Individual nucleotide bases, not base pairs, are critical for triggering site-specific DNA cleavage by vaccinia topoisomerase Journal Article
| Authors: | Tian, L.; Sayer, J. M.; Jerina, D. M.; Shuman, S. |
| Article Title: | Individual nucleotide bases, not base pairs, are critical for triggering site-specific DNA cleavage by vaccinia topoisomerase |
| Abstract: | Vaccinia DNA topoisomerase forms a covalent DNA-(3′-phosphotyrosyl)- enzyme intermediate at a specific target site 5′-C+5C +4C+3T+2T+1p ↓ N-1 in duplex DNA. Here we study the effects of abasic lesions at individual positions of the scissile and nonscissile strands on the rate of single-turnover DNA transesterification and the cleavage-religation equilibrium. The rate of DNA incision was reduced by factors of 350, 250, 60, and 10 when abasic sites replaced the -1N, +1T, +2T, and +4C bases of the scissile strand, but abasic lesions at +5C and +3C had little or no effect. Abasic lesions in the nonscissile strand in lieu of +4G, +3G, +2A, and +1A reduced the rate of cleavage by factors of 130, 150, 10, and 5, whereas abasic lesions at +5G and -1N had no effect. The striking positional asymmetry of abasic interference on the scissile and nonscissile strands highlights the importance of individual bases, not base pairs, in promoting DNA cleavage. The rate of single-turnover DNA religation by the covalent topoisomerase-DNA complex was insensitive to abasic sites within the CCCTT sequence of the scissile strand, but an abasic lesion at the 5′-OH nucleoside (-1N) of the attacking DNA strand slowed the rate of religation by a factor of 600. Nonscissile strand abasic lesions at +1A and -1N slowed the rate of religation by factors of ∼140 and 20, respectively, and strongly skewed the cleavage-religation equilibrium toward the covalent complex. Thus, abasic lesions immediately flanking the cleavage site act as topoisomerase poisons. |
| Keywords: | nonhuman; enzyme activity; dna; molecular sequence data; genetic engineering; substrate specificity; base sequence; base pairing; binding sites; esterification; biochemistry; enzymes; dna cleavage; dna topoisomerase; dna topoisomerases, type i; viral proteins; vaccinia; nucleotides; impurities; topoisomerase; priority journal; article |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 279 |
| Issue: | 38 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2004-09-17 |
| Start Page: | 39718 |
| End Page: | 39726 |
| Language: | English |
| DOI: | 10.1074/jbc.M407376200 |
| PROVIDER: | scopus |
| PUBMED: | 15252055 |
| DOI/URL: | |
| Notes: | J. Biol. Chem. -- Cited By (since 1996):16 -- Export Date: 16 June 2014 -- CODEN: JBCHA -- Source: Scopus |
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