AI Article Synopsis
- Hin recombinase requires negatively supercoiled DNA for effective inversion, as positively supercoiled DNA shows significantly lower activity.
- Both Hin and Fis bind to DNA similarly, regardless of whether the DNA is positively or negatively supercoiled.
- Although Hin can efficiently cleave recombination sites in positively supercoiled DNA, it struggles with the subsequent strand exchange, indicating that the unwinding of the DNA double helix is necessary for this process.
Article Abstract
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.
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| http://dx.doi.org/10.1074/jbc.272.29.18434 | DOI Listing |
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