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J. Biol. Chem., Vol. 277, Issue 18, 15546-15551, May 3, 2002

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Yeast Rev1 Protein Is a G Template-specific DNA Polymerase^*

Lajos Haracska, Satya Prakash, and Louise Prakash

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

Received for publication, December 19, 2001, and in revised form, February 12, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Rev1 protein of Saccharomyces cerevisiae functions with DNA polymerase  in mutagenic trans-lesion synthesis. Because of the reported preferential incorporation of a C residue opposite an abasic site, Rev1 has been referred to as a deoxycytidyltransferase. Here, we use steady-state kinetics to examine nucleotide incorporation by Rev1 opposite undamaged and damaged template residues. We show that Rev1 specifically inserts a C residue opposite template G, and it is ~25-, 40-, and 400-fold less efficient at inserting a C residue opposite an abasic site, an O⁶-methylguanine, and an 8-oxoguanine lesion, respectively. Rev1 misincorporates G, A, and T residues opposite template G with a frequency of ~10³ to 10⁴. Consistent with this finding, Rev1 replicates DNA containing a string of Gs in a template-specific manner, but it has a low processivity incorporating 1.6 nucleotides per DNA binding event on the average. From these observations, we infer that Rev1 is a G template-specific DNA polymerase.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The REV1- and RAD30-encoded proteins of Saccharomyces cerevisiae are members of the Y family of DNA polymerases (1). All of the proteins in this family share five highly conserved motifs from I to V, but they differ in their ability to promote replication through DNA lesions (2, 3). By contrast to the RAD30-encoded DNA polymerase , which incorporates all four deoxynucleotides in a template-specific manner and has the ability to replicate through a variety of DNA lesions (4-7), the Rev1 protein has been reported to preferentially incorporate a C residue opposite an abasic site (8). Rev1 also could incorporate a C residue opposite templates G and A but was only ~20 and 10% as effective, respectively, in these reactions as the insertion opposite an abasic site (8). Because of the preferential ability of Rev1 to insert a C residue opposite an abasic site, a noninformational DNA lesion, this activity has been referred to as a deoxycytidyltransferase.

The Rev1 and Rad30 proteins differ also in the manner in which they contribute to the replication of UV-damaged DNA. Whereas Rev1 functions with the REV3, REV7-encoded DNA polymerase  (9) in the mutagenic bypass of UV lesions, polymerase  (Pol)¹ promotes the error-free bypass of UV-induced cyclobutane pyrimidine dimers. Pol replicates through a cis-syn-thymine-thymine (T-T) dimer with the same efficiency and accuracy as through undamaged Ts (10, 11), and genetic studies in yeast have also indicated a role for Pol in the error-free bypass of cyclobutane dimers formed at 5'-TC-3' and 5'-CC-3' sites (12). Pol, which is a member of the Pol family, promotes lesion bypass by extending from the nucleotide inserted by another DNA polymerase opposite the 3'-T of a T-T dimer or a (6-4)TT photoproduct (13, 14). Although the Rev1 protein is almost as indispensable for UV mutagenesis as is Pol, its C-transferase activity is not needed for this function, because C insertion occurs only rarely opposite the UV lesions in yeast (15, 16) and inactivation of this biochemical activity has no impact on UV mutagenesis.²

The mutagenic bypass of abasic sites also requires Rev1 and Pol (17), and it depends upon the sequential action of two DNA polymerases in which one inserts the nucleotide opposite the abasic site and Pol subsequently extends from the inserted nucleotide (18). Although Rev1 is able to insert a C residue opposite an abasic site in an in vitro reaction, the inactivation of Rev1 C-transferase activity causes no significant reduction in the incidence of mutations resulting from the bypass of abasic sites (18). This result is probably because of the fact that many different polymerases, including Pol and Pol, contribute to the insertion step (18, 19). The indispensability of the Rev1 protein, but not of its C-transferase activity, for the mutagenic bypass of UV lesions as well as that of abasic sites has suggested that the primary role of Rev1 in the mutagenic bypass of these lesions is structural. In this role, Rev1 could act as an intermediary, promoting the assembly of Pol with Pol stalled at a lesion site (18).

To provide for a better understanding of the biochemical activity of Rev1 protein, here we employ steady-state kinetic analyses to examine the efficiency of nucleotide incorporation by this protein on undamaged and damaged DNA templates.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Proteins and DNA Substrates-- S. cerevisiae Rev1 protein in fusion with glutathione S-transferase (8) was expressed in the yeast strain LY2 and purified on a glutathione-Sepharose 4B column followed by MiniS (Amersham Biosciences) chromatography as described previously (18). Oligonucleotides were synthesized by Midland Certified Reagent Co. (Midland, TX). DNA substrates (S1-) were generated by annealing a 52-nucleotide (nt)-long oligonucleotide template 5'-TTC GTA TAA TGC CTA CAC TXG AGT ACC GGA GCA TCG TCG TGA CTG GGA AAA C-3', which contained a G (S1-G), an A (S1-A), a T (S1-T), a C (S1-C), or an abasic site (a tetrahydrofuran moiety) (S1-AP) at position 20 (X) to the 32-nt 5' ³²P-labeled oligonucleotide primer N4456, 5'-GTT TTC CCA GTC ACG ACG ATG CTC CGG TAC TC-3'. DNA substrates (S2-) were generated by annealing a 75-nt oligomer template 5'-AGC TAC CAT GCC TGC CTC AAG AAT TCG TAA XAT GCC TAC ACT GGA GTA CCG GAG CAT CGT CGT GAC TGG GAA AAC-3' containing a G (S2-G), a uracil (S2-U), an abasic site (S2-AP), a 7,8-dihydro-8-oxoguanine (S2-8oxoG), or a O⁶-methylguanine (S2-m6G) at position 31 (X) to the 5'-³²P-labeled oligonucleotide primer N4309, 5'-GTT TTC CCA GTC ACG ACG ATG CTC CGG TAC TCC AGT GTA GGC AT-3'. For DNA substrates shown in Fig. 1B, the 75-nt template used for generating the substrate S2-G was annealed to the 5'-³²P-labeled oligonucleotide primer N4577, 5'-GTT TTC CCA GTC ACG ACG ATG CTC CGG TA-3', yielding substrate S3, or this template was annealed to the 5'-³²P-labeled oligonucleotide primer N4265, 5'-GTT TTC CCA GTC ACG ACG ATG CTC CGG TAC TCC AGT GTA GG-3', yielding substrate S4. S5 DNA substrate (Fig. 2) was generated by annealing a 43-nt oligonucleotide template, 5'-GGG GGG GGG GGG AGT ACC GGA GCA TCG TCG TGA CTG GGA AAA C-3', to the 5'-³²P-labeled oligonucleotide primer N4456.

Deoxynucleotide Incorporation Assays-- A standard primer extension reaction (10 µl) contained 40 mM Tris-HCl (pH 7.5), 8 mM MgCl₂, 1 mM dithiothreitol, 100 µg/ml bovine serum albumin, 10% glycerol, 10 nM 5'-³²P-labeled oligonucleotide primer annealed to an oligonucleotide template, 100 µM of a single or each of all four dNTPs, and 1-5 nM GST-Rev1 protein. In Figs. 1B and 2, instead of the Tris buffer, the reactions were carried out in a phosphate buffer containing 25 mM potassium phosphate (pH 7.4), 6 mM MgCl₂, 1 mM dithiothreitol, 100 µg/ml bovine serum albumin, 10% glycerol. Assays were assembled on ice, incubated at 30 °C for 10 min, and stopped by the addition of loading buffer (40 µl) containing EDTA (20 mM), 95% formamide, 0.3% bromphenol blue, 0.3% cyanol blue. The reaction products were resolved on 10% polyacrylamide gels containing 8 M urea. The quantitation of the results was done using a Molecular Dynamics STORM PhosphorImager and ImageQuant software.

Processivity Assays-- Rev1 (3 nM) was preincubated with the DNA substrate (10 nM) in phosphate buffer that contained no deoxynucleotides for 5 min at 30 °C. Reactions were initiated by adding all four deoxynucleotides at increasing concentration (0.5-100 µM each) or all four deoxynucleotides plus 100-fold excess of nonradiolabeled DNA substrate as a trap followed by 10-min incubation at 30 °C. To demonstrate the effectiveness of the trap, Rev1 was preincubated with the DNA trap and the primer-template substrate before the addition of dNTPs. The processivity of Rev1 at 100 µM dNTP concentration was calculated as described previously (20). Gel band intensities (I_n) of the deoxynucleotide incorporation products were quantitated at each band position (n). Processivity is defined as the probability, P_n, that the enzyme will incorporate the next deoxynucleotide rather than dissociating. The processivity of Rev1 at a particular band position was calculated by using the equation: P_n = (I_n+1 + I_n+2 + ... )/(I_n + I_n+1 + I_n+2 + ... ).

Analysis of Steady-state Kinetics-- Steady-state kinetic analyses for deoxynucleotide incorporation opposite undamaged G, A, T, and C residues or an abasic site, a 8-oxoG, or a m6G were performed as described previously (21, 22). Rev1 (1 nM) was incubated with 10 nM DNA substrate in the presence of increasing concentrations of a single deoxynucleotide for 10 min in a Tris buffer. Gel band intensities of the substrates and products were quantitated by PhosphorImager, and the observed rate of deoxynucleotide incorporation was plotted as a function of dNTP concentration. The data were fit by nonlinear regression using SigmaPlot 5.0 to the Michaelis-Menten equation describing a hyperbola, v = (V_max × [dNTP]/(K_m + [dNTP]). Apparent K_m and V_max steady-state parameters were obtained from the fit and were used to calculate the efficiency of deoxynucleotide incorporation (k_cat/K_m).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Nucleotide Incorporation by Rev1-- First we examined whether Rev1, besides its deoxycytidyltransferase activity, is able to incorporate other nucleotides opposite various undamaged and damaged template residues. DNA substrates containing a different template nucleotide at the primer:template junction were incubated with Rev1 in the presence of just one dNTP (Fig. 1A). Rev1 protein incorporated a C residue across from each of the four undamaged template nucleotides and also with various efficiencies opposite uracil, 8-oxoG, m6G, and abasic site. Whereas the C residue was incorporated most readily opposite template G, Rev1 also incorporated the other nucleotides G, A, and T opposite template G (Fig. 1A, lanes 1-4). Because yeast Rev1 was shown to have only a deoxycytidyltransferase activity in the previous study (8), we examined the dependence of these other nucleotide incorporations by Rev1 on the sequence context and on the Tris buffer we used instead of the phosphate buffer employed in the previous study. The incorporation by Rev1 of each of four deoxynucleotides opposite a template G residue present in two other sequence contexts was examined in phosphate buffer (Fig. 1B). Here also, Rev1 incorporated all four nucleotides opposite template G; however, the level of incorporation of the G, A, and T nucleotides varied depending on the sequence context of the template (Fig. 1B, compare lanes 1-3 with lanes 7-9). Rev1 inserted a C residue opposite uracil, 8-oxoG, and m6G, whereas opposite an abasic site in addition to a C, Rev1 inserted some G and T as well (Fig. 1A).

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Nucleotide incorporation by Rev1 protein. A, Specificity of deoxynucleotide incorporation by Rev1 protein. Nucleotide sequence adjacent to the primer:template junction is shown for each of the DNA substrates. Rev1 (3 nM) was incubated with the DNA substrate (10 nM) in a Tris buffer in the absence (-) or presence of a single (G, A, T, or C) or all four deoxynucleotides (N) (100 µM each) for 10 min at 30 °C. The concentration of product formed (in nM) was determined by phosphorimaging; dashes indicate that no detectable product was formed. B, deoxynucleotide incorporation by Rev1 opposite various template G residues in different sequence contexts. DNA substrates (10 nM) were incubated with Rev1 (5 nM) in a phosphate buffer for 10 min at 30 °C.

DNA Synthesis by Rev1 on a G-containing Template-- Next, we examined whether Rev1 could synthesize DNA on a template containing a string of Gs in a template-specific manner, and we found that Rev1 could in fact synthesize DNA almost to the end of this template, specifically inserting a C residue (Fig. 2A, lanes 5 and 6). This observation raised the possibility that instead of transferring only one nucleotide in a single DNA binding event, Rev1 was able to polymerize DNA, like DNA polymerases, in a G template-dependent manner and could incorporate more than one nucleotide per DNA binding event. We measured the processivity of Rev1 on a DNA substrate containing a string of 11 Gs toward the 5' end of the template right after the primer:template junction (Fig. 2B). Processivity is a measure of how many deoxynucleotides a DNA polymerase incorporates in a single DNA binding event. To ensure that we were observing deoxynucleotide incorporation resulting from a single DNA binding event, we monitored DNA synthesis by Rev1 in the presence of excess of nonradiolabeled DNA substrate as a trap (Fig. 2B). The reactions were performed by first preincubating Rev1 with the radiolabeled DNA substrate without deoxynucleotide. DNA synthesis was initiated by the addition of increasing concentrations of all four deoxynucleotides (Fig. 2B, lanes 1-7) or a mixture of 100-fold excess of the same but nonradiolabeled DNA substrate and all four deoxynucleotides to the reaction (Fig. 2B, lanes 8-14). Under these conditions, any Rev1 molecule that dissociates from the labeled DNA substrate will be trapped by the excess of nonradiolabeled DNA. The effectiveness of the trap was verified by first preincubating Rev1 with the radiolabeled DNA substrate and the excess nonlabeled DNA followed by the addition of deoxynucleotides. The lack of DNA synthesis in this sample shows that the 100-fold excess of nonlabeled DNA is sufficient to trap all Rev1 molecules (Fig. 2B, lane 15). In the presence of saturating nucleotide concentration, Rev1 copied the template DNA, incorporating as many as nine nucleotides in a single DNA binding event (Fig. 2B, lane 14).

View larger version (22K): [in this window] [in a new window]   Fig. 2.   DNA polymerization reaction on a poly(G) DNA template by Rev1. A, specificity of deoxynucleotide incorporation by Rev1 opposite Gs in a poly(G) template. Nucleotide sequence adjacent to the primer:template junction is shown. DNA substrate (10 nM) was incubated with Rev1 (3 nM) in a phosphate buffer in the absence (-) or presence of a single (G, A, T, or C) or all four deoxynucleotides (N) (100 µM each) for 10 min at 30 °C. B, nucleotide incorporation by Rev1 resulting from a single DNA binding event. Rev1 (3 nM) was preincu- bated with the DNA substrate (10 nM) without deoxynucleotides for 5 min at 30 °C. Reactions were initiated by adding all four deoxynucleotides at increasing concentration (0.5-100 µM each) or all four deoxynucleotides plus 100-fold excess of nonradiolabeled DNA substrate as a trap followed by 10-min incubation at 30 °C. As the control to demonstrate the effectiveness of the trap, Rev1 was preincubated with the DNA trap and the primer-template substrate before the addition of dNTPs (lane 15). C, processivity of Rev1. The percentage of Rev1 molecules incorporating at least n deoxynucleotides was calculated from quantitation of the intensity of each of the bands in Fig. 2B, lane 14, as described under "Materials and Methods." The percentage of Rev1 adding at least one deoxynucleotide was set as 100%.

The percentage of active Rev1 molecules was calculated from the intensities of bands in Fig. 2B, lane 14 (see "Materials and Methods"), and the percentage of Rev1 molecules adding at least one deoxynucleotide was set as 100%. The percentage of active Rev1 molecules decreased after the addition of each subsequent nucleotide because of the dissociation of some fraction of Rev1 molecules from DNA (Fig. 2C). The processivity of Rev1, defined quantitatively as the probability (P_n) for each deoxynucleotide incorporation event n that Rev1 will move ahead to incorporate the next nucleotide n + 1 rather than dissociate from the DNA template, on this DNA substrate is 0.61. Thus, after each nucleotide incorporation, on average 61% of the bound Rev1 molecules incorporate at least one additional deoxynucleotide, whereas 39% dissociate from the DNA substrate, which leads to an incorporation of 1.6 nucleotides per DNA binding event on the average. Thus, Rev1 shares the low processivity characteristic of the Y family of DNA polymerases.

Steady-state Kinetic Analysis of Nucleotide Insertion by Rev1 Opposite Undamaged Bases-- Rev1 protein has been designated as a deoxycytidyltransferase based upon its ability, examined at saturating nucleotide concentrations, to insert a C nucleotide more efficiently opposite a template abasic site, a noninstructional lesion, than opposite template G (8). This designation may suggest that Rev1 does not monitor the information of the template strand and is not very efficient at distinguishing between correct and incorrect nucleotide insertion. To characterize further the specificity of the DNA polymerization reaction catalyzed by Rev1, we analyzed the steady-state kinetics of nucleotide incorporation opposite undamaged and damaged template residues by Rev1. The kinetics of insertion of a single deoxynucleotide opposite undamaged G, A, T, or C were determined as a function of deoxynucleotide concentration under steady-state conditions. From the kinetics of deoxynucleotide incorporation, the steady-state apparent K_m and k_cat values for each deoxynucleotide were obtained from the curve fitted to the Michaelis-Menten equation. The frequency of nucleotide misincorporation, f_inc, was calculated as the ratio of the efficiency (k_cat/K_m) of incorrect nucleotide incorporation to the efficiency (k_cat/K_m) of correct nucleotide incorporation (Table I).

As indicated by the k_cat/K_m values, Rev1 incorporates a C opposite template G ~130-500-fold more efficiently than opposite template A, T, or C. Opposite template G, Rev1 incorporates the correct C ~500-, 1000-, or 15000-fold more efficiently than it inserts the incorrect G, T, or A, respectively (Table I). This finding indicates that while inserting nucleotides opposite undamaged template bases, Rev1 uses the information of the template and discriminates between the correct and incorrect nucleotides.

Steady-state Kinetic Analysis of Nucleotide Incorporation by Rev1 Opposite Damaged Bases-- Next, we compared the efficiencies of deoxynucleotide insertion opposite an undamaged G, an abasic site, an m6G, and an 8-oxoG (Fig. 3). As judged from the k_cat/K_m values, Rev1 inserts a G, an A, a T, or a C ~4-, 6-, 3-, 24-fold less efficiently, respectively, opposite an AP site than opposite an undamaged G (Table 1). In another sequence context, Rev1 incorporated a C residue opposite an AP site ~27-fold less efficiently than opposite an undamaged G template residue (Fig. 3 and Table I). Rev1 inserted a C residue opposite an m6G lesion, ~39-fold less efficiently, and opposite an 8-oxoG lesion ~370-fold less efficiently than opposite an undamaged G (Fig. 3 and Table I). Thus, Rev1 is sensitive to DNA lesions, and its activity is inhibited on lesion containing DNA substrates.

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Steady-state kinetic analysis of deoxynucleotide incorporation by Rev1. A, dCTP incorporation opposite an undamaged G, an AP site, an m6G, and an 8-oxoG. Rev1 (1 nM) was incubated with the primer:template DNA (10 nM) and with the indicated concentrations of dCTP for 10 min at 30 °C. B, quantitation of dCTP incorporation reactions. The rate of incorporation is graphed as a function of dCTP concentration, and the data are fit to the Michaelis-Menten equation. The kcat and Km parameters obtained from the fit are listed in Table I.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Here, we show that Rev1 is most efficient at inserting a C opposite template G, and Rev1 replicates a poly(dG) template DNA by specific insertion of Cs, and on this DNA, it incorporates 1.6 nucleotides per DNA binding event on the average. Rev1 misincorporates nucleotides opposite template G with a frequency of ~10³ to 10⁴. In its fidelity and processivity on the G template, Rev1 resembles Pol, which misincorporates nucleotides opposite G with a similar frequency and displays a similar low processivity (20).

In contrast to a previous study that reported that Rev1 was ~5-fold more efficient at inserting a C opposite an abasic site than opposite template G (8), we found that Rev1 is over 20-fold better at inserting C opposite undamaged G than opposite an abasic site. Furthermore, our steady-state kinetic studies show that the more proficient insertion of C opposite template G than that opposite an abasic site results from a reduction in the apparent K_m for the nucleotide, whereas the k_cat stays approximately the same (Table I). Because the previous study was qualitative performed with a single saturating concentration of the nucleotide, one would not have detected the differences in K_m that we report here.

We also examined Rev1 for its ability to insert a C opposite two other DNA lesions, m6G and 8-oxoG. Rev1 inserted a C opposite m6G approximately 40-fold less efficiently than opposite an undamaged G, and in this case as well, the inefficient insertion of C opposite m6G was almost entirely attributed to an increase in the K_m for the nucleotide (Table I). Rev1 was very inefficient at inserting a C opposite 8-oxoG, because it did so ~370-fold less efficiently than the insertion of C opposite template G. This poor nucleotide insertion opposite 8-oxoG results from an ~100-fold increase in the K_m for the nucleotide and from an ~5-fold decrease in the k_cat for the enzyme (Table I). Thus, Rev1 is most conducive for nucleotide incorporation when bound to the template G than when bound to the other undamaged template nucleotides or to DNA lesions. This finding may suggest that Rev1 more readily adopts a configuration suitable for binding the C nucleotide when it is bound to the G template than when bound to the other template nucleotides.

The proficient ability of Rev1 to insert a C opposite template G and to replicate poly(dG) DNA in a template-specific manner implies that Rev1 is a G template-specific DNA polymerase. Although Rev1 can also incorporate a C opposite templates T, A, and C, it does so with a frequency of ~10² to 10³. In this regard also, Rev1 resembles Pol, which misincorporates a C opposite these templates with a similar frequency (20). However, in its specificity for primarily incorporating a C opposite template G, Rev1 is more similar to human Pol (13, 23), another member of the Y polymerase family, than to Pol. Pol is most efficient at inserting the correct nucleotide T opposite template A, and opposite this template, it misincorporates with a frequency of 10⁴ to 10⁶. Although Pol incorporates the correct nucleotides C and G opposite templates G and C, respectively, it is much less efficient and accurate opposite these templates than opposite template A (13). Pol is highly inefficient at inserting nucleotides opposite template T, and moreover, it incorporates a G opposite this template ~10-fold better than an A (13). Thus, Pol is most efficient and accurate on template A, and on the other three template nucleotides, it is much less efficient and accurate. However, in striking contrast to Pol, Rev1 is unable to insert the other three nucleotides with any reasonable efficiency, and its ability to primarily insert a C represents the most extreme deviation from normal polymerase behavior.

Rev1 and Pol resemble each other in their ability to insert nucleotides opposite an abasic site. However, in contrast to the specific insertion of a C opposite this lesion by Rev1, Pol primarily incorporates an A, and to a lesser extent, a G opposite this lesion site (13). The specificity of Rev1 for C insertion opposite an abasic site is enigmatic, because when a pyrimidine is positioned opposite the abasic site, both the pyrimidine and the abasic sugar are extrahelical and the helix collapses (24). By contrast, when an A is positioned opposite the AP site, the DNA retains the B-form, and both the unpaired A and the abasic residues remain intrahelical (24-26). Consequently, most DNA polymerases tend to insert an A opposite an abasic site (27). At low temperatures, a G opposite an abasic site is also predominantly intrahelical, and similar to Pol, Pol inserts a G or an A opposite an abasic site (19).

Pre-steady-state kinetic analyses of Pol have indicated a two-step nucleotide binding mechanism in which the DNA bound polymerase first binds the nucleotide and then undergoes the rate-limiting induced fit conformational change prior to the chemical step of phosphodiester bond formation (28). The preference of Rev1 to insert a C opposite template G and also opposite DNA lesions might suggest that Rev1 undergoes the induced fit conformational change prior to the chemical step of phosphodiester bond formation far more readily when a C nucleotide is bound in the active site of the enzyme. In the presence of nucleotides other than C, such a conformational change may not occur or it becomes too slow. The over 20-fold preferential incorporation of a C opposite template G than opposite an abasic site and even a greater inhibition of C insertion opposite 8-oxoG and m6G lesions additionally suggest that the presence of G in the template is also important for such a putative conformational change to occur.

In conclusion, our results indicate that Rev1 is a G template-specific DNA polymerase. However, because of its inability to incorporate all four deoxynucleotides during the synthesis reaction, we consider it inappropriate to assign it a Greek letter nomenclature used for eukaryotic DNA polymerases.

	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.

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

Published, JBC Papers in Press, February 15, 2002, DOI 10.1074/jbc.M112146200

² L. Haracska, S. Prakash, and L. Prakash, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: Pol, polymerase; nt, nucleotide; AP, abasic site; m6G, O⁶-methylguanine; 8-oxoG, 8-oxoguanine.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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				S. Sabbioneda, B. K. Minesinger, M. Giannattasio, P. Plevani, M. Muzi-Falconi, and S. Jinks-Robertson The 9-1-1 Checkpoint Clamp Physically Interacts with Pol{zeta} and Is Partially Required for Spontaneous Pol{zeta}-dependent Mutagenesis in Saccharomyces cerevisiae J. Biol. Chem., November 18, 2005; 280(46): 38657 - 38665. [Abstract] [Full Text] [PDF]

				N. Acharya, L. Haracska, R. E. Johnson, I. Unk, S. Prakash, and L. Prakash Complex Formation of Yeast Rev1 and Rev7 Proteins: a Novel Role for the Polymerase-Associated Domain Mol. Cell. Biol., November 1, 2005; 25(21): 9734 - 9740. [Abstract] [Full Text] [PDF]

				D. T. Nair, R. E. Johnson, L. Prakash, S. Prakash, and A. K. Aggarwal Rev1 Employs a Novel Mechanism of DNA Synthesis Using a Protein Template Science, September 30, 2005; 309(5744): 2219 - 2222. [Abstract] [Full Text] [PDF]

				W. T. Wolfle, M. T. Washington, E. T. Kool, T. E. Spratt, S. A. Helquist, L. Prakash, and S. Prakash Evidence for a Watson-Crick Hydrogen Bonding Requirement in DNA Synthesis by Human DNA Polymerase {kappa} Mol. Cell. Biol., August 15, 2005; 25(16): 7137 - 7143. [Abstract] [Full Text] [PDF]

				T. Okuda, X. Lin, J. Trang, and S. B. Howell Suppression of hREV1 Expression Reduces the Rate at Which Human Ovarian Carcinoma Cells Acquire Resistance to Cisplatin Mol. Pharmacol., June 1, 2005; 67(6): 1852 - 1860. [Abstract] [Full Text] [PDF]

				J. G. Jansen, A. Tsaalbi-Shtylik, P. Langerak, F. Calléja, C. M. Meijers, H. Jacobs, and N. de Wind The BRCT domain of mammalian Rev1 is involved in regulating DNA translesion synthesis Nucleic Acids Res., January 13, 2005; 33(1): 356 - 365. [Abstract] [Full Text] [PDF]

				M. T. Washington, I. G. Minko, R. E. Johnson, L. Haracska, T. M. Harris, R. S. Lloyd, S. Prakash, and L. Prakash Efficient and Error-Free Replication past a Minor-Groove N2-Guanine Adduct by the Sequential Action of Yeast Rev1 and DNA Polymerase {zeta} Mol. Cell. Biol., August 15, 2004; 24(16): 6900 - 6906. [Abstract] [Full Text] [PDF]

				D. Guo, Z. Xie, H. Shen, B. Zhao, and Z. Wang Translesion synthesis of acetylaminofluorene-dG adducts by DNA polymerase {zeta} is stimulated by yeast Rev1 protein Nucleic Acids Res., February 11, 2004; 32(3): 1122 - 1130. [Abstract] [Full Text] [PDF]

S. Covo, L. Blanco, and Z. Livneh Lesion Bypass by Human DNA Polymerase {micro} Reveals a Template-dependent, Sequence-independent Nucleotidyl Transferase Activity J. Biol. Chem., Janu