A Way Forward
We have seen in the Puzzles how the double helical paradigm for duplex DNA
has been unable to offer helpful insights into a very wide range of
phenomena, comprehensively explored elsewhere (42,64), and how the original
researchers did not offer an explanation of their results. We have seen in
the earlier Parts, culminating in Part 5, that the refinement algorithms and
their mode of use, explicitly or implicitly, whether applied to heavy atom,
MAD, synchrotron or other diffraction, seem set so as to predetermine the
outcome of those studies to a model of plectonaemic
winding first introduced into biological macromolecular studies by Pauling &
Corey in 1951. With the benefit of some 48 years of subsequent research, it
is evident that this model no longer accounts for the practical,
experimental results of biochemists, biophysicists and molecular biologists.
A new model has been described (42,64) and has been found under STM (Puzzle
1, ref.1 & in other studies) and is sketched in the attachment. Hard copies
are available on request.
In order to resolve the difficulty presented to the biological sciences
generally, as encapsulated by the implications of the Cambridge meeting
(79,80,81), there would seem to be at least three lines of investigation to
be followed:
1 We should accept the advice of Crick et al. (83) that the most severe
test of any proposed true side-by-side model would be topological and I
enclose here, and invite researchers to consider, an outline experiment to
do this;
2 We should assemble crystals, under very mild conditions, which have
been formed directly from DNA previously assembled in vivo, and I suggest
here an
outline approach to this, such crystals to be used for x-ray diffraction
studies rather than those crystals grown in vitro; and
3 We start to rework the oligonucleotide structure factor files deposited
at PDB and elsewhere in line with the true side-by-side model, after, or
concurrent with progess with the topological experiment.
1 A Topological Test of the True Side-by-side Model in Covalently
Closed Small Circular DNAs
Drew & Travers (84) have constructed a 169 bp circular, covalently closed
length of duplex DNA. With this length of DNA there can only be a very
limited number of topoisomers present if the ring closure, of itself, could
introduce only a very few superhelical turns. That is, all the 169 bp
molecules would share a range of superhelical turns between about -1 and +1.
Therefore in suitable denaturing conditions, in the absence of any strand
breakage, the 169 bp sequence would be largely separable into complementary
covalently closed single-stranded DNA circles interlinked at very few places
according to the new model. Thus the most direct route to challenging the
DNA double helix is based on topology. The complementary strands in that
169 bp sequence may actually be linked by 0, or perhaps ± 1 superhelical
turns due to the algebra of the enzymic closure reaction used on the 169 bp
linear duplex, and the denatured covalently closed circular strands could
then be examined under AFM to determine their linking number.
Therefore, if the 169 bp circular DNA is denatured under mild conditions,
perhaps using aqueous urea, it could then be decorated with a single-strand
DNA binding protein, such as the product of gene 32 from phage T4 (85), so
as to be rendered easily visible under AFM, and the actual numbers of
crossovers then counted.
According to the double helix model, LK will lie in the range 16 to 18, or
so, while the new model predicts LK is -1, 0 or +1, or so.
A very similar experiment could be carried out with the small circular DNAs
described by Bates & Maxwell (86), where, for the smallest, the double helix
model predicts LK = 9, and the new model predicts LK = -2, or so.
I invite those with suitable facilities to consider conducting such a study.
2 Crystallised short oligonucleotides derived from natural,
duplex DNA formed in vivo
Rhodes & Klug (87) have described the cleavage of high polymer duplex DNA
lying on a flat surface using DNAase 1, the resulting oligonucleotides
having reproducible lengths. Using, say, satellite DNA, perhaps poly d(AT)
from certain crabs, or else poly (dA.dT), the resulting short sequences of
10.5 and 10.0 base pairs, respectively, might lend themselves to easy
crystallisation and subsequent x-ray diffraction. It might be necessary to
chemically fix the two strands together prior to cleavage to minimise the
risk that the short strands, if originally side-by-side, would form a
plectonaemic oligonucleotide after cleavage.
Again, I invite those with suitable resources to consider conducting such a
study.
3 Crystallographers should be sought to rework published
structure factor files
