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Step-wise DNA relaxation and decatenation by NaeI-43K
1Lineberger Comprehensive Cancer Center and Department of Pathology, University of North Carolina Medical School, Chapel Hill, NC 27599-7295, USA.
Nucleic Acids Research
|June 10, 1998
Summary
Nae I-43K functions as a type I topoisomerase, altering DNA linking numbers and decatenating DNA. Its activity switches from processive to distributive based on salt concentration.
Area of Science:
- Molecular Biology
- Enzymology
- Genetics
Background:
- Nae I protein, initially identified as a restriction endonuclease, exhibits DNA relaxation or cleavage.
- The activity of Nae I is dependent on amino acid residue at position 43 (lysine or leucine).
- Nae I-43K displays topoisomerase-like activity, forming transient covalent intermediates with DNA.
Purpose of the Study:
- To further characterize the topoisomerase activity of Nae I-43K.
- To determine the mechanism and processivity of Nae I-43K in DNA relaxation.
- To investigate the ability of Nae I-43K to decatenate DNA.
Main Methods:
- Assessing the change in linking number of supercoiled pBR322 DNA by Nae I-43K.
- Evaluating Nae I-43K activity under varying salt concentrations (low and high).
- Analyzing the decatenation of kinetoplast DNA by Nae I-43K.
Main Results:
- Nae I-43K changes the linking number of negatively supercoiled DNA by one unit, confirming type I topoisomerase activity.
- Positively supercoiled DNA was resistant to Nae I-43K.
- Nae I-43K exhibited processive activity at low salt and switched to distributive activity at high salt, with decatenated nicked circles as products.
Conclusions:
- Nae I-43K is a prokaryotic type I topoisomerase.
- The enzyme's mode of action (processive vs. distributive) is salt-dependent.
- Nae I-43K can cleave DNA opposite a nick, facilitating decatenation.