Ferran Azorín

DDP1, a single-stranded nucleic acid-binding protein of Drosophila, associates with pericentric heterochromatin and is functionally homologous to the yeast Scp160p, which is involved in the control of cell ploidy

The centromeric dodeca‐satellite of Drosophila forms altered DNA structures in vitro in which its purine‐rich strand (G‐strand) forms stable fold‐back structures, while the complementary C‐strand remains unstructured. In this paper, the purification and characterization of DDP1, a single‐stranded DNA‐binding protein of high molecular mass (160 kDa) that specifically binds the unstructured dodeca‐satellite C‐strand, is presented. In polytene chromosomes, DDP1 is found located at the chromocentre associated with the pericentric heterochromatin but its distribution is not constrained to the dodeca‐satellite sequences. DDP1 also localizes to heterochromatin in interphase nuclei of larval neuroblasts. During embryo development, DDP1 becomes nuclear after cellularization, when heterochromatin is fully organized, being also associated with the condensed mitotic chromosomes. In addition to its localization at the chromocentre, in polytene chromosomes, DDP1 is also detected at several sites in the euchromatic arms co‐localizing with the heterochromatin protein HP1. DDP1 is a multi‐KH domain protein homologous to the yeast Scp160 protein that is involved in the control of cell ploidy. Expression of DDP1 complements a Δscp160 deletion in yeast. These results are discussed in view of the possible contribution of DNA structure to the structural organization of pericentric heterochromatin.

Structural polymorphism of homopurine--homopyrimidine sequences: the secondary DNA structure adopted by a d(GA.CT)22 sequence in the presence of zinc ions

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.

Centromeric dodeca-satellite DNA sequences form fold-back structures.

The evolutionarily conserved centromeric dodeca-satellite DNA has an asymmetric distribution of guanine and cytosine residues resulting in one strand being relatively G-rich. This dodeca-satellite G-strand contains a GGGA-tract that is similar to the homopurine tracts found in most telomeric DNA sequences. Here, we show that the dodeca-satellite G-strand forms intramolecular hairpin structures that are stabilized by the formation of non-Watson-Crick G.A pairs as well as regular Watson-Crick G.C pairs. Special stacking interactions are also likely to contribute significantly to the stability of this structure. This hairpin conformation melts at relatively high temperature, around 75 degrees C, and is detected under many different ionic and pH conditions. As judged by electron microscopy visualization, these structures can be formed in a B-DNA environment. Under the same experimental conditions, neither the C-strand nor the double-stranded dodeca-satellite DNA were found to form any unusual DNA structure. A protein activity has been detected that preferentially binds to the single-stranded dodeca-satellite C-strand. The biological relevance of these results is discussed in view of the similarities to telomeric DNA.

Alternating d(GA)n DNA sequences form antiparallel stranded homoduplexes stabilized by the formation of G.A base pairs.

Alternating d(GA)n DNA sequences form antiparallel stranded homoduplexes which are stabilized by the formation of G.A pairs. Three base pairings are known to occur between adenine and guanine: AH+ (anti).G(syn), A(anti).G(anti) and A(syn).G(anti). Protonation of the adenine residues is not involved in the stabilization of this structure, since it is observed at any pH value from 8.3 to 4.5; at pH < or = 4.0 antiparallel stranded d(GA.GA) DNA is destabilized. The results reported in this paper strongly suggest that antiparallel stranded d(GA.GA) homoduplexes are stabilized by the formation of alternating A(anti).G(anti) and G(anti).A(syn) pairs. In this structure, all guanine residues are in the anti conformation with their N7 position freely accessible to DMS methylation. On the other hand, adenines in one strand adopt the anti conformation, with their N7 position also free for reaction, while those of the opposite strand are in the syn conformation, with their N7 position hydrogen bonded to the guanine N1 group of the opposite strand. A regular right‐handed helix can be generated using alternating G(anti).A(syn) and A(anti).G(anti) pairs.

Interaction of DNA with lysine-rich polypeptides and proteins: The influence of polypeptide composition and secondary structure☆

Using X-ray diffraction we have studied fibres obtained from complexes of DNA with lysine-rich polypeptides and with proteins that have different conformations, to ascertain whether the conformations of the polypeptides and the DNA are maintained upon interaction. Substances investigated include N-acetyl-Lys-Ala-Tyr-Ala-Lys-ethylamide, random poly(Leu50, Lys50), sequential poly(Leu-Lys), poly(Val-Lys), poly(Ala-Lys), poly(Lys-Ala-Ala-Lys), poly(Lys-Ala-Ala), poly(Lys-Leu-Ala), poly(Lys-Ala-Gly), protein phi 0 from sea cucumber spermatozoa, histone H1 and two fragments of this protein obtained by chemical cleavage. In general, the B form of DNA with ten base-pairs per helical turn is maintained upon interaction at high levels of humidity. The A form is never observed; it appears to be forbidden in a protein environment. No evidence for transition into any novel DNA conformation has been observed, although the B form is altered in some cases, in particular upon dehydration. Such alteration occurs always in the sense of tightening the double helix, so that the number of base-pairs per helical turn diminishes. The polypeptides may interact with DNA in both the alpha and beta conformations. We have found different types of complexes in which either a monolayer or a double layer of beta-pleated sheets is intercalated between layers of DNA molecules. Alternatively, the polypeptide chain may be wrapped around the DNA, following one of the grooves. The polypeptide conformation may be either maintained or changed upon interaction. The charge density of the polypeptide is an important parameter of the interaction. When it matches the charge density of the DNA, the polypeptide conformation is maintained in most cases; otherwise it is modified. The globular part of histone H1 gives a unique X-ray pattern upon interaction, indicative of a loss of order of DNA in the complex. On the other hand, the C-terminal part of histone H1 gives a very well-ordered complex, similar to a nucleoprotamine, in spite of its lower charge density.

GAGA factor down-regulates its own promoter.

GAGA factor is involved in many nuclear transactions, notably in transcription as an activator inDrosophila. The genomic region corresponding to theTrl promoter has been obtained, and a minimal version of a fully active Trl promoter has been defined using transient transfection assays in S2 cells. DNase I footprinting analysis has shown that this region contains multiple GAGA binding sites, suggesting a potential regulatory role of GAGA on its own promoter. The study shows that GAGA down-regulates Trl expression. The repression does not depend on the GAGA isoform, but binding to DNA is absolutely required. A fragment of the Trl promoter can mediate repression to a heterologous promoter only upon GAGA overexpression in transiently transfected S2 cells. Chromatin immunoprecipitation analysis of S2 cells confirmed that GAGA factors are bound to the Trl promoter over a region of 1.4 kbp. Using a double-stranded RNA interference approach, we show that endogenous GAGA factors limit Trl expression in S2 cells. Our results open the possibility of observing similar GAGA repressive effects on other promoters.

Triple-Stranded DNA

The molecular biology of DNA is determined to a great extent by the chemical properties of its constitutive polynucleotide chains and, in particular, by the stability of the different complexes to which they can give rise. Most frequently, DNA is found in the form of an antiparallel doublestranded association but the formation of DNA complexes containing either three or four strands has also been extensively reported in the literature. The formation of a triple-stranded nucleic acid was first reported in 1957 in the case of the RNA triplex U(A · U) (Felsenfeld et al. 1957) and it was followed by the demonstration that the RNA homopolymers polyI and polyA could also form the triple-stranded helixes I(I · I) and I(A · I) (Rich 1958a,b). In the following decade, formation of triple-stranded conformations was also demonstrated for a variety of RNA and DNA homopolymers, as well as for RNA-DNA hybrids. Recently, triple-stranded DNA has received renewed attention. Most of the recent interest in triple-stranded DNA came after the discovery that DNA triplexes could also be intramolecular. In the case of an intramolecular triplex, the third strand, which associates to the double-stranded DNA fragment, is donated by the same DNA molecule. Intramolecular triplexes are therefore a source of DNA structural polymorphism, which adds to the known capability of the DNA molecule to exist under structurally different double-stranded conformations. On the other hand, in the case of an intermolecular triplex, the third strand is donated by a different DNA or RNA molecule, normally a singlestranded oligomer. Intermolecular triplexes provide a means for the specific recognition of double-stranded DNA by single-stranded DNA or RNA molecules. Here, after a brief introductory summary about the general aspects underlying the formation of triple-stranded DNA, we will review the recent progress on the study of the structural and functional properties of intra- and intermolecular DNA triplexes.

DNA conformation and chromatin organization of a d(CA/GT)30 sequence cloned in SV40 minichromosomes.

The alternating DNA sequence d(CA/GT)n is known to adopt the left‐handed Z‐form in negatively supercoiled DNA in vitro. This element represents a significant fraction of the highly repeated DNA sequences found in eukaryotic genomes. We have cloned an alternating d(CA/GT)30 sequence into SV40 minichromosomes at the unique HpaII restriction site which occurs in the transcriptional leader region of the viral late genes. By comparing the linkage differences of topoisomers obtained from viral DNA with or without the d(CA/GT)30 insert, at various stages of the lytic cycle, we conclude that this sequence does not predominate in the Z‐form in vivo. Furthermore, we find that the d(CA/GT)30 sequence is packaged into nucleosomal core particles and the region of the minichromosomes which contain the d(CA/GT)30 sequence is organized as nucleosomes.

Analysing the contribution of nucleic acids to the structure and properties of centric heterochromatin.

A class of repetitive DNA sequences frequently found at centromeric regions are R/Y-satellites showing an asymmetric distribution of residues resulting in one strand being rich in purines (R-strand) while the complementary strand is pyrimidine-rich (Y-strand). The dodeca-satellite of Drosophila belongs to this class of centromeric satellites. In vitro, the dodeca-satellite forms altered DNA structures in which the R-strand forms very stable intramolecular fold-backs that are stabilised by the formation of tandem G · A mismatches. A single-stranded nucleic acids binding protein, DDP1, binds the unstructured dodeca-satellite Y-strand with high affinity. In polytene chromosomes, DDP1 associates with the heterochromatic chromocenter and, at the euchromatic chromosome arms, co-localises with HP1. DDP1 is a vigilin. Vigilins are highly conserved multi-KH-domain proteins. Scp160p, the vigilin from S. cerevisiae, is involved in the control of ploidy. DDP1 complements a Δscp160 deletion.