Position-specific trapping of topoisomerase I–DNA cleavage complexes by intercalated benzo[
            <i>a</i>
            ]- pyrene diol epoxide adducts at the 6-amino group of adenine

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Vaccinia DNA topoisomerase forms a covalent DNA-(3-phosphotyrosyl)-enzyme intermediate at a specific target site 5-C ؉5 C ؉4 C ؉3 T ؉2 T ؉1 p2N ؊1 in duplex DNA. Here we study the effects of base modifications on the rate and extent of single-turnover DNA transesterification. Chiral trans opened C-10 R and S adducts of benzo[a]pyrene (BP) 7,8-diol 9,10-epoxide were introduced at single N 6 -deoxyadenosine (dA) positions within the 3-sequence of the nonscissile DNA strand. The R and S BPdA adducts intercalate from the major groove on the 5 and 3 sides of the modified base, respectively, and perturb local base stacking. We found that R and S BPdA modifications at ؉1A reduced the transesterification rate by a factor of 700 -1000 without affecting the yield of the covalent topoisomerase-DNA complex. BPdA modifications at ؉2A reduced the extent of transesterification and elicited rate decrements of 200-and 7000-fold for the S and R diastereomers, respectively. In contrast, BPdA adducts at the ؊2 position had no effect on the extent of the reaction and relatively little impact on the rate of cleavage. A more subtle probe of major groove contacts entailed substituting each of the purines of the nonscissile strand with its 8-oxo analog. The ؉3 oxoG modification slowed transesterification 35-fold, whereas other 8-oxo modifications were benign. 8-Oxo substitutions at the ؊1 position in the scissile strand slowed single-turnover cleavage by a factor of six but had an even greater slowing effect on religation, which resulted in an increase in the cleavage equilibrium constant. 2-Aminopurine at positions ؉3, ؉4, or ؉5 in the nonscissile strand had no effect on transesterification per se but had synergistic effects when combined with 8-oxoA at position ؊1 in the scissile strand. These findings illuminate the functional interface of vaccinia topoisomerase with the DNA major groove.Poxvirus DNA topoisomerase is an attractive target for drug therapy of smallpox in light of its unique DNA recognition specificity, compact structure, and distinctive pharmacological sensitivities compared with human topoisomerase I (1-6). Poxvirus topoisomerase, exemplified by the 314-amino acid vaccinia virus enzyme, is a prototype of the type IB DNA topoisomerase family (7-9). Type IB enzymes cleave and rejoin one strand of the DNA duplex through a transient DNA-(3Ј-phosphotyrosyl)-enzyme intermediate.Poxvirus topoisomerases bind and cleave duplex DNA at a pentapyrimidine target sequence, 5Ј-(T/C)CCTTp2 (3). The T2 nucleotide (defined as the ϩ1 nucleotide) is linked to Tyr-274 of the vaccinia enzyme. Four conserved amino acid side chains found in all type IB topoisomerases (Arg-130, Lys-167, Arg-223, and His-265 in the vaccinia enzyme) are responsible for catalyzing the attack by tyrosine on the scissile phosphodiester to form the covalent intermediate (10 -12). The topoisomerase binds to DNA as a C-shaped protein clamp formed by an 80-amino acid N-terminal domain that contacts the DNA major groove and a 234-amino acid C-terminal catalytic domain ...

Section: Discussionmentioning

confidence: 99%

Site-specific DNA Transesterification by Vaccinia Topoisomerase

Yakovleva

Tian

Sayer

et al. 2003

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“…Prior studies have shown that cleavage of Top1 to the 80-kDa form does not prevent its nuclear localization (38) and that 80-kDa Top1 retains its ability to form Top1cc in cells exposed to camptothecin (7). In vitro experiments have further demonstrated that truncated 68-kDa C-terminal recombinant Top1 is still catalytically active (37).…”

Section: Caspase-3-cleaved Top1 Is Preferentially Involved In the Apomentioning

confidence: 99%

“…Although the N-terminal domain of Top1 is dispensable for Top1 catalytic activity (37), it binds to the E3 ubiquitin ligase Topors (47). Top1 ubiquitination and subsequent degradation by the 26 S proteasome (48) have been suggested as an early step in the repair of Top1cc prior to tyrosyl-DNA phosphodiesterase (Tdp1) action (49).…”

Section: Caspase-3-cleaved Top1 Is Preferentially Involved In the Apomentioning

confidence: 99%

Topoisomerase I Requirement for Death Receptor-induced Apoptotic Nuclear Fission

Sordet

Goldman

Redon

et al. 2008

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Topoisomerase I (Top1) is known to relax DNA supercoiling generated by transcription, replication, and chromatin remodeling. However, it can be trapped on DNA as cleavage complexes (Top1cc) by oxidative and carcinogenic DNA lesions, base damage, and camptothecin treatment. We show here that Top1 is also functionally involved in death receptor-induced programmed cell death. In cells exposed to TRAIL or Fas ligand, Top1cc form at the onset of apoptosis. Those apoptotic Top1cc are prevented by caspase inhibition and Bax inactivation, indicating that both caspases and the mitochondrial death pathway are required for their formation. Accordingly, direct activation of the mitochondrial pathway by BH3 mimetic molecules induces apoptotic Top1cc. We also show that TRAIL-induced apoptotic Top1cc are preferentially formed by caspase-3-cleaved Top1 at sites of oxidative DNA lesions with an average of one apoptotic Top1cc/100 kbp. Examination of Top1 knockdown cells treated with TRAIL revealed similar DNA fragmentation but a marked decrease in apoptotic nuclear fission with reduced formation of nuclear bodies. Thus, we propose that Top1 contributes to the full apoptotic responses induced by TRAIL.Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) 2 is a promising therapeutic agent because it induces apoptosis in a wide variety of cancer cells without affecting normal tissues (1, 2). TRAIL belongs to the TNF family of cytokines, including TNF␣ and Fas ligand, which induce apoptosis by binding to their cognate plasma membrane receptors (3). The binding of TRAIL to the DR4 or DR5 receptors and the binding of Fas ligand to Fas receptor cause the intracellular death domains of those receptors to trimerize, which leads to the recruitment of FADD and the activation of caspase-8. Caspase-8 then cleaves and thereby activates caspase-3 either directly (type I cells) or/and indirectly (type II cells) (4) by activating the mitochondrial death pathway through the cleavage of Bid (5). Cleaved Bid binds to and activates the pro-apoptotic Bcl-2 relatives Bax and Bak proteins, causing the release of mitochondrial cytochrome c and the activation of caspase-9 and caspase-3 (6). Activated caspase-3 (and other downstream caspases) cleaves a broad array of intracellular targets including DNA topoisomerase I (Top1) (7). It is also required to induce the controlled rearrangement and degradation of nuclear structures with chromatin condensation, DNA fragmentation, nuclear fission, and release of apoptotic nuclear bodies in the extracellular space (8). TRAIL-induced apoptosis also involves an accumulation of intracellular reactive oxygen species (ROS) (9).Top1 removes DNA superhelical tensions generated during transcription, replication, and chromatin remodeling (10) and is essential in higher eukaryotes (11). It relaxes DNA by forming transient DNA single-strand breaks that are produced as Top1 forms a covalent bond between its active site tyrosine (Tyr 723 ) and a 3Ј-DNA phosphate. These Top1 cleavage complexes (Top1cc) allow con...

“…Differences in the distribution of mutations induced by specific BcPh dA adducts have been observed (64), suggesting that the stereochemistry of these BcPh DEs may influence DNA-metabolic events such as the fidelity of translesion DNA synthesis (62). Although DNA polymerases are obvious targets, the ability of PAH-DE adducts in DNA to interact adversely with other enzymes such as topoisomerases (25,26) may also contribute to their carcinogenic effects. Our present findings that helicases are inhibited by these adducts in a strand-specific and stereospecific manner suggest a further mechanism whereby these lesions could contribute to genetic damage and cell transformation.…”

Section: Effect Of a Bcph Adduct On Blm And Uvrd Helicase Activity-thmentioning

confidence: 99%

“…Polycyclic aromatic hydrocarbon (PAH) 3,4-diol 1,2-epoxide (DE)-DNA adducts have been used by us as tools to probe the interactions between other DNA-processing enzymes such as topoisomerases (25,26) and their DNA substrates as well as to elucidate possible molecular mechanisms for the induction of cancer by these DNA lesions. In this study, we examined the effects of site-specific benzo[c]phenanthrene (BcPh) DE adducts at N 6 of deoxyadenosine (Scheme I) positioned centrally in the double-stranded region of a forked DNA duplex substrate on the unwinding activity catalyzed by WRN helicase.…”

mentioning

confidence: 99%

Inhibition of Werner Syndrome Helicase Activity by Benzo[c]phenanthrene Diol Epoxide dA Adducts in DNA Is Both Strand-and Stereoisomer-dependent

Driscoll

Matson

Sayer

et al. 2003

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Helicases are among the first enzymes to encounter DNA damage during DNA processing within the cell and thus are likely to be targets for the adverse effects of DNA lesions induced by environmental chemicals. Here we examined the effect of cis-and trans-opened 3,4-diol 1,2-epoxide (DE) DNA adducts of benzo[c]phenanthrene (BcPh) at N 6 of adenine on helicase activity. These adducts are derived from the highly tumorigenic (؊)-(1R,2S,3S,4R)-DE as well as its less carcinogenic (؉)-(1S,2R,3R,4S)-DE enantiomer in both of which the benzylic 4-hydroxyl group and epoxide oxygen are trans. The hydrocarbon portions of these adducts intercalate into DNA on the 3 or the 5 side of the adducted deoxyadenosine for the 1S-and 1R-adducts, respectively. These adducts inhibited the human Werner (WRN) syndrome helicase activity in a strand-specific and stereospecific manner. In the strand along which WRN translocates, cis-opened adducts were significantly more effective inhibitors than trans-opened isomers, indicating that WRN unwinding is sensitive to adduct stereochemistry. WRN helicase activity was also inhibited but to a lesser extent by cis-opened BcPh DE adducts in the displaced strand independent of their direction of intercalation, whereas inhibition by the trans-opened stereoisomers in the displaced strand depended on their orientation, such that only adducts oriented toward the advancing helicase inhibited WRN activity. A BcPh DE adduct positioned in the helicase-translocating strand did not sequester WRN, nor affect the rate of ATP hydrolysis relative to an unadducted control. Although the Bloom (BLM) syndrome helicase was also inhibited by a cis-opened adduct in a strand-specific manner, this helicase was not as severely affected as WRN. Because BcPh DEs form substantial amounts of deoxyadenosine adducts at dA, their adverse effects on helicases could contribute to genetic damage and cell transformation induced by these DEs. Thus, the unwinding activity of RecQ helicases is sensitive to the strand, orientation, and stereochemistry of intercalated polycyclic aromatic hydrocarbon adducts.Helicases are enzymes that disrupt complementary strands of duplex DNA in a reaction dependent on nucleoside-5Ј-triphosphate hydrolysis (1-3). Evidence suggests that helicases play important roles in DNA metabolism, and a growing number of helicases have been linked to human disease (4, 5). Pathways of DNA replication, recombination, and repair are affected by DNA lesions, and the effects of helicases on such pathways are probably modulated by their interactions with chemically modified DNA. Although the mechanism for DNA unwinding has been studied for several helicases (reviewed in Refs. 1-3), very little is known regarding how specific covalently linked adducts affect helicase function.RecQ helicases are believed to function in repairing replication forks that have been stalled by DNA damage and may also play a role in the intra-S-phase checkpoint, which delays the replication of damaged DNA, thus permitting repair to occur (6). Only lim...