About ligation

Robert Cowherd rcowherd at mail.med.upenn.edu
Fri Apr 26 14:03:26 EST 1996


>In article <4lou5r$6a at lal.interserv.net>, Robert Cowherd
><rcowherd at mail.med.upenn.edu> wrote:
>
><snip snip snip>
>>
More snipping -- most will hopefully remember or look up the original
posts if interested.

>>In blunt end ligations, you'd prefer the highest ATP, DNA ends and
>>Vmax of ligase that you could get.  15 degrees (the compromise) seems
>>to accomplish that....  

drm21 at mole.bio.cam.ac.uk (David Micklem) wrote:

>This all sounds fine to me, except that NEB state that blunt end 
>ligations are improved by DECREASING the rATP concentration from 1mM to
>50uM!

Very good point.  Obviously I mispoke. I said "In blunt end ligations, 
you'd prefer the highest ATP, DNA ends and Vmax of ligase that you could
get."  If true then why not use 1 or 5 or 10 M ATP instead of 1mM or 50
uM?  What I meant was not so much that one should seek the highest
concentration of ATP but rather the optimal (whatever value that is)
concentration ATP.  Several points:

1.)  It seems to me on fourth, fifth or more thought "highest ATP, DNA
ends and Vmax of ligase" has intrinsically in "Vmax" a qualification of
ATP (a cofactor) concentration as an amount sufficient for Vmax but not
an inhibitory concentration. For many enzymes I imagine that at some 
point an excess of cofactors would/could be inhibitory.  FOR EXAMPLE,
SUPPOSE the four component reaction is not entirely simultaneous (as I
also without absolute knowledge have said otherwise ;-)  -- but very 
nearly so). That is, the two DNA ends must be together simultaneously
with the ligase and the ATP's involvment must occur within some
millionths/ billionths of a second after that event to successfully 
ligate.  Might not ATP binding to the ligase millionths/billionths/
trillionths of a second BEFORE the collision of ends possibly then
inhibit the reaction?)  In such a hypothetical scenario, revising the
ATP concentration (as optimized for sticky end ligations) DOWNWARD
might well be in order.  Of course, I am no "expert" on ligase but 
this is just one possible  way to reconcile the various observations/
suppositions/advisements.  With some imagination, other plausible
scenarios could be envisaged (eg. one end in orientation "A" relative
to the active site - enzyme- ATP - second end) that could also 
explain it.  (BTW, my original post merely relates how it was told 
to me not that I should/want [reminder to my self not to get carried
away by 'possibilities' on this topic] to be an exhaustive apologist
for this perspective or ligase mode of function).

2.)  If NEB, as reported, is correct and that optimal concentration 
of ATP IS 50 uM, then might not my original point about preserving 
the stability of ATP become MORE critical since there is less ATP
available?

>
>Also, as I mentioned before, someone posted data (shock! horror!) that
									                      ^^^^^^^^^^^^^^^^^
                        Bad Scientist. BAAAAD Scientist!

>indicated that blunt end ligations were essentially complete after 1hr
>at room temperature: ie very little difference in number of colonies
>produced afterr 1hr vs overnight ligation.  What I can't remember is
>whether the overnight ligation was at room temperature or 15 degrees...
>
>David
>
>_____________________________________________________________
>D.R.Micklem,
>Wellcome/CRC Institute,       Time flies like an arrow...
>Tennis Court Road,              
>Cambridge CB2 1QR             Fruit flies like a banana.
>UK                             
>Tel: [+44] (0)1223 334129     Email:drm21 at mole.bio.cam.ac.uk
>Fax: [+44] (0)1223 334089               
>_____________________________________________________________

Bob
  <
Robert D. Cowherd
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