About ligation

Zophonias O. Jonsson zjons at vetbio.unizh.ch
Fri Apr 26 16:48:56 EST 1996


In article <4lr6lu$krn at lal.interserv.net>, Robert Cowherd
<rcowherd at mail.med.upenn.edu> wrote:

<snip snip snip>
> >
> ><snip snip snip>
> >
<snip snip snip>

> >>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!
> 
<snip snip snip>

> 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.

<snip snip snip>

> 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?)

I read your vivid imaginary fourth & fifth thoughts with considerable
amusement but alas, logic thinking is not always enough.  Actually people
were considering these things some 20 years ago.  There is an excellent
article in Science 186:790-797 (1974) by I.R. Lehman where the pathways of
DNA ligation are described.

Contrary to your hypothesis DNA ligation is a tree step reaction with some
very  stable intermediates.

1) A Lys residue on the Ligase forms a stable phosphoramide bond to AMP. 
In the case of T4 and eukaryotic ligases the AMP is derived from ATP, but
bacterial ligases use NAD+ for adenylation.  These intermediades are
usually very stable and many Lig preps contain a considerable amount of
the Adenylated form (which killed my dream of crystallyzing a ligase).  I
Don't know the haf time of the T4-AMP bond but I suppose it is stable for
months at -20*C.

2) The second step involves the transfer of the AMP to the 5' side of the
Nick to be closed, forming a pyrophosphate bond between the 5' PO3 and the
AMP.  The third step follows rapidly, but these guys back then still
managed to isolate the intermediate by reacting huge amounts of adenylated
ligase with the DNA at low pH and low temp.

3) The ligase finally catalyses a nucleophilic attack by the 3'-OH on the
pyrophosphate bond, forming ligated DNA and AMP.


That's it.  We have wandered quite far off topic and unfortunately I have
no Idea why high ATP inhibits the ligase.  You'll have to come up with
another theory.  How about interference with DNA binding?

Keep on thinking  ;-)

Zophonias

_____________________________________________________________________
Zophonias O. Jonsson
Institut fur Veterinarbiochemie               Tel: (41-1)-257-54-75
Universitat Zurich-Irchel                     Fax: (41-1)-362-05-01
Winterthurerstrasse 190
CH-8057 Zurich
Switzerland
_____________________________________________________________________



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