Translesion DNA Synthesis by Yeast DNA Polymerase η on Templates Containing N 2-Guanine Adducts of 1,3-Butadiene Metabolites

Proc. Natl. Acad. Sci. U.S.A.

Wolfle

Spratt

et al. 2003

Self Cite

DNA polymerase(Pol ) functions in the proficient bypass of a variety of DNA lesions. Relative to the replicative polymerases, Pol has a greater tolerance for distorted DNA geometries and possesses a low fidelity. X-ray crystal structures and studies with nucleotide analogs have implicated interactions with the DNA minor groove as being crucial for the high fidelity of replicative DNA polymerases. To determine whether Pol also makes such functionally important contacts with the DNA minor groove, here we examine the effects on Pol -catalyzed nucleotide incorporation when 3-deazaguanine, a base analog that lacks the ability to form minor-groove hydrogen bonds with the protein, is substituted for guanine at various positions in the DNA. From these studies, we conclude that Pol makes only a single functional contact with the DNA minor groove at the position of the incoming nucleotide; in this regard, Pol differs from highfidelity DNA polymerases that are unable to replicate through DNA lesions. These results help explain the proficient ability of Pol for bypassing distorting DNA lesions. E ukaryotic DNA polymerase (Pol) is a member of the recently discovered Y family of DNA polymerases. Both yeast and human Pol replicate efficiently and accurately through a cis-syn thymine-thymine (TT) dimer by incorporating As opposite the two Ts of the dimer (1-3), and genetic studies in yeast have also indicated the requirement of Pol for the error-free bypass of cyclobutane dimers formed at TC and CC sequences (4). Pol efficiently bypasses a 7,8-dihydro-8-oxoguanine by preferentially incorporating a C opposite the lesion (5), and it is able to replicate through a variety of other distorting DNA lesions (6-8) but with reduced efficiencies. Mutational inactivation of Pol in humans results in the variant form of xeroderma pigmentosum (9, 10), a genetic disorder characterized by a high incidence of UV-induced skin cancers.Based on the ability of Pol to efficiently incorporate nucleotides opposite distorting DNA lesions, we hypothesized that Pol possesses an active site that is unusually tolerant of geometric distortions in the DNA (11). Because an intolerance of geometric distortions in the DNA plays a crucial role in the high fidelity of replicative DNA polymerases, we predicted that Pol would synthesize DNA with a low fidelity. In fact, steady-state kinetics analyses have shown that both yeast and human Pol misincorporate nucleotides with a frequency of 10 Ϫ2 to 10 Ϫ3 (3, 11), and consistent with this low fidelity, in an in vitro reaction human Pol was found to synthesize DNA in a highly mutagenic manner (12).Crystal structures of ternary complexes of high-fidelity DNA polymerases consisting of the polymerase, double-stranded DNA, and a nucleotide substrate in the active site have revealed specific hydrogen-bonding interactions between the polymerase and the N3 (purine) and O 2 (pyrimidine) hydrogen-bonding acceptors in the minor groove of duplex DNA (13-18). Furthermore, biochemical studies with nucleotide analogs lacking hydrogen...

Section: Dna Polymerasementioning

confidence: 99%

Yeast DNA polymerase η makes functional contacts with the DNA minor groove only at the incoming nucleoside triphosphate

Proc. Natl. Acad. Sci. U.S.A.

Wolfle

Spratt

et al. 2003

Self Cite

“…Yeast and human pol also efficiently bypass a product of oxidative DNA damage, the 7,8-dihydro-8-oxoguanine, and do so in a predominantly error-free manner (34). In addition, several other DNA lesions were reported to be substrates for human (35)(36)(37)(38)(39) and yeast (35,40) pol .…”

mentioning

confidence: 99%

Translesion Synthesis past Acrolein-derived DNA Adduct, γ-Hydroxypropanodeoxyguanosine, by Yeast and Human DNA Polymerase η

Minko

Journal of Biological Chemistry

Kanuri

et al. 2003

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107

-propano-2deoxyguanosine (␥-HOPdG) is a major deoxyguanosine adduct derived from acrolein, a known mutagen. In vitro, this adduct has previously been shown to pose a severe block to translesion synthesis by a number of polymerases (pol). Here we show that both yeast and human pol can incorporate a C opposite ␥-HOPdG at ϳ190-and ϳ100-fold lower efficiency relative to the control deoxyguanosine and extend from a C paired with the adduct at ϳ8-and ϳ19-fold lower efficiency. Although DNA synthesis past ␥-HOPdG by yeast pol was relatively accurate, the human enzyme misincorporated nucleotides opposite the lesion with frequencies of ϳ10 ؊1 to 10 ؊2 . Because ␥-HOPdG can adopt both ring closed and ring opened conformations, comparative replicative bypass studies were also performed with two model adducts, propanodeoxyguanosine and reduced ␥-HOPdG. For both yeast and human pol , the ring open reduced ␥-HOPdG adduct was less blocking than ␥-HOPdG, whereas the ring closed propanodeoxyguanosine adduct was a very strong block. Replication of DNAs containing ␥-HOPdG in wild type and xeroderma pigmentosum variant cells revealed a somewhat decreased mutation frequency in xeroderma pigmentosum variant cells. Collectively, the data suggest that pol might potentially contribute to both error-free and mutagenic bypass of ␥-HOPdG. Acrolein (Fig.

“…Pol can also efficiently replicate through other DNA lesions, such as 8-oxoguanine and O 6 -methylguanine (11,14). Pol, however, is inhibited by the N 2 -guanine adducts of 1,3-butadiene or benzo[a]pyrene diol epoxide (30,37).…”

mentioning

confidence: 99%

Efficient and Error-Free Replication Past a Minor-Groove DNA Adduct by the Sequential Action of Human DNA Polymerases ι and κ

Molecular and Cellular Biology

Minko

Johnson

et al. 2004

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114

127

DNA polymerase (Pol) is a member of the Y family of DNA polymerases, which promote replication through DNA lesions. The role of Pol in lesion bypass, however, has remained unclear. Pol is highly unusual in that it incorporates nucleotides opposite different template bases with very different efficiencies and fidelities. Since interactions of DNA polymerases with the DNA minor groove provide for the nearly equivalent efficiencies and fidelities of nucleotide incorporation opposite each of the four template bases, we considered the possibility that Pol differs from other DNA polymerases in not being as sensitive to distortions of the minor groove at the site of the incipient base pair and that this enables it to incorporate nucleotides opposite highly distorting minor-groove DNA adducts. To check the validity of this idea, we examined whether Pol could incorporate nucleotides opposite the ␥-HOPdG adduct, which is formed from an initial reaction of acrolein with the N 2 of guanine. We show here that Pol incorporates a C opposite this adduct with nearly the same efficiency as it does opposite a nonadducted template G residue. The subsequent extension step, however, is performed by Pol, which efficiently extends from the C incorporated opposite the adduct. Based upon these observations, we suggest that an important biological role of Pol and Pol is to act sequentially to carry out the efficient and accurate bypass of highly distorting minor-groove DNA adducts of the purine bases.Replicative DNA polymerases synthesize DNA with high fidelity, and because of their intolerance of geometric distortions in DNA, they are blocked by DNA lesions. The DNA polymerases belonging to the Y family, however, are low-fidelity enzymes, able to promote replication through DNA lesions. DNA polymerase (Pol) from both the yeast Saccharomyces cerevisiae and humans can efficiently and accurately replicate through a cis-syn thymine-thymine dimer (16,19,43,45), and genetic studies in yeast and humans have also indicated a role for Pol in the error-free replication through cyclobutane dimers formed at 5Ј-TC-3Ј and CC sites (39, 49). Consequently, mutational inactivation of Pol in humans causes the variant form of xeroderma pigmentosum (15, 28), characterized by a greatly enhanced predisposition for sunlight-induced skin cancers. Pol can also efficiently replicate through other DNA lesions, such as 8-oxoguanine and O 6 -methylguanine (11, 14). Pol, however, is inhibited by the N 2 -guanine adducts of 1,3-butadiene or benzo[a]pyrene diol epoxide (30, 37).In addition to Pol, humans contain two other Y-family DNA polymerases, Pol and Pol. By contrast to Pol, which promotes lesion bypass both by efficiently inserting the nucleotide opposite the lesion and by extending from the inserted nucleotide, Pol and Pol are apparently more specialized in their roles in lesion bypass (36). For example, while Pol can incorporate nucleotides opposite the 3Ј T of a (6-4) TT photoproduct or opposite an abasic site, it is unable to carry out the subsequent extensio...