Effect of 2′-5′ phosphodiesters on DNA transesterification by vaccinia topoisomerase Journal Article
| Authors: | Krogh, B. O.; Claeboe, C. D.; Hecht, S. M.; Shuman, S. |
| Article Title: | Effect of 2′-5′ phosphodiesters on DNA transesterification by vaccinia topoisomerase |
| Abstract: | Vaccinia topoisomerase forms a covalent DNA-(3′ -phosphotyrosyl)-enzyme intermediate at a pentapyri-midine target site 5′-CCCTTp ↓ 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 (CCCTT2′p ↓ 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 ↓ N2′ p5′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 ↓ ATpTpC) 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. |
| Keywords: | nonhuman; molecular genetics; metabolism; enzymology; enzyme activity; chemistry; dna; molecular sequence data; kinetics; nucleotide sequence; substrate specificity; vaccinia virus; base sequence; dna metabolism; enzyme specificity; biochemistry; hydrolysis; reaction analysis; enzymes; transesterification; esters; dna topoisomerase; dna topoisomerases, type i; oligodeoxyribonucleotides; vaccinia; virus dna; exodeoxyribonuclease iii; priority journal; article; oligodeoxyribonucleotide; nucleoside diphosphate sugar; nucleoside diphosphate sugars |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 276 |
| Issue: | 24 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2001-06-15 |
| Start Page: | 20907 |
| End Page: | 20912 |
| Language: | English |
| DOI: | 10.1074/jbc.M102312200 |
| PUBMED: | 11399773 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Export Date: 21 May 2015 -- Source: Scopus |
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