Nonpolar nucleobase analogs illuminate requirements for site-specific DNA cleavage by vaccinia topoisomerase Journal Article
| Authors: | Yakovleva, L.; Lai, J.; Kool, E. T.; Shuman, S. |
| Article Title: | Nonpolar nucleobase analogs illuminate requirements for 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 nonpolar pyrimidine isosteres difluorotoluene (F) and monofluorotoluene (D) and the nonpolar purine analog indole at individual positions of the scissile and nonscissile strands on the rate of single-turnover DNA transesterification and the cleavage-religation equilibrium. Comparison of the effects of nonpolar base substitution to the effects of abasic lesions reported previously allowed us to surmise the relative contributions of base-stacking and polar edge interactions to the DNA transesterification reactions. For example, the deleterious effects of eliminating the +2T base on the scissile strand were rectified by introducing the nonpolar F isostere, whereas the requirement for the +1T base was not elided by F substitution. We impute a role for +1T in recruiting the catalytic residue Lys-167 to the active site. Topoisomerase is especially sensitive to suppression of DNA cleavage upon elimination of the +4G and +3G bases of the nonscissile strand. Indole provided little or no gain of function relative to abasic lesions. Inosine substitutions for +4G and +3G had no effect on transesterification rate, implying that the guanine exocyclic amine is not a critical determinant of DNA cleavage. Prior studies of 2-aminopurine and 7-deazaguanine effects had shown that the O6 and N7 of guanine were also not critical. These findings suggest that either the topoisomerase makes functionally redundant contacts with polar atoms (likely via Tyr-136, a residue important for precleavage active site assembly) or that it relies on contacts to N1 or N3 of the purine ring. The cleavage-religation equilibrium is strongly skewed toward trapping of the covalent intermediate by elimination of the +1A base of the nonscissile strand; the reaction equilibrium is restored by +1 indole, signifying that base stacking flanking the nick is critical for the religation step. Our findings highlight base isosteres as valuable tools for the analysis of proteins that act on DNA in a site-specific manner. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. |
| Keywords: | unclassified drug; nonhuman; protein function; protein analysis; protein metabolism; protein binding; substitution reaction; tyrosine; dna; molecular sequence data; guanine; recombinant proteins; vaccinia virus; base sequence; dna flanking region; dna sequence; models, molecular; binding energy; esterification; catalysis; molecular biology; indoles; protein determination; enzymes; transesterification; dna cleavage; dna topoisomerase; dna topoisomerases, type i; oligonucleotides; dna strand; vaccinia topoisomerase; reaction kinetics; indole; nucleic acid base; nucleobase analogs; substitution reactions; 2 aminopurine; inosine; monofluorotoluene; pyrimidine isosteres difluorotoluene; toluene derivative; vaccinium; 2-aminopurine; toluene |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 281 |
| Issue: | 47 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2006-11-24 |
| Start Page: | 35914 |
| End Page: | 35921 |
| Language: | English |
| DOI: | 10.1074/jbc.M608349200 |
| PUBMED: | 17005552 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 4" - "Export Date: 4 June 2012" - "CODEN: JBCHA" - "Source: Scopus" |
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