Identification of the cis-thymine glycol moiety in chemically oxidized and .gamma.-irradiated DNA by HPLC analysis

Frenkel, Krystyna; Goldstein, Mindy S.; Teebor, George W.

doi:10.1021/bi00529a035

Cited by 113 publications

(50 citation statements)

References 31 publications

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“…In the presence of Mn+, the incorporation of one nucleotide opposite the damage residue has been reported for both AP DNA and UV-damaged DNA templates using Pol I (6,26,27). DISCUSSION Thymine glycol and fragmented thymine residues such as urea and Nsubstituted ureas are produced by ionizing radiation (1) and certain chemical reagents (3,11,12,14,15,16,22,28). Although the damaged thymine residues may be removed by base excision-repair, such lesions may be encountered by the replication fork before they are repaired.…”

Section: Effect Of Substitution Of Mn++ For Mg++mentioning

confidence: 99%

Thymine glycols and urea residues in M13 DNA constitute replicative blocksin vitro

Ide¹,

Kow²,

Wallace³

1985

Nucl Acids Res

262

186

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ABSrRACTThymine glycols were produced in M13 DNA in a concentration dependent manner by treating the DNA with osmium tetroxide (0s04). For the formation of urea-containing M13 DNA, OsO 4-oxidized DNA was hydrolyzed in alkali (pH 12) to convert the thymine glycols to urea residues. With both thymine glycol-and urea-containing M13 DNA, DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment was decreased in proportion to the number of damages present in the template DNA. Sequencing gel analysis of the products synthesized by E. coli DNA polymerase I and T4 DNA polymerase showed that DNA synthesis terminated opposite the putative thymine glycol site and at one nucleotide before the putative urea site. Substitution of manganese for magnesium in the reaction mix resulted in increased processivity of DNA synthesis so that a base was incorporated opposite urea. With thymine glycol-containing DNA, processivity in the presence of manganese was strongly dependent on the presence of a pyrimidine 5' to the thymine glycol in the template.

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Section: Effect Of Substitution Of Mn++ For Mg++mentioning

confidence: 99%

Thymine glycols and urea residues in M13 DNA constitute replicative blocksin vitro

Ide¹,

Kow²,

Wallace³

1985

Nucl Acids Res

262

186

View full text Add to dashboard Cite

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“…DNA polymerases kappa and zeta | error-free bypass of thymine glycol | replicative lesion bypass | thymine glycol bypass in humans T hymine glycol (Tg) is the most common oxidation product of thymine (1)(2)(3). Hydroxyl radicals, the principal DNA damaging species formed from aerobic respiration or from exposure to chemical oxidizing agents or ionizing radiation, interact directly with thymine primarily at the 5,6 double bond, generating a thymine glycol.…”

mentioning

confidence: 99%

Error-free replicative bypass of thymine glycol by the combined action of DNA polymerases κ and ζ in human cells

Yoon

Bhatia

Prakash

et al. 2010

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

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Thymine glycol (Tg) is the most common DNA lesion of thymine induced by interaction with reactive oxygen species. Because of the addition of hydroxyl groups at C5 and C6 in a Tg lesion, the damaged base loses its aromatic character and becomes nonplanar; consequently, the C5 methyl group protrudes in an axial direction and that prevents the stacking of the 5′ base above the Tg lesion. Because Tg presents a severe block to continued synthesis by replicative DNA polymerases, we determine here how human cells manage to replicate through this lesion. Using a duplex plasmid system where bidirectional replication ensues from an origin of replication, we show that translesion synthesis (TLS) makes a prominent contribution to Tg bypass and that it occurs in a predominantly error-free fashion. Also, we provide evidence that Polκ and Polζ function together in promoting error-free replication through the lesion, and based on structural and biochemical information, we propose a role for Polκ at the insertion step and of Polζ at the extension step of Tg bypass. We discuss the implications of these observations and suggest that human cells have adapted the TLS machinery to function in a much more error-free fashion than could have been predicted from the intrinsic catalytic efficiencies and fidelities of TLS polymerases.DNA polymerases kappa and zeta | error-free bypass of thymine glycol | replicative lesion bypass | thymine glycol bypass in humans T hymine glycol (Tg) is the most common oxidation product of thymine (1-3). Hydroxyl radicals, the principal DNA damaging species formed from aerobic respiration or from exposure to chemical oxidizing agents or ionizing radiation, interact directly with thymine primarily at the 5,6 double bond, generating a thymine glycol. Although Tg lesions in humans can be removed by base excision repair (BER) involving the action of the DNA glycosylase/AP lyase NTH1 or by nucleotide excision repair (NER) (4, 5), if the damaged base is not removed by these repair processes and goes through the replication fork, that presents a severe block to continued synthesis by eukaryotic replicative DNA polymerases (Pols) (6, 7).Because of the addition of hydroxyl groups at C5 and C6 in a Tg, the damaged base loses its aromatic character and becomes nonplanar (8-10). As a consequence, the C5 methyl group protrudes in an axial direction that prevents the base 5′ to the Tg lesion from stacking above it. Hence a Tg lesion presents a block not only for nucleotide (nt) insertion opposite it but also for the subsequent extension step. Steady-state kinetic analyses of Tg bypass by human translesion (TLS) Pols have indicated that Polη can insert an A opposite Tg with the same catalytic efficiency as for insertion of an A opposite undamaged T; moreover, Polη can also extend from the Tg:A base pair with only an ∼15-fold reduction in efficiency (7). However, Polη incorporates the wrong nts opposite Tg rather frequently as the misincorporation of G or a C residue occurs with only an ∼8-, and 20-fold reduction in effici...

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“…It is estimated that 400 Tgs are formed per cell per day (1,2), and the presence of Tg in DNA has been used as a marker for oxidative stress (1,3). Moreover, Tg is one of the predominant types of base modifications produced by ionizing radiation (4,5), including that used in cancer therapy.…”

mentioning

confidence: 99%

A structural rationale for stalling of a replicative DNA polymerase at the most common oxidative thymine lesion, thymine glycol

Aller

Rould

Hogg³

et al. 2007

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

133

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Thymine glycol (Tg) is a common product of oxidation and ionizing radiation, including that used for cancer treatment. Although Tg is a poor mutagenic lesion, it has been shown to present a strong block to both repair and replicative DNA polymerases. The 2.65-Å crystal structure of a binary complex of the replicative RB69 DNA polymerase with DNA shows that the templating Tg is intrahelical and forms a regular Watson-Crick base pair with the incorporated A. The C5 methyl group protrudes axially from the ring of the damaged pyrimidine and hinders stacking of the adjacent 5 template guanine. The position of the displaced 5 template guanine is such that the next incoming nucleotide cannot be incorporated into the growing primer strand, and it explains why primer extension past the lesion is prohibited even though DNA polymerases can readily incorporate an A across from the Tg lesion.oxidative DNA damage ͉ structure ͉ DNA replication T hymine glycol (5,6-dihydro-5,6-dihydroxythymine; Tg), the most common oxidation product of thymine, is produced endogenously as a consequence of aerobic metabolism or via exogenous factors such as chemical oxidants or ionizing radiation (Fig. 1). It is estimated that 400 Tgs are formed per cell per day (1, 2), and the presence of Tg in DNA has been used as a marker for oxidative stress (1, 3). Moreover, Tg is one of the predominant types of base modifications produced by ionizing radiation (4, 5), including that used in cancer therapy.Of the oxidatively modified DNA bases retaining an intact ring, Tg is thought to induce the most distortion in the regular structure of DNA. Even though there are DNA repair enzymes specialized in excising Tg from the genome, such as human , statistically a few of the damaged bases will evade repair, which means that DNA polymerases will encounter these lesions during replication. Although Tg is a poor mutagenic lesion because it generally pairs with A (9), it has been shown to be a very effective block to DNA replication (10-13). When Tg is encountered as a templating base by replicative or repair polymerases, termination of primer extension occurs immediately past the lesion site with A inserted opposite the Tg. Even in the presence of proofreading, extension proceeds no further. This finding contrasts with the situation observed when the lesion is an abasic site, where, in the presence of proofreading, termination sites are observed one base before the lesion (14). Therefore, it is extension past the Tg⅐adenine pair, rather than insertion across Tg, that constitutes the block to the replicative polymerases. Because Tg is a strong block to repair and replicative DNA polymerases in vitro (10-13), not surprisingly, it is a lethal lesion in vivo (9,(15)(16)(17)(18).Unlike normal DNA bases, Tg is nonplanar because of the loss of aromatic character that accompanies the addition of hydroxyl groups at the 5 and 6 positions of the ring (19). Computational simulations (20,21) predict that the axial orientation of the methyl group with respect to the pyrimidine r...

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Identification of the cis-thymine glycol moiety in chemically oxidized and .gamma.-irradiated DNA by HPLC analysis

Cited by 113 publications

References 31 publications

Thymine glycols and urea residues in M13 DNA constitute replicative blocksin vitro

Thymine glycols and urea residues in M13 DNA constitute replicative blocksin vitro

Error-free replicative bypass of thymine glycol by the combined action of DNA polymerases κ and ζ in human cells

A structural rationale for stalling of a replicative DNA polymerase at the most common oxidative thymine lesion, thymine glycol

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