| Abstract
| - Vaccinia topoisomerase forms a covalent DNA-(3‘-phosphotyrosyl)-enzyme intermediate at apentapyrimidine target site 5‘-CCCTTp↓ in duplex DNA. Here we present experiments that illuminate thecontributions of specific nucleosides and phosphates to site affinity and transesterification. We find thatthe −1 phosphate and −2 nucleoside on the scissile strand (5‘-CCCTTp↓NpN) enhance the rate oftransesterification by factors of 40 and 25, respectively, whereas the DNA segment downstream of the−2 nucleotide makes no significant kinetic contribution. Placement of a 5‘-phosphate/3‘-OH nick at position+2, +3, +4, or +5 within the CCCTT element results in a 5−10-fold reduction in the affinity oftopoisomerase binding to DNA. A nick at the +2 phosphate also slows the rate of transesterification by∼500-fold. This finding, together with earlier studies of the effects of position-specific base and sugarmodifications, points to the +2 Tp nucleotide as being the most critical element of the CCCTT target siteother than the scissile phosphate itself. On the noncleaved strand, the segment downstream of the 3‘-GGGAA element contributes minimally to the rate of transesterification provided that the substrate isotherwise fully base-paired within the 5‘-CCCTT target site. By studying the effects of single nucleotidegaps and missing phosphate nicks within the 3‘-GGGAA sequence, we find that the +1 and +2 adenosinenucleosides enhance the rate of transesterification by 20- and 1000-fold respectively and that the +5phosphate (3‘-GpGGAA) is also important for cleavage. Cumulative functional analyses of the vacciniatopoisomerase−DNA interface are discussed in light of newly available structures for the vaccinia andhuman type IB enzymes.
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