Chemistry Reference
In-Depth Information
Figure 13.4
The three forms of plasmid DNA
for such studies.
3r
This particular form of DNA, which is commonly found in bacte-
ria, is a cyclic supercoiled double strand made by several thousands of base pairs
(Figure 13.4). One single-strand scission unravels the supercoiled DNA (form I) to
a relaxed circular one (nicked, form II), while a second scission on the complemen-
tary strand, within about twelve base pairs from the fi rst one, generates a linear
DNA form (form III). These three DNA forms can be easily separated and quanti-
fi ed by gel electrophoresis, thus allowing a simple and rapid kinetic analysis. Moreo-
ver, supercoiled DNA is somehow more reactive than a short linear DNA fragment
and this makes the study of the reaction easier, even in the presence of relatively
poor catalysts. Such an increased reactivity is due: (a) to the obvious reason that the
cleavage of a single phosphodiester bond in a large DNA molecule is statistically
more likely than in a smaller DNA fragment and (b) to the internal strain resulting
in ground-state destabilization.
3r
Finally, two other peculiar and relevant features of plasmid DNA are: (i) the
possibility to discriminate between a single-strand or a double-strand cleavage
simply by a statistical analysis of the relative abundance of forms II and III produced
in the reaction; (ii) the possibility to enzymatically religate form III or form II giving
both a closed circular form and concatemer DNA which can be easily detected by
gel electrophoresis.
3r
Religation experiments are important in the mechanistic inves-
tigation of DNA cleavage because only fragments that derive from a hydrolytic
process can be religated by the enzyme. Therefore, the success or the failure of this
experiment may give important support to or seriously question the occurrence of
a clean hydrolytic process.
A careful mechanistic investigation becomes extremely important when metal
ions with known redox chemistry are used for the development of artifi cial nucle-
ases. For example, it is well known that Cu(II) and Fe(III) complexes, in the proper
conditions, cleave DNA by an oxidative pathway.
7
This process is so effi cient that it
always competes with the hydrolytic cleavage. Because the reactivity of oxidative
agents depends usually on cofactors such as H
2
O
2
, molecular oxygen and reducing
agents, and may produce diffusible radicals, the absence of externally added cofac-
tors, the insensitivity to radical scavengers and the preservation of reactivity under
anaerobic reactivity are generally considered suffi cient evidence of a hydrolytic
