Structure of a Duplex DNA Containing a Thymine Glycol Residue in Solution

Kung, Hsiang Chuan; Bolton, Philip H.

doi:10.1074/jbc.272.14.9227

Cited by 105 publications

(123 citation statements)

References 40 publications

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“…The Tg lesion is intrahelical in all four complexes, stacking against the 3Ј template base and base-pairing with acyAMP. An NMR structure of a duplex DNA containing Tg suggested that the Tg lesion was largely extrahelical (22), but the differences are likely to be attributable to the fact that the NMR structure was obtained in the absence of a protein. The location of the lesion also differs: it is found within the DNA duplex in the NMR model as opposed to the end of the duplex DNA in the Tg⅐acyAMP crystal structure.…”

Section: Discussionmentioning

confidence: 99%

“…Computational simulations (20,21) predict that the axial orientation of the methyl group with respect to the pyrimidine ring would create steric hindrance with the template base on the 5Ј side of Tg. NMR studies, on the other hand, have suggested that the structural perturbations attributable to the Tg lesion are localized and result in Tg's being extrahelical (22). To date, there is no published structure of any DNA polymerase in complex with DNA containing Tg that can clarify this lesion's ability to impede extension.…”

mentioning

confidence: 98%

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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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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 98%

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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“…Approximately 10 -20% of the damage to DNA induced by ionizing radiation is the result of thymine base oxidation and fragmentation (4). Thymine is an easily oxidized base and is frequently found as a component of clustered lesions (2,3).…”

mentioning

confidence: 99%

Mode of Inhibition of Short-patch Base Excision Repair by Thymine Glycol within Clustered DNA Lesions

Budworth¹,

Dianov²

2003

Journal of Biological Chemistry

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Clustered DNA damage, where two or more lesions are located proximally to each other, is frequently induced by ionizing radiation. Individual base lesions within a cluster are repaired by base excision repair. In this study we addressed the question of how thymine glycol (Tg) within a cluster would affect the repair of opposing lesions by human cell extracts. We have found that Tg located opposite to an abasic site does not affect cleavage of this site by apurinic/apyrimidinic (AP) endonuclease. However, Tg significantly compromised the next step of the repair. Although purified DNA polymerase ␤ was able to incorporate the correct nucleotide (dAMP) opposite to Tg, the rate of incorporation was reduced by 3-fold. Tg does not affect 5-sugar phosphate removal by the 2-deoxyribose-5-phosphate (dRP) lyase activity of DNA polymerase ␤, but further processing of the strand break by purified DNA ligase III was slightly diminished. In agreement with these findings, although an AP site located opposite to Tg was efficiently incised in human cell extract, only a limited amount of fully repaired product was observed, suggesting that such clustered DNA lesions may have a significantly increased lifetime in human cells compared with similar singlestanding lesions.Numerous cytotoxic agents exert their deleterious effects via the formation of lesions and adducts in DNA; these effects may include oxidized purine and pyrimidine residues, abasic sites, and single and double strand breaks. Ionizing radiation induces damage in DNA by direct ionization and through the generation of hydroxyl radicals that attack DNA, resulting in single strand breaks and oxidative damage to sugar and base residues (1). Two or more DNA lesions of the same or different nature may be produced proximal to each other on the same or opposite DNA strands, generally within two helical turns of the DNA. These various types of DNA damage, known as "clustered DNA lesions," may include strand breaks containing damaged DNA termini accompanied by multiple base lesions of varying complexity (2, 3).Approximately 10 -20% of the damage to DNA induced by ionizing radiation is the result of thymine base oxidation and fragmentation (4). Thymine is an easily oxidized base and is frequently found as a component of clustered lesions (2, 3). Individual base damages within a cluster are repaired by base excision repair (BER).1 BER is a multiprotein pathway with a broad substrate specificity that is determined by the damagespecific glycosylases. DNA glycosylases initiate BER by recognizing damaged or abnormal bases and cleaving the glycosylic bond linking the base to the sugar phosphate backbone (5). The majority of the apurinic/apyrimidinic sites (AP sites) formed are further processed by the so-called "short-patch" BER pathway (6). In human cells this pathway is activated by an AP endonuclease (APE1) that introduces a DNA strand break 5Ј to the AP site (7). This strand break cannot be ligated directly; therefore, DNA polymerase ␤ (Pol ␤) first adds one nucleotide to the 3Ј-en...

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“…Given that damage to the mitochondrial genome is believed to occur readily in the oxidative environment of the mitochondrial matrix (37), we assayed Twinkle helicase activity on DNA substrates with a site-and strand-specific cPu or Tg, two structurally distinct oxidative lesions. Tg is characterized by the introduction of two hydroxyl groups to the pyrimidine at positions C5 and C6, causing a significant localized change to B-form DNA and the base to assume an extrahelical position that is more accessible to solvent and other molecules (20). In contrast, the cPu DNA lesion is characterized by the cyclization of deoxyguanine or deoxyadenine such that a second glycosidic bond is made between the base and the corresponding sugar residue, resulting in perturbation of normal helix twist and abnormal base pair stacking (22,23).…”

Section: Discussionmentioning

confidence: 99%

“…Persistence of these free radicals within the mitochondrial matrix can cause a variety of oxidative DNA lesions as well as damage to other macromolecules. A prominent form of oxidative DNA damage is Tg, which causes localized distortion of the DNA double helix (20) and blocks synthesis by a number of DNA polymerases (21). Given that Twinkle is likely to encounter Tg lesions during mtDNA replication, we set out to examine the effect of a single Tg residing in either the helicase-translocating or non-translocating strand within the double-stranded portion of a forked duplex DNA substrate that Twinkle efficiently unwinds.…”

Section: Twinkle Unwinding and Binding Activity On Replicationmentioning

confidence: 99%

Biochemical Characterization of the Human Mitochondrial Replicative Twinkle Helicase

Khan

Crouch

Bharti

et al. 2016

Journal of Biological Chemistry

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Mutations in the c10orf2 gene encoding the human mitochondrial DNA replicative helicase Twinkle are linked to several rare genetic diseases characterized by mitochondrial defects. In this study, we have examined the catalytic activity of Twinkle helicase on model replication fork and DNA repair structures. Although Twinkle behaves as a traditional 5 to 3 helicase on conventional forked duplex substrates, the enzyme efficiently dissociates D-loop DNA substrates irrespective of whether it possesses a 5 or 3 single-stranded tailed invading strand. In contrast, we report for the first time that Twinkle branch-migrates an open-ended mobile three-stranded DNA structure with a strong 5 to 3 directionality preference. To determine how well Twinkle handles potential roadblocks to mtDNA replication, we tested the ability of the helicase to unwind substrates with site-specific oxidative DNA lesions or bound by the mitochondrial transcription factor A. Twinkle helicase is inhibited by DNA damage in a unique manner that is dependent on the type of oxidative lesion and the strand in which it resides. Novel single molecule FRET binding and unwinding assays show an interaction of the excluded strand with Twinkle as well as events corresponding to stepwise unwinding and annealing. TFAM inhibits Twinkle unwinding, suggesting other replisome proteins may be required for efficient removal. These studies shed new insight on the catalytic functions of Twinkle on the key DNA structures it would encounter during replication or possibly repair of the mitochondrial genome and how well it tolerates potential roadblocks to DNA unwinding.

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Structure of a Duplex DNA Containing a Thymine Glycol Residue in Solution

Cited by 105 publications

References 40 publications

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

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

Mode of Inhibition of Short-patch Base Excision Repair by Thymine Glycol within Clustered DNA Lesions

Biochemical Characterization of the Human Mitochondrial Replicative Twinkle Helicase

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