DNA Contacts by Protein Domains of the Molluscum Contagiosum Virus Type-1B Topoisomerase

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 ...

mentioning

confidence: 99%

Site-specific DNA Transesterification by Vaccinia Topoisomerase

Yakovleva

Tian

Sayer

et al. 2003

“…19). This, in turn, appears to cause the free 5Ј-sulfhydryl (which replaces the 5Ј-hydroxyl in this trapped covalent complex; 8,19,[23][24][25][26][27][28] in the nicked DNA strand to shift away from the covalent phosphotyrosine linkage and form a hydrogen bond with the side chain of Asn-722, an interaction not observed in previous topoisomerase I covalent complexes (Figs. 4A and 6).…”

Section: Discussionmentioning

confidence: 75%

“…The G base in italics contained a bridging phosphorothiolate linkage, which upon cleavage by human topoisomerase I generates a free 5Ј-sulfhydryl rather than a hydroxyl (22)(23)(24)(25)(26)(27)(28). This method of trapping human topoisomerase I in covalent complexes with duplex DNA has been used previously in structural studies of this enzyme (8,19), as well as in several detailed mechanistic and biological studies of topoisomerases and related tyrosine recombinases (22)(23)(24)(25)(26)(27)(28). Note that the Ara-C nucleotide is in the intact DNA strand, and the G nucleotide containing the 5Ј-bridging phosphorothiolate linkage is in the scissile DNA strand.…”

Section: Methodsmentioning

confidence: 99%

“…In these trapped covalent protein-DNA complexes, the scissile phosphate contained a bridging phosphorothiolate linkage, which, upon cleavage by topoisomerase I, generates a free 5Ј-sulfhydryl unable to participate in strand religation (8,19,22). The use of 5Ј-bridging phosphorothiolate linkages to trap covalent complexes has been successfully employed to examine several enzymes that form transient 3Ј-phosphotyrosine linkages, including eukaryotic type IB topoisomerases, viral topoisomerases, and bacterial and phage tyrosine recombinases and integrases (8,19,(22)(23)(24)(25)(26)(27)(28). Detailed biochemical studies have shown that the presence of a 5Ј-bridging phosphorothiolate linkage has a marginal effect on the rate of cleavage by such enzymes (down ϳ2-fold), but lowers the rate of religation by at least 10,000-fold (24).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Structural Impact of the Leukemia Drug 1-ॆ-d-Arabinofuranosylcytosine (Ara-C) on the Covalent Human Topoisomerase I-DNA Complex

Chrencik

Burgin²,

Pommier

et al. 2003

1-␤-D-Arabinofuranosylcytosine (Ara-C) is a potent antineoplastic drug used in the treatment of acute leukemia. Previous biochemical studies indicated the incorporation of Ara-C into DNA reduced the catalytic activity of human topoisomerase I by decreasing the rate of single DNA strand religation by the enzyme by 2-3-fold. We present the 3.1 Å crystal structure of human topoisomerase I in covalent complex with an oligonucleotide containing Ara-C at the ؉1 position of the non-scissile DNA strand. The structure reveals that a hydrogen bond formed between the 2-hydroxyl of Ara-C and the O4 of the adjacent ؊1 base 5 to the damage site stabilizes a C3-endo pucker in the Ara-C arabinose ring. The structural distortions at the site of damage are translated across the DNA double helix to the active site of human topoisomerase I. The free sulfhydryl at the 5-end of the nicked DNA strand in this trapped covalent complex is shifted out of alignment with the 3-phosphotyrosine linkage at the catalytic tyrosine 723 residue, producing a geometry not optimal for religation. The subtle structural changes caused by the presence of Ara-C in the DNA duplex may contribute to the cytotoxicity of this leukemia drug by prolonging the lifetime of the covalent human topoisomerase I-DNA complex.Human topoisomerase I solves the DNA topological problems that arise from a wide variety of nuclear processes including replication, transcription, and recombination (1, 2). The enzyme nicks one strand of duplex DNA using a transesterification reaction that produces a transient 3Ј-phosphotyrosine linkage and guides the relaxation of either positive or negative superhelical tension by a proposed "controlled rotation" mechanism (3). The enzyme then catalyzes a second transesterification in which the free hydroxyl at the 5Ј-end of the nicked DNA strand attacks the phosphotyrosine bond, resealing the nick, and releasing a more relaxed DNA molecule. Topoisomerase I plays a vital role in maintaining DNA stability and is known to travel with active replication and transcription complexes in human cells (4,5).Human topoisomerase I is the sole target of the camptothecins (CPT), a potent class of anticancer drugs used to treat late-term solid malignancies (3,4,6). Camptothecin effectively targets the religation phase of topoisomerase I catalysis by stabilizing the covalent protein-DNA complex and trapping the enzyme on DNA (7,8). In this way, CPT converts topoisomerase I into a cellular poison. Human topoisomerase I is also affected by several forms of DNA damage, including abasic lesions, wobble base pairs, and base pair mismatches (9 -13). Such lesions can impact each stage of topoisomerase I's catalytic cycle, including DNA binding, single-strand DNA cleavage, and religation.

“…The poxvirus topoisomerases are two-domain proteins (22)(23)(24)(25) that bind to the 5Ј-(T/C)CCTT-3Ј site as monomers by forming C-shaped clamps around the DNA (22,23,26). Comparison of the vaccinia amino and carboxyl domain structures determined in the absence of DNA (23,25) to the smallpox-topoisomerase/DNA complex (22) allowed the conformational changes that mediate activation to be visualized.…”

mentioning

confidence: 99%

Regulation of Catalysis by the Smallpox Virus Topoisomerase

Hwang

Minkah

Perry

et al. 2006

Self Cite

The poxvirus type IB topoisomerases catalyze relaxation of supercoiled DNA by cleaving and rejoining DNA strands via a pathway involving a covalent phosphotyrosine intermediate. Recently we determined structures of the smallpox virus topoisomerase bound to DNA in covalent and non-covalent DNA complexes using x-ray crystallography. Here we analyzed the effects of twenty-two amino acid substitutions on the topoisomerase activity in vitro in assays of DNA relaxation, single cycle cleavage, and equilibrium cleavage-religation. Alanine substitutions at 14 positions impaired topoisomerase function, marking a channel of functionally important contacts along the protein-DNA interface. Unexpectedly, alanine substitutions at two positions (D168A and E124A) accelerated the forward rate of cleavage. These findings and further analysis indicate that Asp 168 is a key regulator of the active site that maintains an optimal balance among the DNA cleavage, religation, and product release steps. Finally, we report that high level expression of the D168A topoisomerase in Escherichia coli, but not other alanine-substituted enzymes, prevented cell growth. These findings help elucidate the amino acid side chains involved in DNA binding and catalysis and provide guidance for designing topoisomerase poisons for use as smallpox antivirals.All poxviruses encode topoisomerases of the type IB family (1, 2). These enzymes release DNA supercoils via a mechanism involving a 3Ј-phosphotyrosine covalent protein-DNA intermediate (Fig. 1A). In this reaction, the topoisomerase first binds to DNA, then catalyzes nucleophilic attack of an enzyme tyrosine residue on one DNA strand, forming a 3Ј-phosphotyrosine linkage. This resulting DNA strand break allows rotation of the continuous DNA strand around the nick, thereby releasing supercoils (3, 4). The reaction cycle is completed by religation, in which the hydroxyl group of the free DNA 5Ј-end at the nick attacks the phosphotyrosine linkage, rejoining the DNA, followed by product release (5, 6).