replication of plasmids

Robert Whittier rfwhittier at hotmail.com
Fri Jan 14 09:51:39 EST 2000


>Susanne Rohrer wrote:
>>
>>My question: can a plasmid containing 2 identical origins replicate,
>
>No, it can't.
>
>>i.e. could pUC be ligated with itself?
>
>Yes, it can, but the product will not replicate.
>
>-Rafa

Whoa . . .
not so fast there pardner!

Plasmids transformed into recA+ strains naturally form dimers
and trimers, and I have a vague recollection of some paper
in the late 70's that showed you could isolate the monomer
band or the dimer band. Transformed back into a recA- strain,
each would replicate faithfully in whichever form (monomer or
dimer) was used to transform. Transformed back into a recA+
strain, the same spectrum of monomer, dimer, trimer, etc. forms
would reappear.

Of course such multimers formed by RecA-mediated recombination
will be aligned in the same circular orientation. Rafa is right if
Susan used two restriction enzymes to do a forced orientation
cloning. In this case, two plasmid molecules ligating together
could only form in reverse orientation with respect to each other.
Thus the whole plasmid would be a giant palindrome. With gyrase
forming negative supercoils, the dimer would be prone to form
cruciform structures at both junctions, and these will look just
like Holiday structures. So the resolvases that normally complete
recombination events will cut at these structures, leading to
deletions and various bizaar structures. Bottom line: they'll be
lost.

Bob Whittier
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