Site-specific DNA transesterification by vaccinia topoisomerase: role of specific phosphates and nucleosides.
Vaccinia topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a pentapyrimidine target site 5'-CCCTTp downward arrow in duplex DNA. Here we present experiments that illuminate the contributions of specific nucleosides and phosphates to site affinity and transesterification. We find that the -1 phosphate and -2 nucleoside on the scissile strand (5'-CCCTTp / NpN) enhance the rate of transesterification 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 of topoisomerase binding to DNA. A nick at the +2 phosphate also slows the rate of transesterification by approximately 500-fold. This finding, together with earlier studies of the effects of position-specific base and sugar modifications, points to the +2 Tp nucleotide as being the most critical element of the CCCTT target site other 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 is otherwise fully base-paired within the 5'-CCCTT target site. By studying the effects of single nucleotide gaps and missing phosphate nicks within the 3'-GGGAA sequence, we find that the +1 and +2 adenosine nucleosides enhance the rate of transesterification by 20- and 1,000-fold respectively and that the +5 phosphate (3'-GpGGAA) is also important for cleavage. Cumulative functional analyses of the vaccinia topoisomerase-DNA interface are discussed in light of newly available structures for the vaccinia and human type IB enzymes.
Cheng
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Molecular Biology Program, Sloan-Kettering Institute, New York 10021, USA.
Shuman
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D001483:Base Sequence; D002384:Catalysis; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D018736:DNA Adducts / Q000737:chemistry / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000378:metabolism*; D004951:Esterification; D005090:Exodeoxyribonucleases / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000378:metabolism; D009705:Nucleosides / Q000737:chemistry / Q000378:metabolism*; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D014616:Vaccinia virus / Q000201:enzymology*
D018736:DNA Adducts; D009692:Nucleic Acid Heteroduplexes; D009705:Nucleosides; D010710:Phosphates; D004247:DNA; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D004264:DNA Topoisomerases, Type I