Atp

0.16 (12.0)

0.14 (8.9)

0.05 (4.3)

0.02 (2.3)

0.84 (2.6)

0.05 (0.7)

Reprinted with permission from ref. 3.

Reprinted with permission from ref. 3.

Fig. 1. Chemical structures of polyamines and their analogs.

DNA condensation, and aggregation were thought to be the direct result of interactions of polyamines with DNA phosphate groups involving interstrand and intrastrand binding. However, as it became clear that double-stranded DNA exists in different conformations, depending on the relative water content, nature of counter-ions, and the nucleotide sequences involved, the differential interaction of polyamines to different forms of DNA became evident (15). The most prevalent form of DNA is the right-handed B-DNA, but the right-handed A-DNA and the left-handed Z-DNA are also observed in X-ray crystallography and solution studies (16). The preferential binding of spermine and spermidine to A-DNA and conversion of B-DNA to the A-form in the presence of these polyamines was reported as early as 1979 (17). Conversion to A-form was detected by circular dichroism spectroscopy and the effects were observed in water-ethanol mixtures. Interestingly, putrescine, cadaverine, and hexamethylene diamine did not follow the pattern of spermidine and spermine, but stabilized B-DNA.

Molecular modeling studies by Zakrezewska and Pullman (18) indicated that spermine binding depended on DNA conformation and base sequence. A model for the interactions of polyamines with B-DNA simulated by theoretical calculations indicated binding to the minor groove of DNA (18,19). In molecular models of Suwalsky et al. (20) spermine was modeled to bridge two backbone strands across the B-DNA minor groove. However, molecular dynamics modeling by Feuerstein et al. (21) found the major groove of alternating purine/pyrimidine sequence to be the most favorable sites for spermine binding and the association of spermine binding with DNA bending. Binding of spermine along the phosphate backbone was the least favorable interaction. Comparison of d(GC)5-d(GC)5 heteropolymer with the homopolymers (d(G)10-d(C)10 showed continuous interaction with heteropolymer but not homopolymer.

X-ray crystallography of the d(GTGTACAC) octamer duplex showed a unique positioning of spermine in its A-DNA conformation (22). Spermine bound to the floor of the major groove by hydrogen bonding to GTG of one strand, assumed an S shape, and bound to the corresponding bases on the opposite strand. The methylene groups of sper-mine formed a hydrophobic cluster with the methyl group of thymine and the O6 atoms of the guanine of the TGT sequence. The A-DNA dodecamer, d(CCGGGCCCCGG), crystallized in the presence of spermine in three polymorphic forms, illustrating the different modes of spermine binding (23). These different polymorphic forms represent differences in hydration states, widths of major grooves, and the extent of DNA bending. Multiple spermine molecules shield areas of highest phosphate density, allowing crystal-packing forces to condense, bend, and twist duplex DNA. Binding of spermine with A-DNA was classified as backbone/groove, major groove, minor groove, and backbone-only interactions (15). Two amine/imine functionalities of spermine can interact with bases in the deep groove, whereas the remaining two bind to two phosphate groups of the reference duplex and a neighboring duplex in the crystal structure of the dodecamer.

Figure 2 illustrates electron density maps of three different modes of spermine binding to DNA. Although spermine has been found in several A-DNA crystal structures and in a structure of a DNA-RNA hybrid, spermine has been found only rarely in the B-DNA crystal structure.

Spermine binding to the GTG site of a B-DNA hexamer d(CGGTGG)/d(CCACCG) suggests an opening of the base pair T/A resulting in a novel non-Watson-Crick hydrogen bonding scheme between adenine and thymine in the region (24). Figure 3 shows a model of spermine interaction with the hexameric DNA as derived from the crystal structure. Spermine stabilizes the sheared base pair by "pinching together" the minor groove across the G5 and C10 phosphates. Because GTG base triplet appears five times more frequently in regulatory regions, it is suggested that crystallography in the presence of spermine might have captured a general mechanism for protein-DNA recognition, marked by the opening of a base pair.

Although a number of crystallographic and molecular modeling studies indicate site-specific binding of spermine to G/C rich sequences, studies using photoaffinity probes indicate a preferential binding to A-tracts in double-stranded DNA sequences. Lindemose

Spermine Crystals

Fig. 2. Electron density maps of spermine molecules found in the crystal lattice of the Ortho 1 (top) and Ortho 2 (middle and bottom) forms of d(CCGGGCCm5CGG). Broken lines indicate possible hydrogen bonding between spermine amino and imino groups with DNA acceptor atoms. In Ortho1, the single spermine is bound to the terminal guanine, interacting with O-6 and N-7 atoms through the central imino atoms N-5 and N-10. In the second orthorhombic form, Ortho2, one spermine molecule is bound at the opposite end of the helix to G10 and interacts through its terminal amino group nitrogen atom, N-1. Interactions of the central imino group nitrogen atom, N-5, with the adjacent phosphate are ambiguous. The second spermine molecule in Ortho 2 interacts with phosphate groups on opposite sides of the major groove. (Reprinted from ref. 23 with permission from Elsevier.)

Fig. 2. Electron density maps of spermine molecules found in the crystal lattice of the Ortho 1 (top) and Ortho 2 (middle and bottom) forms of d(CCGGGCCm5CGG). Broken lines indicate possible hydrogen bonding between spermine amino and imino groups with DNA acceptor atoms. In Ortho1, the single spermine is bound to the terminal guanine, interacting with O-6 and N-7 atoms through the central imino atoms N-5 and N-10. In the second orthorhombic form, Ortho2, one spermine molecule is bound at the opposite end of the helix to G10 and interacts through its terminal amino group nitrogen atom, N-1. Interactions of the central imino group nitrogen atom, N-5, with the adjacent phosphate are ambiguous. The second spermine molecule in Ortho 2 interacts with phosphate groups on opposite sides of the major groove. (Reprinted from ref. 23 with permission from Elsevier.)

et al. (25) conducted uranyl photocleavage and DNAse I cleavage of E. coli tyr T promoter, cytomegalovirus (CMV) promoter, and other double-stranded DNA containing A/T tracts. In these studies, putrescine, spermidine, and spermine showed preferential binding to A/T sequences, although spermine was 100-fold more efficient than putrescine in

Fig. 3. Symmetry related spermine A molecules (dark bonds) span the minor groove in the vicinity of the a+ phosphates and stabilize the sheared T/A base pair. Thin lines indicate close contacts (<4', after subtracting the van der Waals radii of the phosphates and the amino groups) between anionic phosphates and cationic amino groups. (Reprinted from ref. 24 with permission from Oxford University Press.)

Fig. 3. Symmetry related spermine A molecules (dark bonds) span the minor groove in the vicinity of the a+ phosphates and stabilize the sheared T/A base pair. Thin lines indicate close contacts (<4', after subtracting the van der Waals radii of the phosphates and the amino groups) between anionic phosphates and cationic amino groups. (Reprinted from ref. 24 with permission from Oxford University Press.)

protecting the A/T tract DNA from cleavage. Studies using a photoaffinity polyamine (azidonitrobenzoyl)spermine (ANB-spermine) that could be activated by light indicated preferential binding to the TATA sequence (26). Other studies using imino proton exchange rates and ethidium displacement have also indicated preferential binding to A/T sequences 27-29). Preferential binding of polyamines to the TATA sequence is particularly significant because of the importance of the TATA element in the recognition by RNA polymerase in transcription. Binding of polyamines to the TATA element may act as a mechanism to suppress transcription and this repression might be removed when polyamines are acetylated.

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