Effect of 2'-5' phosphodiesters on DNA transesterification by vaccinia topoisomerase.
Vaccinia topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a pentapyrimidine target site 5'-CCCTTp downward arrow in duplex DNA. By introducing single 2'-5' phosphodiesters in lieu of a standard 3'-5' phosphodiester linkage, we illuminate the contributions of phosphodiester connectivity to DNA transesterification. We find that the DNA cleavage reaction was slowed by more than six orders of magnitude when a 2'-5' linkage was present at the scissile phosphodiester (CCCTT(2')p downward arrow(5')A). Thus, vaccinia topoisomerase is unable to form a DNA-(2'-phosphotyrosyl)-enzyme intermediate. We hypothesize that the altered geometry of the 2'-5' phosphodiester limits the ability of the tyrosine nucleophile to attain a requisite, presumably apical orientation with respect to the 5'-OH leaving group. A 2'-5' phosphodiester located to the 3' side of the cleavage site (CCCTTp downward arrowN(2')p(5')N) reduced the rate of transesterification by a factor of 500. In contrast, 2'-5' phosphodiesters at four other sites in the scissile strand (TpCGCCCTpT downward arrowATpTpC) and five positions in the nonscissile strand (3'-GGGpApApTpApA) had no effect on transesterification rate. The DNAs containing 2'-5' phosphodiesters were protected from digestion by exonuclease III. We found that exonuclease III was consistently arrested at positions 1 and 2 nucleotides prior to the encounter of its active site with the modified 2'-5' phosphodiester and that the 2'-5' linkage itself was poorly hydrolyzed by exonuclease III.
Krogh
B O
BO
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Claeboe
C D
CD
Hecht
S M
SM
Shuman
S
S
D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009702:Nucleoside Diphosphate Sugars; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D014616:Vaccinia virus / Q000201:enzymology*
D009702:Nucleoside Diphosphate Sugars; D009838:Oligodeoxyribonucleotides; D004247:DNA; D004264:DNA Topoisomerases, Type I