NMR Evidence for Syn-Anti Interconversion of a Trans Opened (10R)-dA Adduct of Benzo[a]pyrene (7S,8R)-Diol (9R,10S)-Epoxide in a DNA Duplex

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: Discussionsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Site-specific DNA Transesterification by Vaccinia Topoisomerase

Yakovleva

Tian

Sayer

et al. 2003

“…Although there are cases in which the adduct interacts with the minor (12, 13) or the major groove (14,15) of the double helix, in most cases the interaction occurs through intercalation between the surrounding base pairs (13, 16 -32). In many cases, the intercalation occurs from the major groove side (21,27,(31)(32)(33), resembling the conformation adopted by the cholesterol adducts studied in this paper. Displacement of the opposite base out of the helix is rather common, and in many cases the unpaired base is shifted toward the major groove.…”

Section: Molecular Recognition Properties Of the Damaged Dnas-mentioning

confidence: 67%

Effect of Bulky Lesions on DNA

Gómez‐Pinto

Cubero

Kalko

et al. 2004

The three-dimensional solution structure of two DNA decamers of sequence d(CCACXGGAAC)-(GTTCCG-GTGG) with a modified nucleotide containing a cholesterol derivative (X) in its C1(chol)␣ or C1(chol)␤ diastereoisomer form has been determined by using NMR and restrained molecular dynamics. This DNA derivative is recognized with high efficiency by the UvrB protein, which is part of the bacterial nucleotide excision repair, and the ␣ anomer is repaired more efficiently than the ␤ one. The structures of the two decamers have been determined from accurate distance constraints obtained from a complete relaxation matrix analysis of the NOE intensities and torsion angle constraints derived from J-coupling constants. The structures have been refined with molecular dynamics methods, including explicit solvent and applying the particle mesh Ewald method to properly evaluate the long range electrostatic interactions. These calculations converge to well defined structures whose conformation is intermediate between the A-and B-DNA families as judged by the root mean square deviation but with sugar puckerings and groove shapes corresponding to a distorted B-conformation. Both duplex adducts exhibit intercalation of the cholesterol group from the major groove of the helix and displacement of the guanine base opposite the modified nucleotide. Based on these structures and molecular dynamics calculations, we propose a tentative model for the recognition of damaged DNA substrates by the UvrB protein.DNA is susceptible to a number of lesions, such as those caused by chemicals, ionizing radiation, and ultraviolet light.Failure to repair DNA damage is thought to play a significant role in cancer and aging. Nucleotide excision repair (NER) 1 is a universal DNA repair mechanism that recognizes and removes many structurally diverse DNA lesions. This system repairs the damage in the DNA by a three step mechanism: (i) recognition of the lesion, (ii) dual nicking (3Ј and 5Ј) of the damaged strand around the altered site, and (iii) DNA synthesis and ligation to fill the 12-13-base gap. The NER system in eucaryotes is very complex and involves the action of at least 16 different polypeptides (1). On the contrary, in procaryotic organisms the NER mechanism involves only four specific proteins, UvrA, UvrB, UvrC, and UvrD (in addition to DNA polymerase and ligase). Despite the fact that the general mechanism of the UvrABCD system is known, many key issues remain unclear. The most important one is the determination of the structural characteristics of DNA lesions that trigger the repair system. The wide range of lesions that are recognized by the UvrABCD system suggests that the feature that induces repair is an alteration of the structural characteristics of the damaged DNA rather than a particular chemical structure of the damaging agent (2-4). After the analysis of a large number of lesions that are substrates of the UvrABCD system, van Houten (3) classified them into six categories: (i) covalent damage, (ii) bulky substituents, (iii) localize...

“…BaP DE-dG trans adducts occupy the minor groove, with the hydrocarbon moiety of the 10R and 10S adducts oriented in opposite directions relative to the helix axis (toward the 3Ј and 5Ј ends of the modified strand, respectively) and do not significantly distort the double helix of B-form DNA (28 -30). In contrast, both cis (31) and trans (32)(33)(34)(35) opened dA adducts intercalate between base pairs such that the aromatic moiety of the 10R adducts inserts on the 5Ј side of the adducted base, whereas the aromatic moiety of the 10S adducts inserts on the 3Ј side. Based on the NMR structures, intercalation of the hydrocarbon results in buckling and twisting of the base pairs in the immediate vicinity of the adduct as well as local unwinding and overall bending of the helix axis.…”

mentioning

confidence: 99%

Inhibition of Werner Syndrome Helicase Activity by Benzo[a]pyrene Diol Epoxide Adducts Can Be Overcome by Replication Protein A

Choudhary

Doherty

Handy

et al. 2006

RecQ helicases are believed to function in repairing replication forks 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. Since little is known regarding the effects of DNA damage on RecQ helicases, and because the replication and recombination defects in Werner syndrome cells may reflect abnormal processing of damaged DNA associated with the replication fork, we examined the effects of specific bulky, covalent adducts at N 6 of deoxyadenosine (dA) or N 2 of deoxyguanosine (dG) on Werner (WRN) syndrome helicase activity. The adducts are derived from the optically active 7,8-diol 9,10-epoxide (DE) metabolites of the carcinogen benzo[a]pyrene (BaP). The results demonstrate that WRN helicase activity is inhibited in a strand-specific manner by BaP DE-dG adducts only when on the translocating strand. These adducts either occupy the minor groove without significant perturbation of DNA structure (trans adducts) or cause base displacement at the adduct site (cis adducts). In contrast, helicase activity is only mildly affected by intercalating BaP DE-dA adducts that locally perturb DNA double helical structure. This differs from our previous observation that intercalating dA adducts derived from benzo[c]phenanthrene (BcPh) DEs inhibit WRN activity in a strand-and stereospecific manner. Partial unwinding of the DNA helix at BaP DE-dA adduct sites may make such adducted DNAs more susceptible to the action of helicase than DNA containing the corresponding BcPh DE-dA adducts, which cause little or no destabilization of duplex DNA. The single-stranded DNA binding protein RPA, an auxiliary factor for WRN helicase, enabled the DNA unwinding enzyme to overcome inhibition by either the trans-R or cis-R BaP DE-dG adduct, suggesting that WRN and RPA may function together to unwind duplex DNA harboring specific covalent adducts that otherwise block WRN helicase acting alone.DNA damage evokes a cellular response by a genome surveillance system that senses DNA structural perturbation at the site of the lesion and elicits an appropriate response that may involve direct repair of the lesion, stabilization of the replication fork, or induction of apoptosis (1).Cellular pathways of DNA metabolism are influenced by DNA lesions, and since DNA unwinding enzymes known as helicases are among the first proteins to encounter DNA damage, it is of interest to understand how helicase action is modulated by interaction with chemically modified DNA. Although the mechanism for DNA unwinding has been studied for several helicases (2-4), only limited information is available regarding how specific covalently linked adducts affect helicase function. Of particular interest to us has been the Werner (WRN) 3 helicase that is defective in the premature aging and genome instability disorder Werner syndrome (WS) (5). The WRN helicase belongs to the RecQ family of Superfamily 2 DNA helicases (5) and has been shown to unwind double-stranded DNA in a 3Ј t...