Studies of a positive supercoiling machine. Nucleotide hydrolysis and a multifunctional "latch" in the mechanism of reverse gyrase.
Reverse gyrase, the only topoisomerase known to positively supercoil DNA, has an N-terminal ATPase domain that drives the activity of a topoisomerase domain. This study shows that the N-terminal domain represses topoisomerase activity in the absence of nucleotide, and nucleotide binding is sufficient to relieve the repression. A "latch" region in the N-terminal part was observed to close over the topoisomerase domain in the reverse gyrase crystal structure. Mutants lacking all or part of the latch relax DNA in the absence of nucleotide, indicating that this region mediates topoisomerase repression. The mutants also show altered DNA-dependent ATPase activity, suggesting that the latch may be involved in coupling nucleotide hydrolysis to supercoiling. It is not required for this process, however, because the mutants can still positively supercoil DNA. Nucleotide hydrolysis is essential to the specificity of reverse gyrase for increasing the linking number of DNA. Although with ATP the enzyme performs strand passage always toward increasing linking number, it can increase or decrease the linking number in the presence of a nonhydrolyzable ATP analog. This suggests that the mechanism of reverse gyrase is best described by a combination of recently proposed models.
Rodriguez
A Chapin
AC
Medical Research Council Laboratory of Molecular Biology, Hills Rd., Cambridge CB2 2QH, United Kingdom. acr@mrc-lmb.cam.ac.uk
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D019611:Archaeoglobus fulgidus / Q000201:enzymology; D001483:Base Sequence; D017931:DNA Primers; D026942:DNA Topoisomerases / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D026942:DNA Topoisomerases; C041813:DNA reverse gyrase; D004264:DNA Topoisomerases, Type I