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The genomic distribution of the abundant eukaryotic d(GA x TC)(n) DNA microsatellite suggests that it could contribute to DNA recombination. Here, it is shown that this type of microsatellite DNA sequence enhances DNA recombination in SV40 minichromosomes, the rate of homologous DNA recombination increasing by as much as two orders of magnitude in the presence of a d(GA x TC)(22) sequence. This effect depends on the region of the SV40 genome at which the d(GA x TC)(22) sequence is cloned. It is high when the sequence is located proximal to the SV40 control region but no effect is observed when located 3.5 kb away from the SV40 ori. These results indicate that the recombination potential of d(GA x TC)(n) sequences is likely linked to DNA replication and/or transcription. The potential contribution of the structural properties of d(GA x TC)(n) sequences to this effect is discussed.

The genomic distribution of the abundant eukaryotic d(GA·TC)_n DNA microsatellite suggests that it could contribute to DNA recombination. Here, it is shown that this type of microsatellite DNA sequence enhances DNA recombination in SV40 minichromosomes, the rate of homologous DNA recombination increasing by as much as two orders of magnitude in the presence of a d(GA·TC)₂₂ sequence. This effect depends on the region of the SV40 genome at which the d(GA·TC)₂₂ sequence is cloned. It is high when the sequence is located proximal to the SV40 control region but no effect is observed when located 3.5 kb away from the SV40 ori. These results indicate that the recombination potential of d(GA·TC)_n sequences is likely linked to DNA replication and/or transcription. The potential contribution of the structural properties of d(GA·TC)_n sequences to this effect is discussed.

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.

Alternating d(GA.TC)(n)DNA sequences, which are abundant in eukaryotic genomes, can form altered DNA structures. Depending on the environmental conditions, the formation of (GA.GA) hairpins or [C+T(GA.TC)] and [GA(GA.TC)] intramolecular triplexes was observed in vitro. In vivo, the formation of these non-B-DNA structures would likely require the contribution of specific stabilizing factors. Here, we show that Friend's nuclear extracts are rich in proteins which bind the pyrimidine d(TC)(n)strand but not the purine d(GA)n strand (NOGA proteins). Upon chromatographic fractionation, four major proteins were detected (NOGA1-4) that have been purified and characterized. Purified NOGAs bind single-stranded d(TC)n with high affinity and specificity, showing no significant affinity for either d(GA)n or d(GA.TC)nDNA sequences. We also show that NOGA1, -2 and -3, which constitute the three most abundant and specific NOGA proteins, correspond to the single-stranded nucleic acid binding proteins hnRNP-L, -K and -I, respectively. These results are discussed in the context of the possible contribution of the NOGA proteins to the stabilization of the (GA.GA) and [GA(GA.TC)] conformers of the d(GA.TC)n DNA sequences.

In this paper, we have analysed the conformational behaviour shown by the homopurine–homopyrimidine alternating d(GA.CT)22 sequence cloned into SV40. Our results show that, in the presence of zinc ions, the d(GA.CT)22 sequence adopts an altered secondary DNA structure (*H‐DNA) which differs from either B‐DNA or H‐DNA. Formation of *H‐DNA is facilitated by negative supercoiling and does not appear to require base protonation, since it is induced at neutral pH by approximately 0.4 mM ZnCl2. The patterns of OsO4 and DEPC modification obtained in the presence of zinc are compatible with a homopurine–homopurine–homopyridimine triplex, though other structural models for *H‐DNA are also possible. The hypersensitivity to S1‐cleavage of the d(GA.CT)22 sequence is reinterpreted in terms of the equilibria between the B‐, H‐ and *H‐forms of the sequence. These results reveal the high degree of structural polymorphism shown by homopurine‐homopyrimidine sequences. Its biological relevance is discussed.

Zinc induces transition of a d(GA·CT)₂₂ sequence to a novel DNA conformation, called ^*H-DNA. In this paper, the structural characteristics of this altered DNA conformation are determined. Formation of ^*H-DNA is induced at zinc concentrations higher than 70 µM (Zn/P=2) and it is favoured by negative supercoiling and low ionic strength. Two different structural conformations of the d(GA·CT)₂₂ sequence are observed upon increasing the zinc concentration. At low zinc concentration (Zn/P<15), half of the purine strand falls back upon itself giving rise to the formation of an RRY intramolecular triplex (H^*-triplex). At higher zinc concentration, the complete pyrimidine strand is single-stranded and an RR hairpin is formed (^*H-hairpin). Protection towards dimethylsulphate modification suggests that zinc binds to the N-7 group of guanine residues in the ^*H-hairpin with a higher affinity than in B-DNA. The dissociation constant of the ^*H-zinc complex is estimated to be in the range of 10^-3 M to 10^-4 M. Copyright 1993, 1999 Academic Press

Simple repeating d(GA.TC)n DNA sequences are frequently found at eukaryotic promoters, and in some cases they have been shown to be nucleosome free in vivo. These sequences show a high degree of structural polymorphism and are capable of adopting several types of non-B-DNA conformations. Here we show that the structural versatility of these sequences affects their ability to be packed into nucleosomes. Nucleosome assembly onto short double-stranded DNA fragments carrying d(GA.TC)n sequences of different length (n = 10 and n = 22) is very efficient. However, when the simple repeating sequence is forming a [CT(GA.TC)] triplex, nucleosome assembly is either prevented, as in the case of the d(GA.TC)22 sequence, or results in the destabilization of the triple-stranded conformation, as in the case of the d(GA.TC)10 sequence. Similarly, formation of triple-stranded DNA is hindered when the sequence is organized as nucleosomes. Efficient formation of triplex DNA occurs only at relatively high ionic strength (0.6 M NaCl), when the nucleosome is partially destabilized, and results in the disruption of the nucleosomal particle. These results indicate that nucleosome assembly and triplex formation are competing processes.

The Drosophila GAGA factor binds specifically to simple repeating d(GA.TC)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(GA.TC)]22 intermolecular triplex with similar specificity and affinity as to a regular double-stranded B-form d(GA.TC)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(GA.TC)]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.