JoAnn Sekiguchi

Site-Specific Ribonuclease Activity of Eukaryotic DNA Topoisomerase I

Type I topoisomerases alter DNA topology by cleaving and rejoining one strand of duplex DNA through a covalent protein-DNA intermediate. Here we show that vaccinia topoisomerase, a eukaryotic type IB enzyme, catalyzes site-specific endoribonucleolytic cleavage of an RNA-containing strand. The RNase reaction occurs via transesterification at the scissile ribonucleotide to form a covalent RNA-3-phosphoryl-enzyme intermediate, which is then attacked by the vicinal 2 OH of the ribose sugar to yield a free 2, 3 cyclic phosphate product. Introduction of a single ribonucleoside at the scissile phosphate of an otherwise all-DNA substrate suffices to convert the topoisomerase into an endonuclease. Human topoisomerase I also has endoribonuclease activity. These findings suggest potential roles for topoisomerases in RNA processing.

Mutational analysis of 39 residues of vaccinia DNA topoisomerase identifies Lys-220, Arg-223, and Asn-228 as important for covalent catalysis.

Vaccinia DNA topoisomerase, a 314-amino acid type I enzyme, catalyzes the cleavage and rejoining of DNA strands through a DNA-(3′-phosphotyrosyl)-enzyme intermediate. To identify amino acids that participate in the transesterification reaction, we introduced alanine substitutions at 39 positions within a conserved 57amino acid segment upstream of the active-site tyrosine. Purified wild type and mutant proteins were compared with respect to their activities in relaxing supercoiled DNA. The majority of mutant proteins displayed wild type topoisomerase activity. Mutant enzymes that relaxed DNA at reduced rates were subjected to kinetic analysis of the strand cleavage and religation steps under single-turnover and equilibrium conditions. For the wild type topoisomerase, the observed single-turnover cleavage rate constant (kcl) was 0.29 s−1 and the cleavage-religation equilibrium constant (Kcl) was 0.22. The most dramatic mutational effects were seen with R223A; removal of the basic side chain reduced the rates of cleavage and religation by factors of 10−4.3 and 10−5.0, respectively, and shifted the cleavage-religation equilibrium in favor of the covalently bound state (Kcl = 1). Introduction of lysine at position 223 restored the rate of cleavage to 1/10 that of the wild type enzyme. We conclude that a basic residue is essential for covalent catalysis and suggest that Arg-223 is a constituent of the active site. Modest mutational effects were observed at two other positions (Lys-220 and Asn-228), at which alanine substitutions slowed the rates of strand cleavage by 1 order of magnitude and shifted the equilibrium toward the noncovalently bound state. Arg-223 and Lys-220 are conserved in all members of the eukaryotic type I topoisomerase family; Asn-228 is conserved among the poxvirus enzymes.

Identification of contacts between topoisomerase I and its target DNA by site-specific photocrosslinking.

Vaccinia DNA topoisomerase, a eukaryotic type I enzyme, binds and cleaves duplex DNA at sites containing the sequence 5′‐(T/C)CCTT. We report the identification of Tyr70 as the site of contact between the enzyme and the +4C base of its target site. This was accomplished by UV‐crosslinking topoisomerase to bromocytosine‐substituted DNA, followed by isolation and sequencing of peptide‐DNA photoadducts. A model for the topoisomerase‐DNA interface is proposed, based on the crystal structure of a 9 kDa N‐terminal tryptic fragment. The protein domain fits into the DNA major groove such that Tyr70 is positioned close to the +4C base and Tyr72 is situated near the +3C base. Mutational analysis indicates that Tyr70 and Tyr72 contribute to site recognition during covalent catalysis. We propose, based on this and other studies of the vaccinia protein, that DNA backbone recognition and reaction chemistry are performed by a relatively well‐conserved 20 kDa C‐terminal portion of the vaccinia enzyme, whereas discrimination of the DNA sequence at the cleavage site is accomplished by a separate N‐terminal domain, which is less conserved between viral and cellular proteins. Division of function among distinct structural modules may explain the different site specificities of the eukaryotic type I topoisomerases.

Intramolecular synapsis of duplex DNA by vaccinia topoisomerase

Complexes formed by vaccinia topoisomerase I on plasmid DNA were visualized by electron microscopy. The enzyme formed intramolecular loop structures in which non‐contiguous DNA segments were synapsed within filamentous protein stems. At high enzyme concentrations the DNA appeared to be zipped up within the protein filaments such that the duplex was folded back on itself. Formation of loops and filaments was also observed with an active site mutant, Topo‐Phe274. Binding of Topo‐Phe274 to relaxed DNA circles in solution introduced torsional strain, which, after relaxation by catalytic amounts of wild‐type topoisomerase, resulted in acquisition of negative supercoils. We surmise that the topoisomerase–DNA complex is a plectonemic supercoil in which the two duplexes encompassed by the protein filaments are interwound in a right handed helix. We suggest that topoisomerase‐mediated DNA synapsis plays a role in viral recombination and in packaging of the 200 kbp vaccinia genome during virus assembly.

Kinetic Analysis of DNA and RNA Strand Transfer Reactions Catalyzed by Vaccinia Topoisomerase

Vaccinia topoisomerase binds duplex DNA and forms a covalent DNA-(3′-phosphotyrosyl) protein adduct at the sequence 5′-CCCTT↓. The enzyme reacts readily with a 36-mer CCCTT strand (DNA-p-RNA) composed of DNA 5′ and RNA 3′ of the scissile bond. However, a 36-mer composed of RNA 5′ and DNA 3′ of the scissile phosphate (RNA-p-DNA) is a poor substrate for covalent adduct formation. Vaccinia topoisomerase efficiently transfers covalently held CCCTT-containing DNA to 5′-OH-terminated RNA acceptors; the topoisomerase can therefore be used to tag the 5′ end of RNA in vitro. Religation of the covalently bound CCCTT-containing DNA strand to a 5′-OH-terminated DNA acceptor is efficient and rapid (k rel > 0.5 s−1), provided that the acceptor DNA is capable of base pairing to the noncleaved DNA strand of the topoisomerase-DNA donor complex. The rate of strand transfer to DNA is not detectably affected by base mismatches at the 5′ nucleotide of the acceptor strand. Nucleotide deletions and insertions at the 5′ end of the acceptor slow the rate of religation; the observed hierarchy of reaction rates is as follows: +1 insertion > −1 deletion > +2 insertion ≫ −2 deletion. These findings underscore the importance of a properly positioned 5′-OH terminus in transesterification reaction chemistry, but they also raise the possibility that topoisomerase may generate mutations by sealing DNA molecules with mispaired or unpaired ends.

Covalent DNA Binding by Vaccinia Topoisomerase Results in Unpairing of the Thymine Base 5′ of the Scissile Bond

We have used potassium permanganate to probe contacts between vaccinia DNA topoisomerase and thymine residues in its 5′-CCCTT↓ DNA target site. Two major conclusions emerge from the experiments presented: (i) permanganate oxidation of the +2T base of the scissile strand interferes with topoisomerase binding to DNA, and (ii) the +1T base of the scissile strand becomes unpaired upon formation of the covalent topoisomerase-DNA intermediate. Disruption of T:A base pairing is confined to the +1-position. Covalently bound DNAs that have experienced this structural distortion (such DNAs being marked by oxidation at +1T) are fully capable of being religated. We suggest that a protein-induced DNA conformational change is a component of the strand passage step of the topoisomerase reaction.