DNA Contacts Stimulate Catalysis by a Poxvirus Topoisomerase

Abstract

Eukaryotic type 1B topoisomerases act by forming covalent enzyme-DNA intermediates that transiently nick DNA and thereby release DNA supercoils. Here we present a study of the topoisomerase encoded by the pathogenic poxvirus molluscum contagiosum. Our studies of DNA sites favored for catalysis reveal a larger recognition site than the 5-(T/C)CCTT-3 sequence previously identified for poxvirus topoisomerases. Separate assays of initial DNA binding and covalent complex formation revealed that different DNA sequences were important for each reaction step. The location of the protein-DNA contacts was mapped by analyzing mutant sites and inosine-substituted DNAs. Some of the bases flanking the 5-(T/C)CCTT-3 sequence were selectively important for covalent complex formation but not initial DNA binding. Interactions important for catalysis were probed with 5-bridging phosphorothiolates at the site of strand cleavage, which permitted covalent complex formation but prevented subsequent religation. Kinetic studies revealed that the flanking sequences that promoted recovery of covalent complexes increased initial cleavage instead of inhibiting resealing of the nicked intermediate. These data 1) indicate that previously unidentified DNA contacts can accelerate a step between initial binding and covalent complex formation and 2) help specify models for conformational changes promoting catalysis.Topoisomerases play important roles in DNA replication, recombination, and transcription (1). Type 1B topoisomerases act ( Fig. 1) by first binding double-stranded DNA and then cleaving substrate DNA using a conserved enzyme tyrosine residue, forming a 3Ј-O-phosphotyrosine linkage. The DNA strands then isomerize, either by passing one strand through the break in the other or by rotation of the covalent complex, relaxing the DNA. The broken strand is then resealed by nucleophillic attack of the 5Ј-hydroxyl on the phosphotyrosine bond. Last, the topoisomerase dissociates from substrate.All the poxviruses studied to date encode a type 1B topoisomerase (2-7) that is inferred to be required for viral replication (8). Poxvirus topoisomerases bind to the sequence 5Ј-(T/ C)CCTT-3Ј and cleave just 3Ј of the last T (designated position ϩ1), to form the covalent intermediate (7,9,10). Although many topoisomerases have been found to show some DNA sequence preferences (11)(12)(13)(14), the high degree of specificity seen with the poxvirus topoisomerases is unique.In the case of vaccinia virus, the most thoroughly studied model, the topoisomerase enzyme consists of two domains linked through a trypsin-sensitive bridge (15,16). The N-terminal domain (amino acids 1-80) contacts the 5Ј-(T/C)CCTT-3Ј element on the major groove side of the DNA (15-17). Chemical footprinting and interference studies indicate that the enzyme contacts several phosphates, including the scissile phosphate, on both strands and the minor groove side as well. Recently, the crystal structure of the C-terminal catalytic domain of vaccinia topoisomerase (amino acids 81-314) was det...

Individual Nucleotide Bases, Not Base Pairs, Are Critical for Triggering Site-specific DNA Cleavage by Vaccinia Topoisomerase

“…Available evidence suggests that most of the site specificity is achieved at the level of transesterification chemistry rather than at the noncovalent DNA binding step (24). Whereas the affinity for the target site, the rate of cleavage, and the cleavage equilibrium constant (K cl ) are affected by the nucleotide sequence context surrounding the 5Ј-(C/T)CCTT target site (1,15) in ways that are not well understood in structural terms, the dominant factor triggering the DNA incision reaction is the pentamer 5Ј-CCCTT/3Ј-GGGAA. We have begun to systematically address the features of the individual bases that affect the kinetics of DNA cleavage via position-specific base modifications entailing relatively small additions to, or subtractions from, the standard base structures (11,17), as well as modification by more bulky adducts (16,18).…”

Benzo[c]phenanthrene Adducts and Nogalamycin Inhibit DNA Transesterification by Vaccinia Topoisomerase

“…Topoisomerases encoded by other genera of poxviruses recognize the same DNA target sequence (6 -10). Available structural and biochemical studies suggest that the assembly of a catalytically competent topoisomerase active site is triggered by recognition of the DNA target (11,12).Early studies using nuclease footprinting, modification interference, modification protection, analog substitution, and UV crosslinking techniques suggested that vaccinia topoisomerase makes contact with several nucleotide bases and the sugar-phosphate backbone of DNA within and immediately flanking the CCCTT element (13)(14)(15)(16)(17)(18)(19). Recent studies have focused on delineating the features of the DNA interface that affect the kinetics of transesterification.…”

“…Early studies using nuclease footprinting, modification interference, modification protection, analog substitution, and UV crosslinking techniques suggested that vaccinia topoisomerase makes contact with several nucleotide bases and the sugar-phosphate backbone of DNA within and immediately flanking the CCCTT element (13)(14)(15)(16)(17)(18)(19). Recent studies have focused on delineating the features of the DNA interface that affect the kinetics of transesterification.…”

Vaccinia Topoisomerase Mutants Illuminate Conformational Changes during Closure of the Protein Clamp and Assembly of a Functional Active Site

“…N␦ of Asn-569 is pointed toward the phosphodiester backbone of the scissile strand at a distance of 4.6 Å from the ϩ10 phosphate. A similar scissile strand phosphate contact for Ser-204 of vaccinia topoisomerase is quite plausible given that (i) the footprint of the vaccinia protein on DNA extends 13 bp 5Ј of the cleavage site (9, 41), (ii) both noncovalent and covalent binding of vaccinia topoisomerase to CCCTT-containing DNA are reduced significantly when there are fewer than 10 bp on the 5Ј-side of the cleavage site (42), and (iii) the rate of DNA cleavage is affected by alterations of the ϩ9 and ϩ10 base pairs in the major groove (43).…”