Hin-mediated inversion on positively supercoiled DNA.
Hin recombinase requires negatively supercoiled DNA for an efficient inversion. We have generated positively supercoiled plasmid DNA using reverse gyrase from Sulfolobus shibatae and subjected it to the Hin-mediated inversion reaction. Both Hin and Fis showed the same DNA binding activity regardless of the superhelical handedness of the substrate plasmid. However, inversion activity on positively supercoiled DNA was less than 1% of negatively supercoiled DNA. Assays designed to probe steps in inversion, showed that on positively supercoiled DNA, Hin was able to cleave the recombination sites with the same efficiency shown on negatively supercoiled DNA but was not able to exchange the cleaved DNA. Based on the theoretical differences between positive and negative supercoiling, our data may suggest that unwinding of the double helix at recombination sites is needed after DNA cleavage for strand exchange to occur.
Lim
H M
HM
Department of Biology, College of Natural Sciences, Chungnam National University, 305-764 Taejon, Republic of Korea. hmlim@hanbat.chungnam.ac.kr
Lee
H J
HJ
Jaxel
C
C
Nadal
M
M
D001665:Binding Sites; D007446:Chromosome Inversion; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D015183:Restriction Mapping; D016944:Sulfolobus / Q000201:enzymology
D004278:DNA, Superhelical; D004254:DNA Nucleotidyltransferases; C059997:Hin recombinase; C052380:endodeoxyribonuclease NcoI; D015252:Deoxyribonucleases, Type II Site-Specific; C041813:DNA reverse gyrase; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II