The Drosophila GAGA factor self-oligomerizes both in vivo and in vitro. GAGA oligomerization depends on the presence of the N-terminal POZ domain and the formation of dimers, tetramers, and oligomers of high stoichiometry is observed in vitro. GAGA oligomers bind DNA with high affinity and specificity. As a consequence of its multimeric character, the interaction of GAGA with DNA fragments carrying several GAGA binding sites is multivalent and of higher affinity than its interaction with fragments containing single short sites. A single GAGA oligomer is capable of binding adjacent GAGA binding sites spaced by as many as 20 base pairs. GAGA oligomers are functionally active, being transcriptionally competent in vitro. GAGA-dependent transcription activation depends strongly on the number of GAGA binding sites present in the promoter. The POZ domain is not necessary for in vitro transcription but, in its absence, no synergism is observed on increasing the number of binding sites contained within the promoter. These results are discussed in view of the distribution of GAGA binding sites that, most frequently, form clusters of relatively short sites spaced by small variable distances.

The Drosophila GAGA factor binds specifically to simple repeating d(GA⅐TC) n DNA sequences. These sequences are known to be capable of forming triple-stranded DNA as well as other non-B-DNA conformations. Here, it is shown that GAGA binds to a d[CT(GA⅐TC)] 22 intermolecular triplex with similar specificity and affinity as to a regular double-stranded B-form d(GA⅐TC) 22 sequence. The interaction of GAGA with triplex DNA cannot, however, stimulate transcription in vitro. The affinity of GAGA for triplexes of the purine motif, such as a d[AG(GA⅐TC)] 22 intermolecular triplex, is significantly lower. The DNA binding domain of GAGA is sufficient for efficient binding to triplex DNA. Based on the reported solution structure of the complex of GAGA-DNA binding domain with double-stranded DNA, a model for its interaction with triplex DNA is proposed in which most of the protein-DNA contacts observed in duplex DNA are maintained, especially those occurring through the minor groove. The higher negative charge of the triplex is likely to have also an important contribution to both the specificity and affinity of the interaction.The GAGA factor of Drosophila is a sequence-specific DNAbinding protein that participates in a variety of different chromosomal functions (for reviews see Refs. 1 and 2). GAGA has been shown to stimulate transcription of some developmentally regulated homeotic genes and heat shock genes (3, 4), and GAGA-binding sites are found at the promoters of numerous Drosophila genes (1), suggesting a general role of GAGA in transcription regulation. Although its actual mechanism of action is not yet fully understood, it is believed that, at least to some extent, GAGA functions at the level of chromatin structure, participating in nucleosome remodeling at the promoter regions (5, 6). Consistent with a role at the level of chromatin structure, GAGA is a modifier of position effect variegation (7). A link to chromatin structure is also indicated by its association with heterochromatin (8). Interestingly, GAGA is found associated with the centromeric heterochromatin in mitotic, but not in polytene, chromosomes (9); some GAGA mutants show mitotic defects (10), suggesting a role of GAGA in chromosome condensation and/or segregation.GAGA binds to repeated d(GA⅐TC) n DNA sequences. In general, GAGA-binding sites found at promoters are short, with a consensus of 3.5 dinucleotide repeats, and multiple (1). In some promoters, however, long binding sites are also observed (i.e. hsp26, hsp70, his3, and his4) (1, 11). Moreover, the association of GAGA with heterochromatin occurs mainly through its binding to the (AAGAG) n and (AAGAGAG) n satellites (8), which constitute extremely long arrays of consecutive GAGA-binding sites. Repeated d(GA⅐TC) n DNA sequences are structurally polymorphic being capable of adopting different non-B-DNA conformations (for reviews see Refs. 12 and 13). In particular, they form triple-stranded conformations which, depending on the experimental conditions, are of the d[CT(GA⅐TC)] n or the d[AG(GA⅐T...

Simple repeating d(GA⅐TC)n

The interaction of poly[(G-C)] and poly[d(G-m5C)] with the antitumor antibiotic elsamicin A, which binds to alternating guanine + cytosine tracts in DNA, has been studied under the B and Z conformations. Both the rate and the extent of the B-to-Z transition are diminished by the antibiotic, as inferred by spectroscopic methods under ionic conditions that otherwise favor the left-handed conformation of the polynucleotides. Moreover, elsamicin converts the Z-form DNA back to the B-form. The circular dichroism data indicate that elsamicin binds to poly[d(G-C)] and poly[d(G-m5C)] to form a right-handed bound elsamicin region(s). The transition can be followed by changes of the molar ellipticity at 250 nm, thus providing a convenient wavelength to monitor the Z-to-B conformational change of the polymers as elsamicin is added. The elsamicin A effect might be explained by a model in which the antibiotic binds preferently to a B-form DNA, playing a role as an allosteric effector on the equilibrium between the B and Z conformations, thus favoring the right-handed one.