PROCESO DE ATENCION DE ENFERMERIA

As has already been described, the major and minor grooves differ greatly in their size and hydrogen bonding potential, and hence amount o f hydration. The most important of these factors as to whether a species is a major or minpr groove binder, is the size o f the species in question, and its ability to enter either or both of the grooves well enough to have stabilising interactions. It is found that large molecules such as proteins and oligonucleotides (in forming triplex species) tend to prefer major groove binding, whereas smaller organic compounds show a tendency towards minor groove binding.

1.3.3.1 Minor groove binding

Minor groove binders are generally made up of small aromatic rings such as furans, benzenes and pyrroles joined by bonds which allow rotation between the rings. This freedom o f rotation allows a curved conformation to bç adopted by the compound so that it can fit into the helical groove. Van der Waals interactions can take place between the compound and the groove wall and if the binding is tight enough hydrogen bonds can occur to the groove floor at the points njentioned earlier.

The minor groove does not have a uniform width and is narrpwer at A:T pairs than at G:C pairs. Hence if molecules are tighter in the groove at A:T rich sequences the van der Waals interactions with the sides of the groove walls will be greater.

Electrostatic interactions occur with the floor of the minor groove with hydrogen bonds possible between the sites previously mentioned. However the hydrogen bond between N-3 o f guanine and 0-2 o f cytosine lies in the minor groove and poses a steric block to the approach of molecules into the groove in G;C regions. Thus a second reason exists for species to bind at A:T regions since electrostatic interactions vary with 1/(distance)^ and the approach in G:C sequences is sterically blocked.

Chapter 1 30

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A classic example of cationic minor groove binding is shown by netropsin 3. It is a dicationic species which contains two pyrrole moieties which are able to rotate. A crystal structure of netropsin 3 bound to d(CGCGAATTÇGCG) has been obtained which enables details o f minor groove binding to be ascertained.'17

CH. CH. NH NH .NH NH NH. NH: NH: Netropsin 3

As expected it was found that the compound was bound at t|ie central AATT region and the water in the groove had been displaced from th^t section o f the duplex. The three amide protons point towards the groove and forrp hydrogen bonds to N-3 o f adenine and 0-2 of thymine whilst van der Waals interactions exist with the walls of the groove. The CH groups o f the pyrrole rings act as a brake, and prevent any deeper penetration into the groove (the hydrogen bon4 distances are in

the region o f 3.3-3.8 Â compared to a normal 3 A). As a result o f these factors the pyrrole rings are nearly parallel to the groove walls and twisted by 33° because o f the helical twist o f the DNA. The cationic ends are seen to be interacting with the N-3 of the outer adenines.

Unlike intercalation however there are no noticeable effects on the DNA (i.e. no lengthening measurable by physical methods). There is a slight bending o f the helical axis and groove widening but no change in helical twist is seçn.

Other X-ray diffraction crystal structures have been solve(J which back up what is seen for netropsin 3. Distamycin*^ or Hoescht 33258^" woul(} be equally good examples. Both show a marked curvature and bind at A:T rich sequences.

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1.3.3.2. Major groove binding

The principal determinant in major groove binding is size. The greater width o f the major compared to the minor groove means that larger species are required to bind within it. The most prevalent class is therefore the larger proteins. Because of this increase in size there is an associated increase in the number o f points of interaction between the protein and the DNA. As a result there tçnds to be much more sequence selectivity displayed by these systems. Examples include the large class of activator and repressor proteins and perhaps more obviously the restriction enzymes, such as EcoR l, which cut DNA at specific sequences. This selectivity is vital in vivo for an organism; on a cellular level it is fundamental that the processes that occur do so with an element of control with the responses occurring for the right DNA sequence.

Although the primary sequences of these proteins vary widely it is found that in fact they usually contain one of a limited set o f structural motifs within their three dimensional form which is involved in the protein-DNA interaction. This requirement is most probably made on an electrostatic and geometric basis constrained by the DNA’s shape and potential energy surface.

It is commonly found that proteins have strategically placed lysine and arginine residues, positioned so that they neutralise the negative charge o f the polyphosphate backbone by forming ionic bonds. An example o f this is seen in the crystal structure of Klenow fragment o f DNase I.^° It is seen that the enzyme has an overall anionic electrostatic field but the DNA binds in a “cleft” which has an overall cationic nature.

On a purely size basis a typical a-helix has a diameter of abqut 7 Â, and has a dipole running from its N to C-terminus. This makes it ideally suifed to fit into the major groove of B-DNA. It is worth noting that a [3-sheet has a wi(^th o f only a few angstroms and is geometrically suited to the minor groove.

The binding is envisaged to be a one dimensional random walk along the backbone (non-specific binding) until the energetics at a specific binding site are such that binding is a favourable process. For example, the Klenow fragment has two helices, one o f which sits in the major groove and in doing so it forces the enzyme to proceed along the groove until the preferred binding sequence is obtained.

C h a p t e r 1 32

As Stated previously, the major groove has more potential sites which are hydrogen bond donors or receptors. Since these may be on successive base pairs amino acids such as glutamine and arginine are able to hydrogen bqnd to sequential base pairs simultaneously. Hence, there is more potential for sequence selectivity than exhibited in the minor groove.

Some general points about major groove binding can be gained from looking at the bZIP (basic zipper) family o f proteins.*' These regulator}' proteins bind to DNA as a dimer to an eight base pair long palindromic recognitiop sequence. They consist o f two distinct segments - a leucine zipper and a basic recognition region.

A leucine zipper is a m o tif made up o f two smoothly cgrving a-helices associated in a coiled coil. In a bZIP protein this spans the ( -terminal 30 residues and is held together by repeating leucine residues every seven amino acids.

The basic region occurs from the A-terminal end and consists o f two

unassociated a-helices which bind to DNA in the so-called '‘scissors-grip” mechanism (fig 8).^^ Each helix recognises a specific sequence, for example, in the case o f the protein GCN4,^^ the sequence TGAC. Combining these two h a lf sites we see that the protein GCN4 recognises the overall sequence 5 ’-TGACGTCA - the CRE site (where CRE is the CREB response element protein).

Chapter 1 33

In this case the base pair contacts are via two arginine residues which contact two concurrent nucleic acids at the centre of each half site. There are also found to be present two alanines which have van der Waals interactions with the CH^ group of the central thymine, and a third arginine which contacts the central G of the recognition site (a fourth arginine from the other helix contacts the polyphosphate backbone).

It is seen that the DNA backbone is distorted and is bent at an angle 20° towards the leucine zipper with a concomitant underwinding o f the DNA at the centre of the binding site. This large DNA distortion means that the protein GCN4 is not only specific for the CRE site. A similar site - the API site - is also found to be a substrate for the protein. This is a similar sequence to the CRE sequence but differs in one base pair at the centre (5’-TGACCTCA).

Point mutations on the DNA and protein have been used fo investigate the binding.^'* It was seen that the thymine van der Waals interactions \yere essential and a change to uracil stopped all binding. Similarly all the amino acids at the binding site were seen to be essential although binding could be altered by increasing the steric bulk of the amino acids at relevant points.

Both these families of proteins show us that the factors that are essential for binding in the major groove are the same as for the minor groove. The main difference, however, is that there is a much larger scope for contacts in the major groove and, therefore, the opportunities for sequence selectivity arc increased. This is both due to the size o f the major groove itself and, more fundamentally, the increased potential for interaction o f species with it because of their larger size and the fact that they contain, in general, more functionalities than do minor groove binders.