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Molecular and Cellular Biology, October 2005, p. 8748-8754, Vol. 25, No. 19
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.19.8748-8754.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Human DNA Polymerase Promotes Replication through a Ring-Closed Minor-Groove Adduct That Adopts a syn Conformation in DNA

William T. Wolfle,¹ Robert E. Johnson,¹ Irina G. Minko,² R. Stephen Lloyd,² Satya Prakash,¹ and Louise Prakash^1*

Sealy Center for Molecular Science, University of Texas Medical Branch at Galveston, 6.104 Blocker Medical Research Building, 11th and Mechanic Streets, Galveston, Texas 77555-1061,¹ Center for Research in Occupational and Environmental Toxicology, Oregon Health and Science University, Portland, Oregon 97239²

Received 16 June 2005/ Returned for modification 7 July 2005/ Accepted 11 July 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Acrolein, an ,ß-unsaturated aldehyde, is generated in vivo as the end product of lipid peroxidation and from oxidation of polyamines. The reaction of acrolein with the N² group of guanine in DNA leads to the formation of a cyclic adduct, -hydroxy-1,N²-propano-2'-deoxyguanosine (-HOPdG). Previously, we have shown that proficient replication through the -HOPdG adduct can be mediated by the sequential action of human DNA polymerases (Pols) and , in which Pol incorporates either pyrimidine opposite -HOPdG, but Pol extends only from the cytosine. Since -HOPdG can adopt either a ring-closed cyclic form or a ring-opened form in DNA, to better understand the mechanisms that Pols and employ to promote replication through this lesion, we have examined the ability of these polymerases to replicate through the structural analogs of -HOPdG that are permanently either ring closed or ring opened. Our studies with these model adducts show that whereas the ring-opened form of -HOPdG is not inhibitory to synthesis by human Pols , , or , only Pol is able to incorporate nucleotides opposite the ring-closed form, which is known to adopt a syn conformation in DNA. From these studies, we infer that (i) Pols , , and have the ability to proficiently replicate through minor-groove DNA lesions that do not perturb the Watson-Crick hydrogen bonding of the template base with the incoming nucleotide, and (ii) Pol can accommodate a minor-groove-adducted template purine which adopts a syn conformation in DNA and forms a Hoogsteen base pair with the incoming nucleotide.

Top Abstract Introduction Materials and Methods Results Discussion References

 
DNA polymerases of the Y family promote replication through distorting DNA lesions. DNA polymerase (Pol) is distinct from other members of this family by virtue of its proficient ability to replicate through cyclobutane pyrimidine dimers, and Pol from both Saccharomyces cerevisiae and humans incorporates an A opposite the 3' T and the 5' T of a cis-syn TT dimer with the same efficiency and fidelity as it does opposite the corresponding undamaged T's (5, 8, 22, 25). This unique proficiency of Pol derives from an open active site that is able to accommodate both nucleotides of the cyclobutane pyrimidine dimer (20). Inactivation of Pol in humans causes the variant form of xeroderma pigmentosum (4, 14) characterized by a high incidence of sunlight-induced skin cancers.

In addition to Pol, humans have two other Y family DNA polymerases, Pol and Pol. Although Pols , , and are all low-fidelity DNA polymerases, they differ in their nucleotide incorporation specificities opposite different template bases. Similar to high-fidelity replicative polymerases, Pols and incorporate nucleotides opposite the four template bases with nearly equivalent efficiencies and fidelities (6, 8, 23), while Pol incorporates nucleotides opposite the four template bases with very different efficiencies and fidelities (2, 7, 19, 21). Pol exhibits the highest efficiency and fidelity opposite template A; opposite template G, however, it incorporates nucleotides with a lower efficiency and fidelity than it does opposite template A. Relative to template purines, Pol is highly inefficient at inserting the correct nucleotide opposite template pyrimidines.

A major insight into the unusual nucleotide incorporation pattern of Pol has emerged from the recently determined crystal structure of the ternary complex of Pol bound to the template-primer junction of DNA and an incoming nucleotide (16). In the structure, the template A adopts a syn conformation and forms a Hoogsteen base pair with the incoming T which remains in the anti conformation. The ability of Pol to utilize Hoogsteen base pairing for synthesis opposite template A provides an explanation for the higher efficiency and fidelity of nucleotide incorporation opposite template purines relative to pyrimidines, since only purine bases have a Hoogsteen edge and can participate in hydrogen bonding with the incoming pyrimidine nucleotide that retains the anti conformation.

The ability of Pol to accommodate the template purines in syn conformation provides for an elegant mechanism for replicating through the minor-groove adducts of purines since the distorting lesion would then be displaced into the major groove, where there is much less steric interference. In keeping with this proposal, we have recently shown that Pol is able to promote efficient replication through the N²-guanine acrolein adduct by incorporating nucleotides opposite the lesion site (24).

The reaction of acrolein, an ,ß-unsaturated aldehyde, with the N² of a guanine followed by ring closure at N1 results in the formation of the cyclic adduct -hydroxy-1,N²-propano-2'-deoxyguanosine (-HOPdG) (Fig. 1). -HOPdG presents a strong block to synthesis by replicative DNA polymerases (9), and synthesis by Pol is also inhibited opposite this lesion, particularly at the nucleotide incorporation step (15). Efficient and error-free replication through this lesion, however, can be mediated by the sequential action of Pols and , in which Pol incorporates the nucleotide opposite the lesion site, and Pol performs the subsequent extension step (24).

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FIG. 1. Structure of acrolein and related deoxyguanine adducts.

As a free nucleoside and in single-stranded DNA, -HOPdG exists predominantly in the ring-closed form (12, 17), whereas in duplex DNA, the exocyclic ring opens to form N²-(3-oxo-propyl)-2'-deoxyguanosine (Fig. 1) when -HOPdG is paired with a C. In the ring-opened conformation, the adducted G forms a normal Watson-Crick base pair with the C, and the N²-propyl chain stays in the minor groove, pointing toward the solvent (1, 10, 11). From these studies, it has been hypothesized that -HOPdG would adopt the closed cyclic form when it is a template base at the replication fork, whereas the incorporation of a C opposite -HOPdG would trigger the change from the closed cyclic form to the open conformation (1).

To delineate the mechanisms of Pol- and Pol-mediated replication through the -HOPdG adduct, we have utilized two model N²-dG adducts that structurally resemble either the ring-opened or the ring-closed form of -HOPdG. In the presence of a reducing agent, -HOPdG can be trapped as the N²-(3-hydroxy propyl)-2'-deoxyguanosine adduct, (r) -HOPdG, which permanently stays in the ring-opened conformation (Fig. 1). By contrast, another structural analog of -HOPdG, 1,N²-propano-2'-deoxyguanosine (PdG), permanently stays in the ring-closed form because of the lack of the hydroxyl group (Fig. 1). The nuclear magnetic resonance studies of a PdG present in the duplex DNA have shown that the adducted base adopts a syn conformation and forms a PdG(syn) · dC⁺(anti) Hoogsteen base pair with the opposing C (18, 27).

The propensity of PdG to adopt a syn conformation in DNA has suggested that -HOPdG present in the ring-closed form at the template site would similarly assume a syn conformation in DNA. -HOPdG and PdG, however, would differ in the conformations they adopt following their pairing with a C residue. Since the pairing with a C would shift the equilibrium of the -HOPdG to the ring-opened form, that would enable the formation of a normal -HOPdG(anti) · dC(anti) Watson-Crick base pair, whereas for the ring-closed PdG, the PdG(syn) · dC⁺(anti) pair would stay in the Hoogsteen configuration. In the ring-opened reduced -HOPdG, the adducted G would retain the anti conformation and the ability to form a normal Watson-Crick base pair with a C.

Here we show that Pol efficiently incorporates nucleotides opposite the PdG lesion, but neither it nor Pol promotes the subsequent extension reaction. By contrast, (r) -HOPdG is not inhibitory to replication by either of these polymerases, and they both can carry out efficient nucleotide insertion and extension reactions opposite from this lesion. From these observations, we conclude that Pol's ability to incorporate nucleotides opposite -HOPdG derives from its unique ability to accommodate a Hoogsteen base pair in its active site. However, for Pol to promote the subsequent extension reaction, the -HOPdG has to adopt a ring-opened configuration able to form a normal Watson-Crick -HOPdG(anti) · dC(anti) base pair at the primer terminus.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Purification of DNA polymerases. Yeast strain BJ5464 was transformed with either plasmid pPOL42 or plasmid pPOL114, which carries the genes encoding wild-type human Pol or Pol, respectively, fused in frame with glutathione S-transferase (2, 3, 6, 7). Yeast strain BJ5464 harboring either pPOL42 or pPOL114 was grown and harvested by centrifugation at 4°C. The yeast cells were resuspended in cell breakage buffer (50 mM Tris-HCl [pH 7.5], 10% sucrose, 300 mM NaCl, 1 mM EDTA, 10 mM ß-mercaptoethanol, 0.5 mM benzamidine, 0.5 mM phenylmethylsulfonyl fluoride, and 5 µg/µl each of aprotinin, chymostatin, pepstatin A, and leupeptin) and lysed by a French press prior to centrifugation (100,000 x g, 60 min). The resulting cell extract was passed over a glutathione-Sepharose 4B column (Amersham Pharmacia) at 4°C, and the column was subsequently washed with 10 column volumes of cell breakage buffer containing 1 M NaCl. The column was then equilibrated three times with 1 column volume of elution buffer (100 mM Tris-HCl [pH 8.0], 100 mM NaCl, 10% glycerol, 0.01% Nonidet P-40), the glutathione S-transferase tag was cleaved with PreScission protease (Amersham Pharmacia), and the polymerase batch eluted. This resulted in a seven-amino-acid leader peptide remaining attached to full-length Pol or Pol. Protein concentrations were determined by the Bio-Rad protein assay using bovine serum albumin as a standard and through UV absorbance at 280 nM under denaturing conditions (8 M urea) using the molar extinction coefficient calculated from the amino acid composition. The purified polymerases were stored in 5-µl aliquots at –80°C.

Nucleotides and DNA substrates. 5'-end-labeled deoxynucleoside triphosphates (dNTPs) (100 mM) were purchased from Roche Diagnostics and stored in aliquots at –20°C. Synthetic oligodeoxynucleotide templates and primers were used to prepare the nondamaged DNA substrates. The modified DNA substrates were constructed as described previously (9, 15). Template sequences were as follows: 5'-GCT AGC GAG TCC GCG CCA AGC TTG GGC TGC AGC AGG TC-3', where the underlined G indicates either an undamaged G, PdG, or (r) -HOPdG. The standing-start DNA substrates contain the primer 5'-A GCC CAA GCT TGG CGC GGA CT-3' annealed to the template. To assay extension from a C · G or T · G primer terminal base pair, the primer oligonucleotide 5'-GCC CAA GCT TGG CGC GGA CTC-3' or 5'-GCC CAA GCT TGG CGC GGA CTT-3' was used. 5'-end ³²P-labeled primers (1.0 µM) were annealed to templates (1.5 µM) in 50 mM Tris HCl (pH 7.5) and 100 mM NaCl by heating to 95°C for 2 min, followed by slow cooling to room temperature.

DNA polymerase assays. The standard DNA polymerase reaction mixture contained 25 mM Tris HCl (pH 7.5), 5 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, 5 mM MgCl₂, 10 nM DNA substrate, 1 nM Pol or Pol, unless otherwise noted, and 50 µM of each of four dNTPs. Escherichia coli Pol I (Roche Diagnostics), 3'-to-5' exonuclease-deficient Klenow fragment (New England BioLabs, Inc.), and yeast Pol (a generous gift from Peter Burgers, Washington University) were used at final concentrations of 0.15 nM, 0.15 nM, and 1 nM, respectively. Reactions were carried out at 24°C and subsequently quenched with 4 volumes of 95% formamide loading buffer (95% formamide, 0.3% bromphenol blue, 0.3% xylene cyanole) and placed on ice. Quenched reactions were heat denatured at 95°C for 3 min, and product formation was then monitored using 12% polyacrylamide gel electrophoresis (8 M urea), followed by PhosphorImager analysis (Molecular Dynamics).

For the steady-state kinetic analyses, the standard DNA polymerase reaction conditions were used with the exceptions that a single dNTP was used and concentrations were in a range appropriate for K_m determinations. Product formation was monitored using 12% polyacrylamide-8 M urea gel electrophoresis, and the respective gel band intensities were quantified using a PhosphorImager (Molecular Dynamics). Deoxynucleotide incorporation was measured at multiple time points for each dNTP concentration, and the observed rate of nucleotide incorporation was determined by linear regression. Values of the steady-state parameters k_cat and K_m for nucleotide incorporation were then determined from the best fit to the Michaelis-Menten equation (Sigma Plot 7.0). The reported kinetic parameters for each substrate are the average of at least three independent experiments.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Efficient replication through the ring-opened reduced -HOPdG by DNA polymerases and . Since -HOPdG can adopt either the ring-opened conformation or the closed cyclic conformation, we first determined whether (r) -HOPdG is inhibitory to synthesis by DNA polymerases and . As shown in Fig. 2, in the presence of all four dNTPs, both polymerases replicated through the lesion nearly as well as they did through the undamaged G. However, (r) -HOPdG presents a severe block to synthesis by Escherichia coli Pol I, the Klenow fragment lacking the 3'-to-5' proofreading exonuclease activity, and yeast Pol.

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FIG. 2. DNA synthesis by E. coli Pol I, 3'-to-5' exonuclease-deficient Klenow fragment (KF exo–), yeast Pol, human Pol, and human Pol on templates containing an unmodified G or (r) -HOPdG. Pol I (0.15 nM), KF exo– (0.15 nM), Pol (1 nM), Pol (1 nM), and Pol (1 nM) were incubated with DNA substrate (10 nM) in the presence of all four dNTPs (50 µM each) for 20 min at 24°C. The poor incorporation opposite template C by Pol reflects the lowered efficiency of nucleotide incorporation opposite pyrimidines by this polymerase. ND, nondamaged DNA.

As determined quantitatively from single-nucleotide incorporation reactions, the nucleotide insertion patterns of Pols and opposite (r) -HOPdG were the same as those opposite an undamaged G. Thus, Pol incorporated a C or a T opposite (r) -HOPdG as it did opposite the undamaged G (Fig. 3A), and Pol predominantly inserted a C in both cases (data not shown).

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FIG. 3. Translesion DNA synthesis by Pol on templates containing an (r) -HOPdG or a PdG. (A) Deoxynucleotide incorporation by Pol opposite (r) -HOPdG and opposite PdG, which is an analog of the ring-closed form of -HOPdG. ND, undamaged DNA. (B) Extension by Pol from a C opposite an (r) -HOPdG or a PdG template at the primer-template junction. Pol (1 nM) was incubated with DNA (10 nM) and with 50 µM of dGTP. Portions of the DNA templates and primers used to determine the insertion (A) and extension (B) propensity of Pol are shown. The position of the modified G is indicated by an asterisk. ND, nondamaged DNA.

Next, we quantified the efficiency of nucleotide incorporation reactions opposite (r) -HOPdG and opposite the corresponding undamaged G by Pols and using steady-state kinetic analyses. As shown in Table 1, Pol incorporates a C or a T opposite (r) -HOPdG with the same efficiency as it does opposite the undamaged G, and the incorporation of a C is approximately threefold more efficient than the incorporation of a T. Pol is also not inhibited by (r) -HOPdG, and it inserts a C opposite the lesion nearly as efficiently as opposite the undamaged G template (Table 2). Steady-state kinetic analyses have also indicated that both Pol and Pol extend from the (r) -HOPdG · C primer terminus as efficiently as from the G · C terminus (Tables 1 and 2). As Pol also inserts a T opposite this lesion, we determined the efficiency of extension from the (r) -HOPdG · T primer terminus by steady-state kinetic analyses. However, Pol was severely inhibited in extending from such a primer terminus, since no significant extension was detected even with very high dNTP concentrations (data not shown). Pol, on the other hand, extended from the (r) -HOPdG · T primer terminus as efficiently as it did from the G · T terminus (Table 2); the efficiency of extension from both of these primer termini, however, was 20-fold lower than from the normal G · C primer terminus.

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TABLE 1. Steady-state kinetic parameters of nucleotide insertion and extension reactions opposite (r) -HOPdG and PdG template residues by human Pol

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TABLE 2. Steady-state kinetic parameters of nucleotide insertion and extension reactions opposite (r) -HOPdG and PdG template residues by human Pol

Efficient nucleotide incorporation by Pol opposite a structural analog of the ring-closed form of -HOPdG, the PdG adduct. To determine if Pol can incorporate nucleotides opposite the closed cyclic form of -HOPdG, we used a DNA substrate containing PdG at the template site. As judged from single-nucleotide incorporation assays, Pol incorporated a C or a T opposite PdG as it did opposite the undamaged G (Fig. 3A), and as determined from steady-state kinetic analyses, the efficiencies of C and T incorporation opposite PdG were quite similar to that opposite the undamaged G (Table 1). Pol, however, was unable to promote the subsequent extension reaction, since following the PdG · C primer terminus, no significant G incorporation was detected opposite template C (Fig. 3B and Table 1).

The ring-closed PdG adduct is inhibitory to replication by Pol. PdG is very inhibitory to replication by Pol at both the nucleotide incorporation and extension steps. In single-nucleotide incorporation experiments, no significant incorporation of any of the four nucleotides was observed opposite this lesion (data not shown), and the lack of any significant C incorporation was verified by steady-state kinetic analyses (Table 2). Also, steady-state kinetic analyses indicated that Pol is highly inefficient at promoting extension from the PdG · C primer terminus (Table 2).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Here we show that (r) -HOPdG is not inhibitory to synthesis by Pol or Pol at either the nucleotide insertion or the extension step, and our previous studies with yeast and human Pol have indicated that this polymerase is only modestly affected by this adduct at either of these steps (15). All three Y family polymerases can then replicate across (r) -HOPdG quite efficiently and incorporate opposite the lesion the same nucleotides as they do opposite an undamaged G. The ability of Y family polymerases to proficiently replicate through the ring-opened reduced -HOPdG stands in sharp contrast to the inability of high-fidelity repair and replicative polymerases, such as E. coli Pol I and yeast Pol, to replicate through this lesion. Presumably these differences are manifestations of the constrained active site of high-fidelity DNA polymerases and occur because these enzymes participate in extensive hydrogen bonding interactions with the DNA minor groove. The Y family polymerases, on the other hand, have a much less constrained active site and are much more limited in their interactions with the DNA minor groove (see reference 26 for discussion).

By contrast to the proficient ability of Pols , , and to replicate through the permanently ring-opened reduced form of -HOPdG, which can participate in normal Watson-Crick hydrogen bonding, -HOPdG is inhibitory to synthesis by these polymerases. Opposite -HOPdG, yeast and human Pol are inhibited at the nucleotide incorporation step 100- to 200-fold, whereas the inhibition at the extension step is not that significant (15). Pol, however, can proficiently incorporate nucleotides opposite -HOPdG but is inhibited at the extension step, whereas Pol is inhibited at the nucleotide incorporation step but not at the extension step (24). The observations made here with the permanently ring-closed PdG and the ring-opened reduced -HOPdG enable us to outline the mechanism of translesion synthesis opposite a -HOPdG lesion that can adopt alternative configurations depending upon whether the lesion is at the template site or present in the template at the primer terminus.

At the replication fork, -HOPdG, when at the template site, would adopt the closed 1,N²-exocyclic form, unable to participate in normal Watson-Crick pairing with a C; and as for PdG, we expect the ring-closed form of -HOPdG to also assume a syn conformation in DNA. We presume that the inability of Pol or Pol to accommodate the -HOPdG in a syn conformation in their respective active sites accounts for their inefficient nucleotide incorporation ability opposite this lesion, whereas the ability of Pol to proficiently incorporate nucleotides opposite -HOPdG derives from its unique ability to accommodate the adducted template purine base in the syn conformation in its active site. In the syn conformation, -HOPdG could pair with the incoming C by Hoogsteen base pairing (Fig. 4A), forming a structure similar to that seen for the PdG · C pair (18, 27). The incorporation of a T opposite -HOPdG in the syn configuration could occur via the formation of a single hydrogen bond between the N7 of -HOPdG and the N3 of T.

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FIG. 4. Conformations adopted by -HOPdG. (A) Conformation of -HOPdG when at the template site. As a template residue, -HOPdG would be predominantly in the closed cyclic form, adopting a syn conformation and forming a Hoogsteen base pair with the incorporated C. (B) Conformation adopted by -HOPdG at the primer terminus. The incorporation of a C opposite -HOPdG would trigger the change from the ring-closed form of the adduct to the ring-opened configuration, able to form a normal Watson-Crick -HOPdG · C base pair.

All three polymerases, , , and , are strongly inhibited in extending from the PdG · C primer terminus (reference 15 and this study). This suggests that all of these Y family polymerases are unable to accommodate the PdG(syn) · dC(anti) Hoogsteen base pair when it is at the primer terminus. Thus, although Pol could accommodate a PdG(syn) at the template site in its active site and incorporate a C or a T opposite from it with almost the same efficiency as that opposite an undamaged G, once the lesion moves to the primer terminal position, it can no longer be accommodated in Pol's active site.

The proficient ability of Pol to extend from the -HOPdG · C primer terminus but not from the PdG · C terminus could derive from the potential ability of -HOPdG to adopt the ring-opened conformation upon the incorporation of dCMP (Fig. 4B), whereas PdG would remain in the ring-closed form. By contrast to the proficient extension from the -HOPdG · C primer terminus, Pol is inhibited in extending from the -HOPdG · T primer terminus, but it can extend from the (r) -HOPdG · T primer terminus nearly as well as from a G · T primer terminus. We suggest that whereas the presence of dCMP opposite -HOPdG at the primer terminus in the Pol active site triggers the chemical reaction of ring opening (Fig. 4B), the presence of dTMP does not trigger as efficient a ring opening reaction. Such a mechanism of a cytosine-catalyzed ring opening reaction has been adduced from nuclear magnetic resonance studies of a malondialdehyde 1,N²-exocyclic guanine adduct (13).

The mechanism of proficient and accurate bypassing of -HOPdG by the combined action of Pols and can be formulated as shown in Fig. 4 and 5. Pol can incorporate a C or a T opposite -HOPdG because of its ability to accommodate the closed cyclic form in the syn conformation in its active site. Incorporation of dCMP, but not of dTMP, opposite -HOPdG by Pol triggers an efficient change to a ring-opened conformation able to form a normal Watson-Crick hydrogen-bonded pair with the C (Fig. 4B) from which Pol proficiently extends (Fig. 5).

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FIG. 5. Model for translesion synthesis through -HOPdG by the sequential action of Pols and . Pol incorporates a C or a T opposite -HOPdG present in the ring-closed syn conformation. Incorporation of a C, but not of a T, induces the chemical transformation of the ring-closed -HOPdG to the ring-opened form, which can participate in normal Watson-Crick base pairing with the incorporated C. Pol proficiently extends from this base pair. Pol, however, is inhibited from extending the -HOPdG · T base pair because the pairing with a T does not elicit as efficient a ring opening reaction as that elicited from the -HOPdG · C base pair, and Pol is unable to extend from the -HOPdG(syn) · T(anti) base pair.

It is curious, however, that although Pol can proficiently extend from the C opposite the ring-opened reduced -HOPdG, it is inhibited in extending from a C opposite -HOPdG, whereas Pol can extend in both cases. Although it is not clear why the -HOPdG · C primer terminus is inhibitory to extension by Pol, one possibility is that at the -HOPdG · C primer terminus, the transition of -HOPdG from the syn (ring-closed) to the anti (ring-opened) conformation is impeded in the Pol active site but not in the Pol active site.

Our observation that the three Y family polymerases, , , and , can proficiently replicate through the ring-opened reduced form of -HOPdG indicates that they can all withstand the minor-groove distortions imposed by this adduct when it is at the template site or when it is paired at the primer terminus. For lesions such as the cyclic form of -HOPdG, however, which can adopt the ring-closed form or the ring-opened form, depending upon whether the lesion is located at the template site or at the primer terminus, the lesion bypass reaction entails the sequential action of two different Y family polymerases, Pols and , at the nucleotide incorporation and extension steps, respectively (Fig. 5).

The fact that Y family polymerases are not very efficient at dNTP incorporation, even opposite the undamaged template nucleotides, raises the question of how they promote effective lesion bypass during DNA replication. We suggest that interactions with protein components of the replication fork enhance their nucleotide incorporation efficiency. In support of this view, we have shown previously that interactions with PCNA stimulate the DNA synthetic activity of Y family polymerases. For both human Pols and , PCNA enhances the efficiency of nucleotide incorporation 50- to 200-fold, and that results from a decrease in the apparent K_m for dNTPs of nearly the same magnitude (2, 3). PCNA also stimulates nucleotide incorporation opposite DNA lesions by these polymerases to the same extent as that stimulated opposite undamaged templates (2, 3). Additional protein-protein interactions could lead to further activation of these polymerases.

 
This work was supported by National Institute of Environmental Health Sciences grants ES012411 (L.P., principal investigator) and ES 05355 (Michael Stone, principal investigator; R.S.L., principal investigator for Project 2).

We acknowledge Pam Tamura and Albena Kozekova for synthesis of -HOPdG-adducted oligodeoxynucleotides, and we thank Thomas M. Harris (Department of Chemistry, Center in Molecular Toxicology, Vanderbilt University, Nashville, TN) for providing us with these oligonucleotides. We thank Lawrence J. Marnett and James N. Riggins (Department of Biochemistry and Chemistry, Center in Molecular Toxicology, Vanderbilt University, Nashville, TN) for a generous gift of PdG-adducted oligodeoxynucleotides.

 
* Corresponding author. Mailing address: Sealy Center for Molecular Science, University of Texas Medical Branch at Galveston, 6.104 Blocker Medical Research Building, 11th and Mechanic Streets, Galveston, TX 77555-1061. Phone: (409) 747-8601. Fax: (409) 747-8608. E-mail: l.prakash{at}utmb.edu.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. de los Santos, C., T. Zaliznyak, and F. Johnson. 2001. NMR characterization of a DNA duplex containing the major acrolein-derived deoxyguanosine adduct -OH-1,-N²-propano-2'-deoxyguanosine. J. Biol. Chem. 276:9077-9082.[Abstract/Free Full Text]
2
2. Haracska, L., R. E. Johnson, I. Unk, B. B. Phillips, J. Hurwitz, L. Prakash, and S. Prakash. 2001. Targeting of human DNA polymerase to the replication machinery via interaction with PCNA. Proc. Natl. Acad. Sci. USA 98:14256-14261.[Abstract/Free Full Text]
3
3. Haracska, L., I. Unk, R. E. Johnson, B. B. Phillips, J. Hurwitz, L. Prakash, and S. Prakash. 2002. Stimulation of DNA synthesis activity of human DNA polymerase by PCNA. Mol. Cell. Biol. 22:784-791.[Abstract/Free Full Text]
4
4. Johnson, R. E., C. M. Kondratick, S. Prakash, and L. Prakash. 1999. hRAD30 mutations in the variant form of xeroderma pigmentosum. Science 285:263-265.[Abstract/Free Full Text]
5
5. Johnson, R. E., S. Prakash, and L. Prakash. 1999. Efficient bypass of a thymine-thymine dimer by yeast DNA polymerase, Pol. Science 283:1001-1004.[Abstract/Free Full Text]
6
6. Johnson, R. E., S. Prakash, and L. Prakash. 2000. The human DINB1 gene encodes the DNA polymerase Pol. Proc. Natl. Acad. Sci. USA 97:3838-3843.[Abstract/Free Full Text]
7
7. Johnson, R. E., M. T. Washington, L. Haracska, S. Prakash, and L. Prakash. 2000. Eukaryotic polymerases and act sequentially to bypass DNA lesions. Nature 406:1015-1019.[CrossRef][Medline]
8
8. Johnson, R. E., M. T. Washington, S. Prakash, and L. Prakash. 2000. Fidelity of human DNA polymerase . J. Biol. Chem. 275:7447-7450.[Abstract/Free Full Text]
9
9. Kanuri, M., I. G. Minko, L. V. Nechev, T. M. Harris, C. M. Harris, and R. S. Lloyd. 2002. Error prone translesion synthesis past -hydroypropanodeoxyguanosine, the primary acrolein-derived adduct in mammalian cells. J. Biol. Chem. 277:18257-18265.[Abstract/Free Full Text]
10
10. Kim, H.-Y. H., M. Voehler, T. M. Harris, and M. P. Stone. 2002. Detection of an interchain carbinolamine cross-link formed in a CpG sequence by the acrolein DNA adduct -OH-1,N²-propano-2'-deoxyguanosine. J. Am. Chem. Soc. 124:9324-9325.[CrossRef][Medline]
11
11. Kozekov, I. D., L. V. Nechev, M. S. Moseley, C. M. Harris, C. J. Rizzo, M. P. Stone, and T. M. Harris. 2003. DNA interchain cross-links formed by acrolein and crotonaldehyde. J. Am. Chem. Soc. 125:50-61.[CrossRef][Medline]
12
12. Kurtz, A. J., and R. S. Lloyd. 2003. 1,N²-deoxyguanosine adducts of acrolein, crotonaldehyde, and trans-4-hydroxynonenal cross-link to peptides via Schiff base linkage. J. Biol. Chem. 278:5970-5976.[Abstract/Free Full Text]
13
13. Mao, H., N. C. Schnetz-Boutaud, J. P. Weisenseel, L. J. Marnett, and M. P. Stone. 1999. Duplex DNA catalyzes the chemical rearrangement of a malondialdehyde deoxyguanosine adduct. Proc. Natl. Acad. Sci. USA 96:6615-6620.[Abstract/Free Full Text]
14
14. Masutani, C., R. Kusumoto, A. Yamada, N. Dohmae, M. Yokoi, M. Yuasa, M. Araki, S. Iwai, K. Takio, and F. Hanaoka. 1999. The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase . Nature 399:700-704.[CrossRef][Medline]
15
15. Minko, I. G., M. T. Washington, M. Kanuri, L. Prakash, S. Prakash, and R. S. Lloyd. 2003. Translesion synthesis past acrolein-derived DNA adduct, -hydroxypropanodeoxyguanosine, by yeast and human DNA polymerase . J. Biol. Chem. 278:784-790.[Abstract/Free Full Text]
16
16. Nair, D. T., R. E. Johnson, S. Prakash, L. Prakash, and A. K. Aggarwal. 2004. Replication by human DNA polymerase occurs via Hoogsteen base-pairing. Nature 430:377-380.[CrossRef][Medline]
17
17. Nechev, L. V., C. M. Harris, and T. M. Harris. 2000. Synthesis of nucleosides and oligonucleotides containing adducts of acrolein and vinyl chloride. Chem. Res. Toxicol. 13:421-429.[CrossRef][Medline]
18
18. Singh, U. S., J. G. Moe, G. R. Reddy, J. P. Weisenseel, L. J. Marnett, and M. P. Stone. 1993. 1H NMR of an oligodeoxynucleotide containing a propanodeoxyguanosine adduct positioned in a (CG)³ frameshift hotspot of Salmonella typhimurium hisD3052: Hoogsteen base-pairing at pH 5.8. Chem. Res. Toxicol. 6:825-836.[CrossRef][Medline]
19
19. Tissier, A., J. P. McDonald, E. G. Frank, and R. Woodgate. 2000. Pol, a remarkably error-prone human DNA polymerase. Genes Dev. 14:1642-1650.[Abstract/Free Full Text]
20
20. Trincao, J., R. E. Johnson, C. R. Escalante, S. Prakash, L. Prakash, and A. K. Aggarwal. 2001. Structure of the catalytic core of S. cerevisiae DNA polymerase : implications for translesion DNA synthesis. Mol. Cell 8:417-426.[CrossRef][Medline]
21
21. Washington, M. T., R. E. Johnson, L. Prakash, and S. Prakash. 2004. Human DNA polymerase utilizes different nucleotide incorporation mechanisms dependent upon the template base. Mol. Cell. Biol. 24:936-943.[Abstract/Free Full Text]
22
22. Washington, M. T., R. E. Johnson, S. Prakash, and L. Prakash. 2000. Accuracy of thymine-thymine dimer bypass by Saccharomyces cerevisiae DNA polymerase . Proc. Natl. Acad. Sci. USA 97:3094-3099.[Abstract/Free Full Text]
23
23. Washington, M. T., R. E. Johnson, S. Prakash, and L. Prakash. 1999. Fidelity and processivity of Saccharomyces cerevisiae DNA polymerase . J. Biol. Chem. 274:36835-36838.[Abstract/Free Full Text]
24
24. Washington, M. T., I. G. Minko, R. E. Johnson, W. T. Wolfle, T. M. Harris, R. S. Lloyd, S. Prakash, and L. Prakash. 2004. Efficient and error-free replication past a minor-groove DNA adduct by the sequential action of human DNA polymerases and . Mol. Cell. Biol. 24:5687-5693.[Abstract/Free Full Text]
25
25. Washington, M. T., L. Prakash, and S. Prakash. 2003. Mechanism of nucleotide incorporation opposite a thymine-thymine dimer by yeast DNA polymerase . Proc. Natl. Acad. Sci. USA 100:12093-12098.[Abstract/Free Full Text]
26
26. Washington, M. T., W. T. Wolfle, T. E. Spratt, L. Prakash, and S. Prakash. 2003. Yeast DNA polymerase makes functional contacts with the DNA minor groove only at the incoming nucleoside triphosphate. Proc. Natl. Acad. Sci. USA 100:5113-5118.[Abstract/Free Full Text]
27
27. Weisenseel, J. P., G. R. Reddy, L. J. Marnett, and M. P. Stone. 2002. Structure of an oligodeoxynucleotide containing a 1,N²-propanodeoxyguanosine adduct positioned in a palindrome derived from the Salmonella typhimurium hisD3052 gene: Hoogsteen pairing at pH 5.2. Chem. Res. Toxicol. 15:127-139.[CrossRef][Medline]

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Molecular and Cellular Biology, October 2005, p. 8748-8754, Vol. 25, No. 19
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.19.8748-8754.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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