Topoisomerase I mediated TA cloning

See also TOPO TA cloning.

Topoisomerase site

Topoisomerase

   Topoisomerase recognition site
   ---------
      Topoisomerase nick site
            |
            V
5' C C C T T N N N N N N
3' G G G A A N N N N N N
          ^
          |
   Restriction enzyme must nick here

Offset nicking enzyme

• Nt.BstNB I: offset nicking enzyme from NEB.

   Nt.BstNB I recognition site 
   ---------
             Nt.BstNB I nick site
                    |
                    V
5' G A G T C N N N N N 3'
3' C T C A G N N N N N 5'

Site for topoisomerase-mediated cloning

Upstream of the BioBricks prefix

   Topoisomerase recognition site
   ---------
            |
            V
5' C C C T T N N N G A C T C 3'
3' G G G A A N N N C T G A G 5'
          ^
          |
                   ---------
                   Nt.BstNB I recognition site

Downstream of BioBricks suffix

   Nt.BstNB I recognition site
   ---------
                    |
                    V
5' G A G T C N N N A A G G G 3'
3' C T C A G N N N T T C C C 5'
                  ^
                  |
                   ---------
                   Topoisomerase recognition site

Possible enzymes to generate 3' T overhang

None of these enzymes will work because it appears as if the topoisomerase enzyme needs some duplex DNA in order to function.

Each can accommodate the topoisomerase site CCCTT

• BmrI
  • offset cutter that leaves 3' single base overhang
  • two sites in sopC (incD) repeat region of BBa_I50000
  • no sites in BBa_I50020
  • no sites in BBa_P1010, BBa_P1000, BBa_B0055, BBa_B0054
  • pretty high activity in all 4 NEB buffers
• BciVI
  • offset cutter that leaves 3' single base overhang
  • no sites in BBa_I50000
  • one site in BBa_I50020
  • no sites in BBa_P1010, BBa_P1000, BBa_B0055, BBa_B0054
  • Austin says this is a very bad enzyme.
• XcmI
  • long recognition sequence with internal N₉ that leaves 3' single base overhang
  • no sites in BBa_I50000
  • no sites in BBa_I50020
  • no sites in BBa_P1010, BBa_P1000, BBa_B0055, BBa_B0054
  • 100% activity only in NEBBuffer 2

Topoisomerase sites internal to vector components are not important because topoisomerase only operates near double stranded breaks.

Based on the preexistence of sites in the designed parts, XcmI looks like a good bet but it is not as robust to buffer conditions as BmrI.

Topoisomerase I

Most of the papers reference Topoisomerase I from vaccinia. It seems to be available commercially at Epicentre. html protocol

• Anselm Levskaya - I've gotten Vaccinia WR strain isolate and am presently cloning the topoisomerase IB gene out. Classically this is purified by using a phosphocellulose column but I'm going to just try a His-Tagged approach since it's a small protein.

Vector preparation

Reference

1. Heyman JA, Cornthwaite J, Foncerrada L, Gilmore JR, Gontang E, Hartman KJ, Hernandez CL, Hood R, Hull HM, Lee WY, Marcil R, Marsh EJ, Mudd KM, Patino MJ, Purcell TJ, Rowland JJ, Sindici ML, and Hoeffler JP. Genome-scale cloning and expression of individual open reading frames using topoisomerase I-mediated ligation. Genome Res. 1999 Apr;9(4):383-92.

• Vectors pcDNA3.1/GS and pYES2/GS

               v
              HindIII site
              -----------
5' N N N N N  A A G C T T  N N N N N 3'
3' N N N N N  T T C G A A  N N N N N 5'
              -----------
              HindIII site
                       ^

• Cut with HindIII

5' C C C T T  A 
3' G G G A A  T T C G A

             A G C T T  A A G G G 3'
                     A  T T C C C 5'

• Ligate on oligos (TOPO-H and TOPO-4)
  • TOPO-H destroys the HindIII site
  • TOPO-4 provides recessed end
  • In the drawings below, " | " refers to a break in the backbone on that strand.

                  TOPO-H oligo
                  -----------------------------------------------
5' N N N N N  A   A G C T   C G C C C T T A T T C C G A T A G T G
3' N N N N N  T   T C G A   G C G G G A
                  -------   -----------
         HindIII overhang   TOPO-4 oligo


                        TOPO-4 oligo   HindIII overhang
                        ------------   -------
                        A G G G C  G   A G C T T   N N N N N 3'
G T G A T A C C T T A T T C C C G  C   T C G A A   N N N N N 5'
------------------------------------------------
TOPO-H oligo

• Purify and cut again with HindIII to remove circular vector.
• Add TOPO-5 oligo and topoisomerase.
  • Vaccinia topoisomerase I cleaves after and remains covalently attached to second T in CCCTT sequence.

                                           Topoisomerase nick
                                           v
                  TOPO-H oligo
                  -------------------------------------------------
5' N N N N N  A   A G C T   C G C C C T   T A T T C C G A T A G T G        3'
3' N N N N N  T   T C G A   G C G G G A | A T A A G G C T A T C A C A A C  5'
                  -------   -----------   -------------------------------
         HindIII overhang   TOPO-4 oligo  TOPO-5 oligo


TOPO-5 oligo                      TOPO-4 oligo   HindIII overhang
-------------------------------   ------------   -------
C A A C A C T A T C G G A A T A | A G G G C  G   A G C T   T  N N N N N  3'
        G T G A T A C C T T A T   T C C C G  C   T C G A   A  N N N N N  5'
        ------------------------------------------------
        TOPO-H oligo
                             ^
                             Topoisomerase nick

• Add TOPO-10X stop buffer
• Purify away free oligonucleotides and unbound topoisomerase I

Questions

1. The authors add a primer TOPO-5 into the mix when they add the isomerase. It seems like this primer should not be necessary. Does the topoisomerase I "need" some duplex DNA extending out from where it will nick in order to cleave the backbone? Austin thinks most enzymes do need duplex DNA.
2. How much of a performance hit will we take if we don't do all the purification steps they do ... it kind of seems like a lot of work (even though you only have to prep the vector once for multiple reactions).
3. Can a restriction enzyme and topoisomerase I cleave simultaneously or will they occlude one another?
4. Does TOPO TA cloning have a better success rate than TA cloning or is it just simpler?

Notes

This approach to topoisomerase I mediated cloning has NOT been tested. This project is still a work in progress.

Additional references

Topoisomerase I

2. Shuman S and Moss B. Identification of a vaccinia virus gene encoding a type I DNA topoisomerase. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7478-82. DOI:10.1073/pnas.84.21.7478 |
3. Shuman S and Prescott J. Specific DNA cleavage and binding by vaccinia virus DNA topoisomerase I. J Biol Chem. 1990 Oct 15;265(29):17826-36.
4. Shuman S. DNA strand transfer reactions catalyzed by vaccinia topoisomerase I. J Biol Chem. 1992 Apr 25;267(12):8620-7.
5. Shuman S. Recombination mediated by vaccinia virus DNA topoisomerase I in Escherichia coli is sequence specific. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10104-8. DOI:10.1073/pnas.88.22.10104 |
6. Shuman S. Two classes of DNA end-joining reactions catalyzed by vaccinia topoisomerase I. J Biol Chem. 1992 Aug 25;267(24):16755-8.
7. Shuman S. DNA strand transfer reactions catalyzed by vaccinia topoisomerase I. J Biol Chem. 1992 Apr 25;267(12):8620-7.
8. Shuman S. Novel approach to molecular cloning and polynucleotide synthesis using vaccinia DNA topoisomerase. J Biol Chem. 1994 Dec 23;269(51):32678-84.