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I have to use the SacII restriction enzyme, which requires multiple recognition sites to efficiently cut DNA, for a DNA assembly. From my understanding, multiple copies of DNA holding only a single restriction site can still lead to digestion through bridging interactions between molecules and enzyme. However, this appears to require digestion optimization I would rather avoid. Can I place two SacII sites in tandem on my insert DNA to enhance my digestion? How far should these be spaced to allow efficient enzyme interaction between the sites?

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    $\begingroup$ You've got a very nice answer from @acvill, but for future reference the NEB website is a good first place to look when you have questions about the specifics of RE usage (and for many other enzymes as well). $\endgroup$
    – tyersome
    Nov 12 '21 at 22:20
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    $\begingroup$ Hi @tyersome, that's where I started but they are not clear on design. $\endgroup$
    – JEJS
    Nov 13 '21 at 9:27
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In its active form, SacII (also called Cfr42I) is a homotetramer that must bind two copies of its recognition sequence in order to efficiently hydrolyze its substrate.1 Upon binding two sites on a single molecule, the interstitial sequence is "looped out" and cleaved. The efficiency of cleavage is a function of the length of the intervening sequence due to the sterics of DNA bending (see resources concerning the persistence length of DNA 2 to better understand the energetics at play). One study on the effects of DNA tension on the cutting frequency of two-site restriction endonucleases (including SacII) suggests that the optimal loop length is hundreds of bp.3

In each case the DNA template contained multiple recognition sites for the enzymes being tested with separations ranging from ≈200 to 1,000 bp, predicted by theory to be favorable for looping.

A different study notes that EcoRII, another two-site enzyme, can cleave its target with as little as 10 bp between the 5′-cytosines of its two recognition sites.4

As an alternative to designing a construct with multiple SacII sites, you could supplement your digestion with a dsDNA oligonucleotide containing one SacII recognition site.3

...stimulation of activity upon addition of short oligonucleotide duplexes containing the recognition sequence has been reported for Eco57I, HpaII, Cfr9I, and SacII. Such stimulation provides evidence that an enzyme complex can bind in trans (i.e., across two sites on different molecules).

If you go this route, I would recommend first incubating SacII with your construct for some time, then add the oligos and continue incubation. This way, SacII will associate with the cut site on your target, and you will reduce the frequency of events where SacII binds and cuts two oligos at once, which would reduce the available oligo concentration and possibly lower your target cleavage efficiency.


References

  1. Gasiunas G, Sasnauskas G, Tamulaitis G, Urbanke C, Razaniene D, Siksnys V. Tetrameric restriction enzymes: expansion to the GIY-YIG nuclease family. Nucleic Acids Res. 2008 Feb;36(3):938-49.
  2. Manning GS. The persistence length of DNA is reached from the persistence length of its null isomer through an internal electrostatic stretching force. Biophys J. 2006 Nov 15;91(10):3607-16.
  3. Gemmen GJ, Millin R, Smith DE. Tension-dependent DNA cleavage by restriction endonucleases: two-site enzymes are "switched off" at low force. Proc Natl Acad Sci U S A. 2006 Aug 1;103(31):11555-60.
  4. Reuter M, Kupper D, Meisel A, Schroeder C, Krüger DH. Cooperative binding properties of restriction endonuclease EcoRII with DNA recognition sites. J Biol Chem. 1998 Apr 3;273(14):8294-300.
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  • $\begingroup$ Thank you @acvill! I think I better go with the dsDNA oligos in this case, it's a shame Benchling doesn't highlight these enzymes. $\endgroup$
    – JEJS
    Nov 13 '21 at 9:26
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    $\begingroup$ @JEJS I've updated my answer with a methodological tip. I hope your experiment goes well. $\endgroup$
    – acvill
    Nov 13 '21 at 15:48
  • $\begingroup$ Oh great thanks @acvill! $\endgroup$
    – JEJS
    Nov 14 '21 at 12:12
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    $\begingroup$ Update for future reference: I actually had no problem with standard digestion conditions being 1ug DNA (~170 pmols) to 1ul SacII. However, I did leave digestion for 16+ hours which I imagine to be critical. $\endgroup$
    – JEJS
    Nov 16 '21 at 9:34

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