Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Johnson, R. E. Articles by Prakash, L. Search for Related Content PubMed PubMed Citation Articles by Johnson, R. E. Articles by Prakash, L.

 Previous Article  |  Next Article 

Molecular and Cellular Biology, May 2001, p. 3558-3563, Vol. 21, No. 10
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.10.3558-3563.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Role of DNA Polymerase  in the Bypass of a (6-4) TT Photoproduct

Robert E. Johnson, Lajos Haracska, Satya Prakash, and Louise Prakash^*

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

Received 10 January 2001/Returned for modification 14 February 2001/Accepted 20 February 2001

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion References

UV light-induced DNA lesions block the normal replication machinery. Eukaryotic cells possess DNA polymerase  (Pol), which has the ability to replicate past a cis-syn thymine-thymine (TT) dimer efficiently and accurately, and mutations in human Pol result in the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Here, we test Pol for its ability to bypass a (6-4) TT lesion which distorts the DNA helix to a much greater extent than a cis-syn TT dimer. Opposite the 3' T of a (6-4) TT photoproduct, both yeast and human Pol preferentially insert a G residue, but they are unable to extend from the inserted nucleotide. DNA Pol, essential for UV induced mutagenesis, efficiently extends from the G residue inserted opposite the 3' T of the (6-4) TT lesion by Pol, and Pol inserts the correct nucleotide A opposite the 5' T of the lesion. Thus, the efficient bypass of the (6-4) TT photoproduct is achieved by the combined action of Pol and Pol, wherein Pol inserts a nucleotide opposite the 3' T of the lesion and Pol extends from it. These biochemical observations are in concert with genetic studies in yeast indicating that mutations occur predominantly at the 3' T of the (6-4) TT photoproduct and that these mutations frequently exhibit a 3' TC change that would result from the insertion of a G opposite the 3' T of the (6-4) TT lesion.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

The RAD30 gene of Saccharomyces cerevisiae functions in error-free bypass of UV-damaged DNA, and RAD30-encoded DNA polymerase  (Pol) replicates through a cis-syn thymine-thymine (TT) dimer with the same efficiency and accuracy as through undamaged T's (13, 27). Both yeast and human Pol efficiently insert two A's opposite the two T's of the dimer and extend from the resulting primer (15, 27). In yeast as well as humans, inactivation of Pol results in UV hypermutability (26, 29), and defects in Pol in humans cause the variant form of xeroderma pigmentosum (XP-V) (12, 22). As a consequence of UV hypermutability, XP-V individuals suffer from a high incidence of skin cancers.

Pol is unique among eukaryotic DNA polymerases in its ability to replicate through lesions that distort the DNA helix. Thus, in addition to a cis-syn TT dimer, Pol replicates through an 8-oxoguanine (8oxoG) (11) or an O⁶-methylguanine (m⁶G) lesion (10). Although the template strand is highly distorted in the vicinity of the lesion in the 8oxoG · C base pair, yeast Pol (yPol) efficiently replicates through this lesion by inserting a C opposite the lesion and then extending from the resulting base pair (11), and Pol bypasses an m⁶G lesion by inserting a C or T opposite the lesion (10). The ability of Pol to bypass lesions which distort the DNA helix suggests an unusual tolerance of its active site for geometric distortions in DNA. Consistent with this idea, both yPol and human Pol (hPol) are low-fidelity enzymes, misincorporating nucleotides opposite nondamaged template bases with a frequency of 10² to 10³ (15, 28).

In addition to cis-syn cyclobutane pyrimidine dimers, UV light elicits the formation of pyrimidine (6-4) pyrimidinone photoproducts. By contrast to a cis-syn TT dimer, which has only a modest effect on DNA structure and which does not affect the ability of the two T's in the dimer to base pair with A's, a (6-4) TT photoproduct induces a large structural distortion in DNA, and the 3' T in the (6-4) lesion is oriented perpendicular to the 5' T (17). Nuclear magnetic resonance studies have, however, indicated that the 3' T in the (6-4) lesion can hydrogen bond with a G residue (20). Here we test the ability of Pol to replicate through a (6-4) TT lesion. We find that although Pol does not bypass this lesion, it is nevertheless able to insert a G residue opposite the 3' T of the lesion. DNA Pol, essential for the mutagenic bypass of DNA lesions, efficiently extends from the resulting base pair by incorporating the correct nucleotide A opposite the 5' T of the lesion. Thus, the sequential action of DNA polymerases  and  coordinates the mutagenic bypass of a (6-4) TT lesion.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Enzyme purification. The yeast RAD30 and human RAD30A genes were cloned in frame with the glutathione S-transferase (GST) gene in the overexpression plasmid pBJ760 (14), generating plasmids pBJ763 and pBJ765, respectively. Yeast and human Pol were purified from S. cerevisiae BJ5464 harboring either pBJ763 or pBJ765 as described elsewhere (13, 15), except that protein was batch eluted from glutathione-Sepharose 4B by cleavage of the GST tag on Pol by treatment with thrombin for 4 h at 4°C. Cleavage of the GST portion by thrombin leaves an eight-amino-acid leader peptide on the N terminus of yeast and human Pol. All subsequent purification steps were done as described elsewhere (13, 15).

yPol was purified from S. cerevisiae Sc334 harboring plasmid pGST-REV3 and pREV7 as described previously (14) except that a Mini-Q column step was added. Protein eluted from glutathione-Sepharose 4B was dialyzed against buffer A (25 mM NaPO₄ [pH 7.4], 100 mM NaCl, 10% glycerol, 0.01% NP-40, 5 mM dithiothreitol, 0.5 mM EDTA), loaded onto a Mini-Q PC 2.3/3 column (Pharmacia), and washed with 20 column volumes buffer A before elution of the protein with a 2.4-ml 100 to 500 mM NaCl gradient in buffer A. The GST-Rev3/Rev7-containing fractions were pooled and concentrated in buffer A containing 200 mM NaCl and 50% glycerol and stored at 20°C.

DNA substrates. The 75-nucleotide (nt) template (5'-AGCAAGTCA CCAATGTCT AAGAGTTCG TATTATGCC TACACTGGA GTACCGGAG CATCGTCGT GACTGGGAA AAC-3') either containing or not containing a (6-4) TT photoproduct at the underlined position was derived from the 10-nt oligomer 5' CGTATTATGC 3' by ligation to flanking 25- and 40-nt oligomers. The (6-4) TT photoproduct was introduced into the 10-nt oligomer by irradiation with 254-nm-wavelength UV light and was purified by high-pressure liquid chromatography. The (6-4) TT photoproduct was identified by its unique absorption at 326-nm light (19). For bypass assays and for steady-state kinetic analyses of insertion of nucleotides opposite the 3' T of the (6-4) photoproduct or a nondamaged T residue, the primer N4309 (5' GTTTTCCCAG TCACGACGAT GCTCCGGTAC TCCAGTGTAG GCAT 3') was annealed to the 75-nt template. For steady-state kinetic analyses of primer extension from an A or a G residue opposite the 3' T of the (6-4) photoproduct in the template, oligomers 5' GTTTTCCCAG TCACGACGAT GCTCCGGTAC TCCAGTGTAG GCATA 3' and 5' GTTTTCCCAG TCACGACGAT GCTCCGGTAC TCCAGTGTAG GCATG 3' respectively, were used.

DNA polymerase assays. For synthesis assays on damaged and nondamaged DNAs (Fig. 1), the standard DNA polymerase reaction (5 µl) was used; the mixture contained 25 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 1 mM dithiothreitol, 100 µg of bovine serum albumin/ml, 10% glycerol, 100 µM each deoxynucleoside triphosphate (dNTP) (dGTP, dATP, dTTP, and dCTP), 10 nM 5'-³²P-labeled oligonucleotide primer annealed to an oligonucleotide DNA template, 1 nM yeast or human Pol, and 1.8 nM yPol. Reactions were carried out at 37°C for 5 min and terminated by the addition of 50 mM EDTA. DNA products were precipitated with 6 volumes of ice-cold acetone, dried under vacuum, resuspended in loading buffer (95% formamide, 0.05% cyanol blue, 0.05% bromophenol blue), and then resolved on 10% polyacrylamide gels containing 8 M urea. Gels were dried before autoradiography at 70°C.

Steady-state kinetic analyses. To determine the efficiency and fidelity of deoxynucleotide incorporation by yeast and human Pol at the 3' T of a (6-4) TT photoproduct or an undamaged T, the standard DNA polymerase assay was employed except that 0.5 nM hPol or yPol was used, and only a single deoxynucleotide was included at the concentrations indicated in the figures and figure legends. Reactions were carried out at 30°C for 5 min. Gel band intensities of the substrate and products of the deoxynucleotide incorporation reactions were quantitated using a PhosphorImager and ImageQuant software (Molecular Dynamics). The observed rate of deoxynucleotide incorporation, v_obs, was determined by dividing the amount of product formed by the reaction time. v_obs was plotted as a function of the deoxynucleotide concentration, and the data were fit to the Michaelis-Menten equation describing a hyperbola: v_obs = (V_max × [dNTP])/(K_m + [dNTP]). From the best fit curve, the apparent K_m and V_max steady-state kinetic parameters for the incorporation of each deoxynucleotide were obtained and used to calculate the relative efficiency of deoxynucleotide incorporation, f_inc, using the following equation:

f_inc = (V_max/K_m)_{G, A, T, or C} / (V_max/K_m)_A (7, 9, 24).

To determine the efficiency of nucleotide incorporation at the 5' T of the (6-4) TT photoproduct or an undamaged TT sequence by Pol, following a correctly base-paired or mispaired primer terminus, the standard DNA polymerase assay was used except that reaction mixtures contained 40 mM Tris-HCl (pH 7.5), 5 nM Pol, and 20 nM DNA substrate, and the reactions were carried out for 3 min at 30°C. The dNTP concentration was varied from 0 to 25 µM for dATP and from 0 to 500 µM for dGTP, dTTP, or dCTP.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Bypass of a (6-4) TT photoproduct by the combined action of Pol and Pol. The bypass of a (6-4) TT photoproduct was examined in standing start reactions using a 75-nt template containing the lesion 45 nt from the 3' end and primed with a 5'-³²P-labeled 44-nt oligomer. As shown in Fig. 1, neither yeast or human Pol could replicate through the (6-4) TT lesion. Both polymerases, could insert a deoxynucleotide opposite the 3' T of the lesion (Fig. 1, lanes 5 and 6), but neither could extend past this site. Extensive genetic studies in yeast have indicated the requirement of Pol in the mutagenic bypass of DNA lesions, including those induced by UV light. Pol, however, does not bypass the (6-4) TT photoproduct; moreover, it does not even insert a nucleotide opposite the 3' T of the lesion (Fig. 1, lane 7). Efficient bypass of the (6-4) TT lesion, however, occurs when Pol is combined with Pol (Fig. 1, lanes 8 and 9).

View larger version (54K): [in this window] [in a new window]   FIG. 1.   Bypass of the (6-4) TT photoproduct by the combined action of Pol and Pol. Lanes 1 to 3, undamaged DNA; lanes 4 to 9, (6-4) TT photoproduct-containing DNA. Positions of the two T's in the undamaged or the (6-4) TT photoproduct-containing template are indicated on the right. hPol (1 nM), yPol (1 nM), yPol (1.8 nM), or either yeast or human Pol combined with yPol was incubated with the DNA substrate for 5 min at 37°C in the presence of 100 µM each of the four dNTPs.

Preferential incorporation of a G residue opposite the 3' T of the (6-4) TT photoproduct by human and yeast Pol. To examine the efficiency (V_max/K_m) of nucleotide incorporation opposite the 3' T of the (6-4) lesion by human and yeast Pol, we measured the kinetics of insertion for each deoxynucleotide under steady-state conditions. Figure 2 shows the incorporation pattern of each deoxnucleotide by human Pol opposite a nondamaged T residue (Fig. 2A) and opposite the 3' T of the (6-4) TT photoproduct (Fig. 2B). Opposite the nondamaged T residue, hPol inserts the correct A residue with a high efficiency (Table 1). Relative to the incorporation of A, hPol misincorporates a G, a T, or a C opposite the nondamaged T template with frequencies of ~3 × 10³ to 1 × 10². However, opposite the 3' T the (6-4) TT lesion, hPol is almost 450-fold less efficient at incorporating an A than on the equivalent T residue in the nondamaged template, and T and C are incorporated even less well than an A opposite the 3' T of the lesion. hPol preferentially inserts a G opposite the 3' T of the (6-4) TT lesion, as hPol is eightfold more efficient at inserting a G opposite the 3' T of the (6-4) TT lesion than it is at inserting an A opposite this site (Table 1). Relative to the insertion of a G or an A opposite the nondamaged T template, hPol inserts a G opposite the 3' T of the (6-4) TT lesion about 2-fold better or 55-fold less well, respectively (Table 1). The insertion of G opposite the 3' T of this lesion by hPol has been reported, but in the absence of any kinetic analyses, the efficiency with which hPol inserted this or other nucleotides could not be evaluated (31). yPol is also highly inefficient at inserting an A opposite the 3' T of the (6-4) TT lesion, and compared to the insertion of an A opposite nondamaged T, yPol incorporates an A opposite the 3' T of the (6-4) lesion over 700-fold less well (Table 2). yPol also preferentially inserts a G residue opposite the 3'T of the 6-4 lesion, and the yeast enzyme is 1.5-fold more or ~100-fold less efficient at inserting a G opposite the lesion than it is at inserting a G or an A opposite the nondamaged T, respectively (Table 2).

View larger version (49K): [in this window] [in a new window]   FIG. 2.   Nucleotide incorporation by hPol opposite the 3' T residue in a nondamaged template or a (6-4) TT photoproduct-containing template. (A) Incorporation of nucleotides opposite the nondamaged T residue; (B) incorporation opposite the equivalent 3' T residue of a (6-4) TT photoproduct. A portion of each primer:template substrate is shown at the top. hPol (0.5 nM) was incubated with DNA substrate (10 nM) and the indicated concentrations of dNTPs for 5 min at 30°C.

View this table: [in this window] [in a new window]   TABLE 1.   Steady-state kinetic parameters for nucleotide incorporation opposite the 3'T of the (6-4) TT photoproduct by hPol

View this table: [in this window] [in a new window]   TABLE 2.   Steady-state kinetic parameters for nucleotide incorporation opposite the 3' T of the (6-4) TT photoproduct by yPol

Nucleotide incorporation opposite the 5' T of the (6-4) TT photoproduct by Pol. Since Pol is able to extend from the nucleotide inserted by Pol opposite the 3' T of the (6-4) TT lesion (Fig. 1, lanes 8 and 9), we next examined the relative efficiency of each nucleotide incorporation by Pol opposite the 5' T of the nondamaged or (6-4) TT photoproduct using steady-state kinetic assays. Since Pol preferentially inserts a G residue opposite the 3' T of the (6-4) TT lesion, we compared the efficiency of incorporation of nucleotides following a primer in which a G (Fig. 3A) or an A (Fig. 3B) is paired with the 3' T of the lesion. When extending from the A · T or the G · T base pair on nondamaged TT sequence, Pol incorporates the correct A opposite the 5' T with high efficiency and misincorporates nucleotides with a frequency of ~10³ to 10⁴ (Table 3). Pol, however, is somewhat more accurate in inserting nucleotides opposite the 5' T of the (6-4) TT photoproduct when extending from a G opposite the 3' T of the lesion than when extending from an A opposite the 3' T of the lesion (Table 3). Furthermore, Pol is about threefold more efficient at incorporating an A when G is paired with the 3' T of the (6-4) lesion than when an A is paired with the 3' T of the lesion and is almost fourfold more efficient at incorporating an A following the G opposite the (6-4) lesion than from the G · T base pair in the nondamaged template (Table 3).

View larger version (48K): [in this window] [in a new window]   FIG. 3.   Nucleotide incorporation opposite the 5' T of the (6-4) TT photoproduct by yPol. (A) Nucleotide incorporation following a G residue opposite the 3' T of the (6-4) photoproduct; (B) nucleotide incorporation following an A residue opposite the 3' T of a (6-4) photoproduct. yPol (5 nM) was incubated for 3 min at 30°C with the primer:template DNA substrate (20 nM) and with the indicated concentrations of dNTPs.

View this table: [in this window] [in a new window]   TABLE 3.   Steady-state kinetic parameters for nucleotide incorporation opposite the 5' T of the (6-4) TT photoproduct by yPol

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Biochemical studies with yeast and human Pol have indicated a role for this enzyme in the error-free bypass of a cis-syn TT dimer. Both enzymes replicate through this lesion by inserting As opposite the two T's of the dimer, and they do so with the same efficiency and fidelity as when replicating through undamaged T's. In addition to the TT dimer, UV light also induces the formation of cyclobutane dimers at the CC and TC sites. Because of the rapid deamination of C to U, in vitro bypass studies with these lesions are difficult to perform; genetic studies in yeast, however, have implicated Pol in the error-free bypass of cyclobutane dimers at the CC and TC sites as well (30).

By contrast to a cis-syn cyclobutane pyrimidine dimer, which has only a modest effect on the DNA structure, a (6-4) TT photoproduct induces a large structural distortion, leading to a 44° bend in the DNA helix; moreover, the 3' T in the (6-4) lesion is held perpendicular to the 5' T (16). Nuclear magnetic resonance studies have shown that the O₂ carbonyl of the 3' T in the (6-4) TT lesion cannot hydrogen bond with the amino proton of an opposed A residue (17). The O₂ carbonyl of the 3' T residue, however, can form hydrogen bonds with the imino and amino protons of the opposed G residue (20). The 5' T at the (6-4) lesion maintains normal hydrogen bonding interactions with the A residue in the complementary strand (20).

In UV-irradiated DNA, the (6-4) TT photoproduct is formed much less frequently than the cis-syn TT dimer; the (6-4) lesion, however, is more mutagenic than the dimer (1, 2, 4, 5). Experiments in S. cerevisiae with single-stranded or gapped duplex vectors that carried a cis-syn TT dimer or a (6-4) TT photoproduct at a unique site have indicated that by contrast to a cis-syn dimer, which is replicated very accurately (0.4% targeted mutations), a (6-4) TT photoproduct induces mutations in 30 to 40% of the replicated plasmid molecules, and as many as 50% of these mutations are 3' TC substitutions (8). Such mutations would arise from the incorporation of a G opposite the 3' T of the (6-4) TT lesion. In concurrence with these genetic observations, we show here that the bypass of a (6-4) TT lesion is accomplished by the combined action of Pol and Pol, wherein Pol inserts a G opposite the 3' T of the (6-4) lesion and Pol extends from the resulting base pair.

Our steady-state kinetic analyses indicate that both yeast and human Pol incorporate a G opposite the 3' T of the (6-4) TT lesion about eightfold more efficiently than an A. Thus, in spite of the large distortion of the DNA duplex, Pol is able to insert a G opposite the 3' T of the lesion; Pol, however, does not extend from the ensuing base pair. The ability of Pol to preferentially insert a G rather than an A opposite the 3' T of the lesion supports the view that although Pol is rather insensitive even to a major geometric distortion such as that conferred upon DNA by the (6-4) lesion, it prefers to insert nucleotides opposite DNA lesions where some base pairing is possible. Our previous observations that Pol inserts A's opposite the two T's in the dimer which form correct base pairs, and that it inserts a C rather than an A opposite an 8-oxoG lesion, also concur with this view. Thus, although the 8-oxoG · A base pair has the correct Watson-Crick geometry, this base pairing involves the same two hydrogen bonds as in the T · A base pair, whereas in the 8-oxoG · C base pair, in spite of the very considerable distortion of the template, the base pairing involves the same three hydrogen bonds as in the G · C base pair (18, 21, 23, 25).

DNA Pol is essential for the mutagenic bypass of UV induced DNA lesions. By itself, Pol bypasses a cis-syn TT dimer quite poorly, and it does not bypass a (6-4) TT lesion. This is because Pol is highly inefficient at inserting nucleotides opposite the 3' T of either of these lesions; Pol, however, is very adept at extending from nucleotides inserted opposite the 3' T of either lesion by another DNA polymerase (14). Interestingly, Pol extends from a G opposite the 3' T of the (6-4) TT lesion fourfold more efficiently than it extends from a G opposite the nondamaged T; importantly, Pol incorporates the correct nucleotide A opposite the 5' T of the (6-4) lesion, whereas the wrong nucleotides are incorporated very poorly, with a frequency of ~5 × 10⁴. Thus, although Pol is quite accurate in inserting the correct nucleotide opposite the 5' T of the lesion, its contribution to mutagenesis emanating from the bypass of (6-4) TT lesions would derive from its ability to efficiently extend from the G nucleotide inserted opposite the 3' T by Pol. The accurate insertion of an A opposite the 5' T of the (6-4) lesion by Pol explains the genetic observation that mutations occur predominantly at the 3' site of the (6-4) TT lesion and not at the 5' site, and the insertion of a G opposite the 3' T of the (6-4) TT lesion by Pol accounts for the 3' TC substitutions that occur at this lesion site (8).

The (6-4) photoproduct is formed much more frequently at the TC and CC sequences than at the TT site (1-6). At TC, the (6-4) lesion is formed almost as frequently as the cyclobutane dimer, whereas at CC, dimer formation predominates over the (6-4) lesion. At TT sites, the (6-4) lesion is formed even much less frequently than at CC sites. Genetic studies in yeast indicating a role of Pol in the error-free bypass of UV lesions at the TC and CC sites (30) have raised the possibility that in addition to its role in the error-free bypass of cis-syn CC and TC dimers, Pol contributes also to the error-free bypass of (6-4) lesions at these sites.

Similar to the bypass of a cis-syn TT dimer, we presume that Pol bypasses a cis-syn CC or TC dimer by incorporating the correct nucleotides opposite the two residues of the dimer. Opposite the CC or TC (6-4) lesion, we expect Pol to insert a G opposite the 3' C of the lesion because the (6-4) lesion at these sites is structurally very similar to that at the TT site, and the O₂ carbonyl of the 3' C in the lesion is expected to form hydrogen bonds with a G. The insertion of a G opposite the 3' C of the (6-4) lesion by Pol, followed by extension by the incorporation of the correct nucleotide opposite the 5' nucleotide of the lesion, would then promote error-free bypass of (6-4) TC and CC lesions.

	ACKNOWLEDGMENTS

We thank M. T. Washington for helpful discussions.

This work was supported by National Institutes of Health grant GM19261. The (6-4) TT photoproduct containing DNA was constructed in the Synthetic Organic Chemistry Core Laboratory, supported by NIEHS Center grant P30-ESO6676, and we are grateful to Richard Hodge for providing this DNA.

	FOOTNOTES

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

	REFERENCES

Top Abstract Introduction Materials and Methods Results Discussion References

1.	Armstrong, J. D., and B. A. Kunz. 1990. Site and strand specificity of UVB mutagenesis in the SUP4-o gene of yeast. Proc. Natl Acad. Sci. USA 87:9005-9009[Abstract/Free Full Text].
2.	Bourre, F., G. Renault, and A. Sarasin. 1987. Sequence effect on alkali-sensitive sites in UV-irradiated SV40 DNA. Nucleic Acids Res. 15:8861-8875[Abstract/Free Full Text].
3.	Brash, D. E. 1997. Sunlight and the onset of skin cancer. Trends Genet. 13:410-414[CrossRef][Medline].
4.	Brash, D. E., and W. A. Haseltine. 1982. UV-induced mutation hotspots occur at DNA damage hotspots. Nature 298:189-192[CrossRef][Medline].
5.	Brash, D. E., S. Seetharam, K. H. Kraemer, M. M. Seidman, and A. Bredberg. 1987. Photoproduct frequency is not the major determinant of UV base substitution hot spots or cold spots in human cells. Proc. Natl. Acad. Sci. USA 84:3782-3786[Abstract/Free Full Text].
6.	Canella, K. A., and M. M. Seidman. 2000. Mutation spectra in supF: approaches to elucidating sequence context effects. Mutat. Res. 450:61-73[Medline].
7.	Creighton, S., L. B. Bloom, and M. F. Goodman. 1995. Gel fidelity assay measuring nucleotide misinsertion, exonucleolytic proofreading, and lesion bypass efficiencies. Methods Enzymal. 262:232-256.
8.	Gibbs, P. E. M., A. Borden, and C. W. Lawrence. 1995. The T-T pyrimidine (6-4) pyrimidinone UV photoproduct is much less mutagenic in yeast than in Escherichia coli. Nucleic Acids Res. 23:1919-1922[Abstract/Free Full Text].
9.	Goodman, M. F., S. Creighton, L. B. Bloom, and J. Petruska. 1993. Biochemical basis of DNA replication fidelity. Crit. Rev. Biochemi. Mol. Biol. 28:83-126.
10.	Haracska, L., S. Prakash, and L. Prakash. 2000. Replication past O6-methylguanine by yeast and human DNA polymerase . Mol. Cell. Biol. 20:8001-8007[Abstract/Free Full Text].
11.	Haracska, L., S.-L. Yu, R. E. Johnson, L. Prakash, and S. Prakash. 2000. Efficient and accurate replication in the presence of 7,8-dihydro-8-oxoguanine by DNA polymerase . Nat. Genet. 25:458-461[CrossRef][Medline].
12.	Johnson, R. E., C. M. Kondratick, S. Prakash, and L. Prakash. 1999. hRAD30 mutations in the variant form of xeroderm pigmentosum. Science 285:263-265[Abstract/Free Full Text].
13.	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].
14.	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].
15.	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].
16.	Kim, J.-K., and B.-S. Choi. 1995. The solution structure of DNA duplex-decamer containing the (6-4) photoproduct of thymidyl(3'5')thymidine by NMR and relaxation matrix refinement. Eur. J. Biochem. 228:849-854[Medline].
17.	Kim, J.-K., D. Patel, and B.-S. Choi. 1995. Contrasting structural impacts induced by cis-syn cyclobutane dimer and (6-4) adduct in DNA duplex decamers: implication in mutagenesis and repair activity. Photochem. Photobiol. 62:44-50[Medline].
18.	Kouchakdjian, M., V. Bodepudi, S. Shibutani, M. Eisenberg, F. Johnson, A. P. Grollman, and D. J. Patel. 1991. NMR structural studies of the ionizing radiation adduct 7-hydro-8-oxodeoxyguanosine (-8-oxo-7H-dG) opposite deoxyadenosine in a DNA duplex. 8-oxo-7H-dG(syn).dA(anti) alignment at lesion site. Biochemistry 30:1403-1412[CrossRef][Medline].
19.	LeClerc, J. E., A. Borden, and C. W. Lawrence. 1991. The thymine-thymine pyrimidine-pyrimidone (6-4) ultraviolet light photoproduct is highly mutagenic and specially induces 3' thymine-to-cytosine transitions in Escherichia coli. Proc. Natl. Acad. Sci. USA 88:9685-9689[Abstract/Free Full Text].
20.	Lee, J.-H., G.-S. Hwang, and B.-S. Choi. 1999. Solution structure of a DNA decamer duplex containing the stable 3' T · G base pair of the pyrimidine(6-4)pyrimidone photoproduct [(6-4) adduct]: implications for the highly specific 3' T  C transition of the (6-4) adduct. Proc. Natl. Acad. Sci. USA 96:6632-6636[Abstract/Free Full Text].
21.	Lipscomb, L. A., M. E. Peek, M. L. Morningstar, S. M. Verghis, E. M. Miller, A. Rich, J. M. Essignman, and L. D. Williams. 1995. X-ray structure of a DNA decamer containing 7,8-dihydro-8-oxoguanine. Proc. Natl. Acad. Sci. USA 92:719-723[Abstract/Free Full Text].
22.	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].
23.	McAuley-Hecht, K. E., G. A. Leonard, N. J. Gibson, J. B. Thomson, W. P. Watson, W. N. Hunter, and T. Brown. 1994. Crystal structure of a DNA duplex containing 8-hydroxydeoxyguanine-adenine base pairs. Biochemistry 33:10266-10270[CrossRef][Medline].
24.	Mendelman, L. V., J. Petruska, and M. F. Goodman. 1990. Base mispair extension kinetics. Comparison of DNA polymerase  and reverse transcriptase. J. Biol. Chem. 265:2338-2346[Abstract/Free Full Text].
25.	Oda, Y., S. Uesugi, M. Ikehara, S. Nishimura, Y. Kawase, H. Ishikawa, H. Inoue, and E. Ohtsuka. 1991. NMR studies of a DNA containing 8-hydroxydeoxyguanosine. Nucleic Acids Res. 19:1407-1412[Abstract/Free Full Text].
26.	Wang, Y.-C., V. M. Maher, D. L. Mitchell, and J. J. McCormick. 1993. Evidence from mutation spectra that the UV hypermutability of xeroderma pigmentosum variant cells reflects abnormal, error-prone replication on a template containing photoproducts. Mol. Cell. Biol. 13:4276-4283[Abstract/Free Full Text].
27.	Washington, M. T., R. E. Johnson, S. Prakash, and L. Prakash. 2000. Accuracy of thymine-thymine dimer bypass by Saccharomyces cerevisiae DNA polymerase . Proc. Nat. Acad. Sci. USA 97:3094-3099[Abstract/Free Full Text].
28.	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].
29.	Waters, H. L., S. Seetharam, M. M. Seidman, and K. H. Kraemer. 1993. Ultraviolet hypermutability of a shuttle vector propagated in xeroderma pigmentosum variant cells. J. Investig. Dermatol. 101:744-748[CrossRef][Medline].
30.	Yu, S.-L., R. E. Johnson, S. Prakash, and L. Prakash. 2001. Requirement of DNA polymerase  for error-free bypass of UV-induced CC and TC photoproducts. Mol. Cell. Biol. 21:185-188[Abstract/Free Full Text].
31.	Zhang, Y., F. Yuan, X. Wu, O. Rechkoblit, J.-S. Taylor, M. E. Geachintov, and Z. Wang. 2000. Error-prone lesion bypass by human DNA polymerase . Nucleic Acids Res. 28:4717-4724[Abstract/Free Full Text].

---

Molecular and Cellular Biology, May 2001, p. 3558-3563, Vol. 21, No. 10
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.10.3558-3563.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

This article has been cited by other articles:

• Auerbach, P. A., Demple, B. (2009). Roles of Rev1, Pol {zeta}, Pol32 and Pol {eta} in the bypass of chromosomal abasic sites in Saccharomyces cerevisiae. Mutagenesis 0: gep045v1-gep045 [Abstract] [Full Text]  
• Acharya, N., Johnson, R. E., Pages, V., Prakash, L., Prakash, S. (2009). Yeast Rev1 protein promotes complex formation of DNA polymerase {zeta} with Pol32 subunit of DNA polymerase {delta}. Proc. Natl. Acad. Sci. USA 106: 9631-9636 [Abstract] [Full Text]  
• Stone, J. E., Kissling, G. E., Lujan, S. A., Rogozin, I. B., Stith, C. M., Burgers, P. M. J., Kunkel, T. A. (2009). Low-fidelity DNA synthesis by the L979F mutator derivative of Saccharomyces cerevisiae DNA polymerase {zeta}. Nucleic Acids Res 37: 3774-3787 [Abstract] [Full Text]  
• Szuts, D., Marcus, A. P., Himoto, M., Iwai, S., Sale, J. E. (2008). REV1 restrains DNA polymerase {zeta} to ensure frame fidelity during translesion synthesis of UV photoproducts in vivo. Nucleic Acids Res 36: 6767-6780 [Abstract] [Full Text]  
• Pages, V., Bresson, A., Acharya, N., Prakash, S., Fuchs, R. P., Prakash, L. (2008). Requirement of Rad5 for DNA Polymerase {zeta}-Dependent Translesion Synthesis in Saccharomyces cerevisiae. Genetics 180: 73-82 [Abstract] [Full Text]  
• Sugasawa, K. (2008). Xeroderma pigmentosum genes: functions inside and outside DNA repair. Carcinogenesis 29: 455-465 [Abstract] [Full Text]  
• Acharya, N., Haracska, L., Prakash, S., Prakash, L. (2007). Complex Formation of Yeast Rev1 with DNA Polymerase {eta}. Mol. Cell. Biol. 27: 8401-8408 [Abstract] [Full Text]  
• Santa Maria, S. R., Gangavarapu, V., Johnson, R. E., Prakash, L., Prakash, S. (2007). Requirement of Nse1, a Subunit of the Smc5-Smc6 Complex, for Rad52-Dependent Postreplication Repair of UV-Damaged DNA in Saccharomyces cerevisiae. Mol. Cell. Biol. 27: 8409-8418 [Abstract] [Full Text]  
• Gangavarapu, V., Prakash, S., Prakash, L. (2007). Requirement of RAD52 Group Genes for Postreplication Repair of UV-Damaged DNA in Saccharomyces cerevisiae. Mol. Cell. Biol. 27: 7758-7764 [Abstract] [Full Text]  
• Gangavarapu, V., Haracska, L., Unk, I., Johnson, R. E., Prakash, S., Prakash, L. (2006). Mms2-Ubc13-Dependent and -Independent Roles of Rad5 Ubiquitin Ligase in Postreplication Repair and Translesion DNA Synthesis in Saccharomyces cerevisiae. Mol. Cell. Biol. 26: 7783-7790 [Abstract] [Full Text]  
• Abdulovic, A. L., Jinks-Robertson, S. (2006). The in Vivo Characterization of Translesion Synthesis Across UV-Induced Lesions in Saccharomyces cerevisiae: Insights Into Pol{zeta}- and Pol{eta}-Dependent Frameshift Mutagenesis. Genetics 172: 1487-1498 [Abstract] [Full Text]  
• Wolfle, W. T., Johnson, R. E., Minko, I. G., Lloyd, R. S., Prakash, S., Prakash, L. (2006). Replication past a trans-4-Hydroxynonenal Minor-Groove Adduct by the Sequential Action of Human DNA Polymerases {iota} and {kappa}. Mol. Cell. Biol. 26: 381-386 [Abstract] [Full Text]  
• Chiapperino, D., Cai, M., Sayer, J. M., Yagi, H., Kroth, H., Masutani, C., Hanaoka, F., Jerina, D. M., Cheh, A. M. (2005). Error-prone Translesion Synthesis by Human DNA Polymerase {eta} on DNA-containing Deoxyadenosine Adducts of 7,8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. J. Biol. Chem. 280: 39684-39692 [Abstract] [Full Text]  
• Gibbs, P. E. M., McDonald, J., Woodgate, R., Lawrence, C. W. (2005). The Relative Roles in Vivo of Saccharomyces cerevisiae Pol {eta}, Pol {zeta}, Rev1 Protein and Pol32 in the Bypass and Mutation Induction of an Abasic Site, T-T (6-4) Photoadduct and T-T cis-syn Cyclobutane Dimer. Genetics 169: 575-582 [Abstract] [Full Text]  
• Nakajima, S., Lan, L., Kanno, S.-i., Takao, M., Yamamoto, K., Eker, A. P. M., Yasui, A. (2004). UV Light-induced DNA Damage and Tolerance for the Survival of Nucleotide Excision Repair-deficient Human Cells. J. Biol. Chem. 279: 46674-46677 [Abstract] [Full Text]  
• Washington, M. T., Minko, I. G., Johnson, R. E., Haracska, L., Harris, T. M., Lloyd, R. S., Prakash, S., Prakash, L. (2004). 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. 24: 6900-6906 [Abstract] [Full Text]  
• Pessoa-Brandao, L., Sclafani, R. A. (2004). CDC7/DBF4 Functions in the Translesion Synthesis Branch of the RAD6 Epistasis Group in Saccharomyces cerevisiae. Genetics 167: 1597-1610 [Abstract] [Full Text]  
• Haracska, L., Torres-Ramos, C. A., Johnson, R. E., Prakash, S., Prakash, L. (2004). Opposing Effects of Ubiquitin Conjugation and SUMO Modification of PCNA on Replicational Bypass of DNA Lesions in Saccharomyces cerevisiae. Mol. Cell. Biol. 24: 4267-4274 [Abstract] [Full Text]  
• Culligan, K., Tissier, A., Britt, A. (2004). ATR Regulates a G2-Phase Cell-Cycle Checkpoint in Arabidopsis thaliana. Plant Cell 16: 1091-1104 [Abstract] [Full Text]  
• Washington, M. T., Prakash, L., Prakash, S. (2003). Mechanism of nucleotide incorporation opposite a thymine-thymine dimer by yeast DNA polymerase {eta}. Proc. Natl. Acad. Sci. USA 100: 12093-12098 [Abstract] [Full Text]  
• Washington, M. T., Helquist, S. A., Kool, E. T., Prakash, L., Prakash, S. (2003). Requirement of Watson-Crick Hydrogen Bonding for DNA Synthesis by Yeast DNA Polymerase {eta}. Mol. Cell. Biol. 23: 5107-5112 [Abstract] [Full Text]  
• Yang, I.-Y., Miller, H., Wang, Z., Frank, E. G., Ohmori, H., Hanaoka, F., Moriya, M. (2003). Mammalian Translesion DNA Synthesis across an Acrolein-derived Deoxyguanosine Adduct. PARTICIPATION OF DNA POLYMERASE eta IN ERROR-PRONE SYNTHESIS IN HUMAN CELLS. J. Biol. Chem. 278: 13989-13994 [Abstract] [Full Text]  
• Masuda, Y., Ohmae, M., Masuda, K., Kamiya, K. (2003). Structure and Enzymatic Properties of a Stable Complex of the Human REV1 and REV7 Proteins. J. Biol. Chem. 278: 12356-12360 [Abstract] [Full Text]  
• Lee, D.-H., Pfeifer, G. P. (2003). Deamination of 5-Methylcytosines within Cyclobutane Pyrimidine Dimers Is an Important Component of UVB Mutagenesis. J. Biol. Chem. 278: 10314-10321 [Abstract] [Full Text]  
• Haracska, L., Prakash, S., Prakash, L. (2003). Yeast DNA Polymerase {zeta} Is an Efficient Extender of Primer Ends Opposite from 7,8-Dihydro-8-Oxoguanine and O6-Methylguanine. Mol. Cell. Biol. 23: 1453-1459 [Abstract] [Full Text]  
• Wang, A., Gu, J., Judson-Kremer, K., Powell, K.L., Mistry, H., Simhambhatla, P., Aldaz, C. M., Gaddis, S., MacLeod, M. C. (2003). Response of human mammary epithelial cells to DNA damage induced by BPDE: involvement of novel regulatory pathways. Carcinogenesis 24: 225-234 [Abstract] [Full Text]  
• Johnson, R. E., Yu, S.-L., Prakash, S., Prakash, L. (2003). Yeast DNA polymerase zeta (zeta ) is essential for error-free replication past thymine glycol. Genes Dev. 17: 77-87 [Abstract] [Full Text]  
• Servant, L., Cazaux, C., Bieth, A., Iwai, S., Hanaoka, F., Hoffmann, J.-S. (2002). A Role for DNA Polymerase beta in Mutagenic UV Lesion Bypass. J. Biol. Chem. 277: 50046-50053 [Abstract] [Full Text]  
• Haracska, L., Prakash, L., Prakash, S. (2002). Role of human DNA polymerase kappa as an extender in translesion synthesis. Proc. Natl. Acad. Sci. USA 99: 16000-16005 [Abstract] [Full Text]  
• Prakash, S., Prakash, L. (2002). Translesion DNA synthesis in eukaryotes: A one- or two-polymerase affair. Genes Dev. 16: 1872-1883 [Full Text]  
• Haracska, L., Prakash, S., Prakash, L. (2002). Yeast Rev1 Protein Is a G Template-specific DNA Polymerase. J. Biol. Chem. 277: 15546-15551 [Abstract] [Full Text]  
• Van Sloun, P. P. H., Varlet, I., Sonneveld, E., Boei, J. J. W. A., Romeijn, R. J., Eeken, J. C. J., De Wind, N. (2002). Involvement of Mouse Rev3 in Tolerance of Endogenous and Exogenous DNA Damage. Mol. Cell. Biol. 22: 2159-2169 [Abstract] [Full Text]  
• Torres-Ramos, C. A., Prakash, S., Prakash, L. (2002). Requirement of RAD5 and MMS2 for Postreplication Repair of UV-Damaged DNA in Saccharomyces cerevisiae. Mol. Cell. Biol. 22: 2419-2426 [Abstract] [Full Text]  
• Huang, M.-E., Rio, A.-G., Galibert, M.-D., Galibert, F. (2002). Pol32, a Subunit of Saccharomyces cerevisiae DNA Polymerase {delta}, Suppresses Genomic Deletions and Is Involved in the Mutagenic Bypass Pathway. Genetics 160: 1409-1422 [Abstract] [Full Text]  
• Chiapperino, D., Kroth, H., Kramarczuk, I. H., Sayer, J. M., Masutani, C., Hanaoka, F., Jerina, D. M., Cheh, A. M. (2002). Preferential Misincorporation of Purine Nucleotides by Human DNA Polymerase eta Opposite Benzo[a]pyrene 7,8-Diol 9,10-Epoxide Deoxyguanosine Adducts. J. Biol. Chem. 277: 11765-11771 [Abstract] [Full Text]  
• Zhang, H., Siede, W. (2002). UV-induced T->C transition at a TT photoproduct site is dependent on Saccharomyces cerevisiae polymerase {eta}in vivo. Nucleic Acids Res 30: 1262-1267 [Abstract] [Full Text]  
• Ulrich, H. D. (2002). Degradation or Maintenance: Actions of the Ubiquitin System on Eukaryotic Chromatin. Eukaryot Cell 1: 1-10 [Full Text]  
• Haracska, L., Johnson, R. E., Unk, I., Phillips, B. B., Hurwitz, J., Prakash, L., Prakash, S. (2001). Targeting of human DNA polymerase iota to the replication machinery via interaction with PCNA. Proc. Natl. Acad. Sci. USA 10.1073/pnas.261560798v1 [Abstract] [Full Text]  
• Haracska, L., Johnson, R. E., Unk, I., Phillips, B., Hurwitz, J., Prakash, L., Prakash, S. (2001). Physical and Functional Interactions of Human DNA Polymerase {eta} with PCNA. Mol. Cell. Biol. 21: 7199-7206 [Abstract] [Full Text]  
• Washington, M. T., Johnson, R. E., Prakash, L., Prakash, S. (2001). Accuracy of lesion bypass by yeast and human DNA polymerase eta. Proc. Natl. Acad. Sci. USA 98: 8355-8360 [Abstract] [Full Text]  
• You, Y.-H., Lee, D.-H., Yoon, J.-H., Nakajima, S., Yasui, A., Pfeifer, G. P. (2001). Cyclobutane Pyrimidine Dimers Are Responsible for the Vast Majority of Mutations Induced by UVB Irradiation in Mammalian Cells. J. Biol. Chem. 276: 44688-44694 [Abstract] [Full Text]  
• Washington, M. T., Johnson, R. E., Prakash, L., Prakash, S. (2002). Human DINB1-encoded DNA polymerase kappa is a promiscuous extender of mispaired primer termini. Proc. Natl. Acad. Sci. USA 99: 1910-1914 [Abstract] [Full Text]  
• Haracska, L., Johnson, R. E., Unk, I., Phillips, B. B., Hurwitz, J., Prakash, L., Prakash, S. (2001). Targeting of human DNA polymerase iota to the replication machinery via interaction with PCNA. Proc. Natl. Acad. Sci. USA 98: 14256-14261 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Johnson, R. E. Articles by Prakash, L. Search for Related Content PubMed PubMed Citation Articles by Johnson, R. E. Articles by Prakash, L.