pol {beta} (22), where replacing the primary lysine nucleophile (Lys-72) with alanine also did not completely inactivate dRP lyase activity.



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  		FIG. 2.
Analysis of purified pol IV. a, analysis by SDS-PAGE. Pol IV was purified as described under "Experimental Procedures." Lane 1, 1.5 µg of purified pol IV; lane 2, molecular weight markers. b, processivity analysis. The percentages of termination probabilities were quantified as described under "Experimental Procedures"; the values are an average from three independent determinations. 5 nuc gap, five-nucleotide gap.

 



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  		FIG. 3.
Gap filling by pol IV. Reactions contained 200 nM non-gap template-primer (T/P) or a five-nucleotide gap-containing DNA with a hydroxyl group (5'OH), a phosphate (5'P), or a tetrahydrofuran (5' THF) on the 5'-end of the gap and 5 nM pol IV and were performed as described under "Experimental Procedures." On the left is the sequence of the extended primer.

 



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  		FIG. 4.
Removal of a dRP group by pol IV. a, substrate used for measuring dRP lyase activity prepared as described (10). b, resolution of products of dRP lyase reaction. The reactions were conducted as described under "Experimental Procedures." UDG, uracil DNA glycosylase; C, control; Wt, wild-type; KK/AA, K247A/K248A. c, trapping of polymerase-DNA complexes with sodium borohydride. Lane1, DNA only; lane 2, pol IV; lane 3, pol IV K247A/K248A; lane 4, pol {beta}.

 
Incorporation of Ribonucleoside Triphosphates by Pol IV— Most DNA polymerases discriminate by factors of 104-106 against incorporation of NTPs as compared with dNTPs (23). Exceptions include pol µ (20) and TdT (21), which discriminate against rNTPs by only ~10-fold. Because both of these enzymes are X family members and because rNTP discrimination has not been examined for yeast pol IV, we performed steady state kinetic analyses to determine the efficiency of dNTP and rNTP incorporation into DNA by pol IV. In contrast with pol {lambda} and pol {beta}, which favor incorporation of dATP over rATP by factors approaching 10,000-fold, pol IV incorporates rATP and rCTP only 46-fold and 11-fold less efficiently than it incorporates dATP and dCTP, respectively (Table I). This limited discrimination against rNTP incorporation is similar to that reported previously for pol µ and TdT (15, 24). Upon insertion of a rNTP, further extension by pol IV in the presence of all four rNTPs is inefficient (data not shown). Also notable among the kinetic results is the apparent Km for dATP incorporation by pol IV, which is 23-fold higher than that for incorporation by pol {lambda}, consistent with the possibility that pol IV, like pol µ and TdT, may have a lower dNTP binding affinity than does pol {lambda} (12).


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  		TABLE I
Sugar discrimination by family X DNA polymerases

 
Fidelity of DNA Synthesis by Pol IV—Another property that distinguishes mammalian X family polymerases is their fidelity. On one extreme is TdT, which is a template-independent polymerase. On the other end is pol {beta}, which, although less accurate than the major replicative polymerases in the B family, is considerably more accurate than the translesion synthesis polymerases in the Y family (25). Compared with pol {beta}, pol {lambda} has only slightly lower average base substitution fidelity, but it is generally much less accurate for single base deletion errors (26). In fact, its deletion rate exceeds that of the family Y polymerases. Two studies indicate that pol µ is also prone to generate errors involving misalignment of DNA strands (27). This property indicates that pol {lambda} and pol µ can more readily use template-primers with unpaired bases at and/or near the 3' terminus, perhaps as needed for their roles in NHEJ of substrates that have limited base-pairing homology.

Because yeast pol IV is also implicated in NHEJ (14, 15) and perhaps in BER by virtue of its dRP lyase activity, we determined the base substitution and insertion-deletion fidelity of pol IV. We used an assay that scores a broad range of errors including base substitutions, deletions, and additions of single or multiple nucleotides during synthesis to fill in a 407-nucleotide, single-strand gap in the lacZ{alpha} complementation sequence of the M13mp2 DNA (20). Synthesis by pol IV filled in the gap to completion as determined by analysis of the reaction products by agarose gel electrophoresis (20). These gap-filled reaction products were introduced into E. coli, which were plated to obtain M13mp2 plaques from which the fraction of lacZ{alpha} mutants among total plaques was scored. Pol IV generated mutants with a frequency of 34%. With this same assay, human pol {lambda} generated a 21% frequency and human pol {beta} generated a 3.5% frequency (26). To determine the error specificity of pol IV, DNA from 139 independent lacZ{alpha} mutants was isolated and sequenced. Pol IV primarily generated single base substitutions and single base deletions (Table II), which were distributed throughout the template but with some clustering (Fig. 5). From the frequency and error specificity information, rates were calculated (26) for various errors by type and location (Table II), and these rates were compared with rates for pol {lambda} and pol {beta} from an earlier study (26) using the same approach.


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  		TABLE II
Single-base error rates of X family polymerases

The analysis is based on a total of 139 mutants (56,573 sequenced bases). The total number of changes was 430 and, among these, 202, 178, 10 and 5 were single-base deletions, single-base substitutions, single-base additions, and two-base deletions, respectively. Thirty-five mutants contained other changes, which included closely spaced substitutions, substitution-deletions, substitution-additions, large deletions, and even more complex changes. Error rates were calculated as described under "Experimental Procedures." The number of template sites for calculating error rates were as stated in Ohashi et al. (17).

 



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  		FIG. 5.
Mutation spectrum of pol IV. Five lines of the primary wild-type DNA sequence of the lacZ {alpha} gene, carried in M13mp2, are shown. Position +1 is the first transcribed base. Arrow indicates the direction of synthesis. Letters below the line of the target sequence indicate the new base found in the viral template strand DNA sequence. Deletion of a base is indicated by an open triangle above the line of the sequence, and addition of a base is indicated below the line of the sequence by the symbol {Lambda} above the added base.

 
Yeast pol IV generates T to G transversions (T·dCMP mismatch) at unusually high rates (Table II). The majority (70 of 82) of these substitutions are flanked by 5' template G (Fig. 5; for examples, see template positions –67, 70 and 103), suggesting involvement of a transiently misaligned mutational intermediate (discussed further below). Transient substrate misalignment may also explain the unusually high rate for A to G transitions (Table II), because 25 of 30 of these errors were again flanked by a 5' template G (Fig. 5). Error rates are also extremely high for single-base deletions (Table II). The majority of these occurred in short repeat sequences, again suggesting involvement of misaligned intermediates. Most of the pol IV error rates are higher than those for pol {beta} (Table II). Depending on the type of error, differences are from ~10-fold to >100-fold (e.g. for deletion of non-iterated bases or the T to G transversion at position –67 in the lacZ gene). The fidelity of pol IV is generally more similar to that of human pol {lambda}, but even here differences are apparent, e.g. in the rates for T to G substitutions and in the ratio of base substitutions to deletions (Table II).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The specific combination of yeast pol IV properties described here is unique in comparison to mammalian family X DNA polymerases (Table III). The closest match is to pol {lambda}, with which Pol IV shares the highest homology. For example, pol IV and pol {lambda} both contain a large number of positively charged residues in their 8-kDa domains (Fig. 1A), residues that in pol {lambda} interact with the 5'-phosphate in a short gap in DNA. Despite this, but consistent with an earlier observation (18), gap-filling synthesis by pol IV is not stimulated by a phosphate on the 5'-end of the gap, whereas pol {lambda} activity is stimulated. This difference suggests that pol IV and pol {lambda} may participate in repairing different substrates in vivo. The observation that gap filling synthesis by pol IV is stimulated by the presence of an abasic site on the 5'-end of the gap and the fact that pol IV has dRP lyase activity suggest involvement in repairing DNA with damaged ends, e.g. during NHEJ or BER. Nonetheless, any such roles are not essential for survival, because yeast cells lacking pol IV are not hypersensitive to monofunctional alkylating agents like methylmethane sulfonate (28–30), and they can repair double strand breaks in DNA (19).


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  		TABLE III
Properties of family X polymerases

 
The traditional classification of polymerases as DNA and RNA polymerases is based on their specificity for dNTPs and rNTPs, respectively. Most DNA polymerases have very high sugar selectivity and primarily incorporate dNTPs despite much higher rNTP pools in cells relative to dNTP pools (31, 32). Our data indicate that yeast pol IV is an exception, being the second template-dependent polymerase of the X family shown to incorporate rNTPs relatively efficiently (Table I). This property was unexpected, because the closest mammalian relative of pol IV is pol {lambda}, and pol {lambda} (20, 30), like pol {beta} (15), strongly discriminates against rNTPs. Instead, pol IV sugar discrimination resembles that of pol µ and TdT. In pol {beta}, it has been suggested that rNTPs are excluded due to a steric clash of the 2'-OH with the peptide backbone of residues Tyr-271, Phe-272, and Thr-273. The same can be inferred for pol {lambda}, which conserves these residues. However, the side chains equivalent to the Tyr-271 and Phe-272 of pol {beta} are different in pol IV, pol µ, and TdT, likely contributing to their low sugar discrimination. As proposed for pol µ (15), the ability of pol IV to incorporate rNTPs could allow the enzyme to conduct gap-filling synthesis during NHEJ at the stages of the cell cycle when the dNTP concentrations are low but rNTP concentrations are high. This could be particularly important for pol IV, whose affinity for dNTPs may not be as high as that of pol {lambda} but resembles that of pol µ (Table I). The incorporated ribonucleotide may subsequently be excised by sequential action of RNase HII and Rad27p (33). Thus, closely related enzymes like pol IV and pol {lambda} may have evolved different strategies to perform synthesis at low concentration of nucleotide precursors. Alternatively, incorporation of a ribonucleotide(s) could have a signaling function, e.g. marking a site for repair or other cellular processes. For example, initiation of replication-coupled recombination that leads to mating type switching in Schizosaccharomyces pombe depends on an imprint of one or two ribonucleotide residues (34).

The overall fidelity with which yeast pol IV synthesizes DNA is very low, much lower than that of the yeast replicative DNA polymerases {alpha} (35), {delta} (36), and {epsilon} (37, 38). As suggested previously for human pol {lambda} (26, 39), this low fidelity may be a hallmark of family X polymerases that participate in joining broken DNA ends that can align with minimal base pair homology. This hypothesis for the biological advantage of relaxing substrate specificity in order to deal with DNA damage (e.g. lesions or broken DNA) is somewhat different from that for family Y DNA polymerases such as pol {eta}, pol {kappa}, and pol {iota} that are implicated in translesion DNA synthesis. Those polymerases are also very inaccurate, but they generate all 12 base-base mismatches at high rates by directly misinserting nucleotides in a variety of sequence contexts, a promiscuity suggested to reflect the relaxed active site geometry required to bypass lesions. In contrast, most of the errors generated by yeast pol IV are single base deletions and T to C and A to G substitutions in very specific sequence contexts (Table II and Fig. 5). These errors can all be rationalized by several non-exclusive models of substrate misalignments involving DNA strand slippage and/or dNTP misalignment in the polymerase active site (reviewed in Ref. 25). Particularly notable here is the fact that the error specificity of yeast pol IV (Fig. 5 and Table II) is unique among all DNA polymerases examined to date with the M13mp2 fidelity assay. For example, some pol IV error rates are in common with those of pol {lambda} (for insertion/deletions, Table II, bottom), whereas others are not, and at least one pol IV error rate (T to G at position 103) is in common with those of pol {beta}, whereas most are not. Thus, for fidelity as for the other properties reported here, yeast pol IV appears to be a biochemical hybrid that interacts with template-primers somewhat differently than its biologically more specialized mammalian homologs. That such differences may be relevant to biological function is suggested by recent evidence that differences in template strand interactions near the active sites of TdT, pol {lambda}, and pol µ may help to explain their different roles in NHEJ in vivo.2


   FOOTNOTES
 
* 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. Back

{ddagger} To whom correspondence should be addressed. Tel.: 919-541-2644; Fax: 919-541-7613; E-mail: kunkel{at}niehs.nih.gov.

1 The abbreviations used are: pol, polymerase; BER, base excision repair; BRCT, breast cancer susceptibility gene 1 C terminus; dRP, 5'-2-deoxyribose-5-phosphate; NHEJ, non-homologous end joining. Back

2 S. A. Nick McElhinny, Jody M. Havener, M. Garcia-Diaz, R. Juarez, K. Bebenek, B. L. Kee, L. Blanco, T. A. Kunkel, and D. A Ramsden, submitted for publication. Back


   ACKNOWLEDGMENTS
 
We thank Dmitry Gordenin and William Beard for critically reading the manuscript and providing helpful comments and Dinh Nguyen for assistance in DNA sequencing.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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