Variola virus topoisomerase: DNA cleavage specificity and distribution of sites in Poxvirus genomes

Abstract

Topoisomerase enzymes regulate superhelical tension in DNA resulting from transcription, replication, repair, and other molecular transactions. Poxviruses encode an unusual type IB topoisomerase that acts only at conserved DNA sequences containing the core pentanucleotide 5'-(T/C)CCTT-3'. In X-ray structures of the variola virus topoisomerase bound to DNA, protein-DNA contacts were found to extend beyond the core pentanucleotide, indicating that the full recognition site has not yet been fully defined in functional studies. Here we report quantitation of DNA cleavage rates for an optimized 13 bp site and for all possible single base substitutions (40 total sites), with the goals of understanding the molecular mechanism of recognition and mapping topoisomerase sites in poxvirus genome sequences. The data allow a precise definition of enzyme-DNA interactions and the energetic contributions of each. We then used the resulting "action matrix" to show that favorable topoisomerase sites are distributed all along the length of poxvirus DNA sequences, consistent with a requirement for local release of superhelical tension in constrained topological domains. In orthopox genomes, an additional central cluster of sites was also evident. A negative correlation of predicted topoisomerase sites was seen relative to early terminators, but no correlation was seen with early or late promoters. These data define the full variola virus topoisomerase recognition site and provide a new window on topoisomerase function in vivo.

Insights from the Structure of a Smallpox Virus Topoisomerase-DNA Transition State Mimic

“…The sequence of the -3 to -1 region of the vTopIB target site has been shown to have minimal effects on non-covalent binding of the enzyme, but strongly influences the rate of DNA cleavage (Hwang et al, 1999; Hwang et al, 1998; Minkah et al, 2007; Shuman and Prescott, 1990). These sequence preferences can be readily explained by the observed α10a contacts.…”

“…The variola virus topoisomerase (which differs by only three amino acids from the vaccinia virus protein) has also been well-studied, including a detailed analysis of sequence preferences in the regions flanking the core pentanucleotide motif (Minkah et al, 2007). An analysis of TopIB cleavage sites has revealed a distribution throughout the poxvirus genomes, with a central clustering of sites observed only for the orthopox viruses (Minkah et al, 2007). The poxvirus topoisomerases are part a broader family of small type IB enzymes which includes the topoisomerases from mimi virus and a number of eubacteria (Benarroch et al, 2006; Krogh and Shuman, 2002a).…”

Topoisomerases and site-specific recombinases: similarities in structure and mechanism

“…3a-b) opposite the catalytic domain. Interestingly, even with homologous DNA-binding domains, TopIB enzymes of some viral origins have DNA sequence preferences (Shuman and Prescott, 1990; Perry et al, 2006; Minkah et al, 2007), while others, e.g. human topoisomerase I, are non-sequence specific (Redinbo et al, 1998; Champoux, 2001).…”

Diverse Energetic Effects of Charge Reversal Mutations of Poxvirus Topoisomerase IB

“…The DNA sequences used in the current study contain a T at the −1 position relative to the cleavage site. Although previous work has shown that an A at this position accelerates cleavage by about 10-fold relative to T, this substitution does not change the cleavage equilibrium and it facilitates singleturnover kinetic measurements by hand mixing (9, 13). The nature of the base at the -1 position has no bearing on the interpretations of this study.…”