Nucleotide variation and molecular structure of the heterochromatic repetitive AluI DNA in the brine shrimp Artemia franciscana

Summary It has been suggested that DNA bending could play a role in the regulation of gene expression, chromosome segregation, specific recombination and/or DNA packaging. We have previously demonstrated that an Alul DNA family of repeats is the major component of constitutive heterochromatin in the brine shrimp A. franciscana. By the analysis of cloned oligomeric (monomer to hexamer) heterochromatic fragments we verified that the repetitive AluI DNA shows a stable curvature that determines a solenoidal geometry to the double helix. This particular structure could be of relevant importance in conferring the characteristic heterochromatic condensation. In this paper we evaluate how the point mutations that occurred during the evolution of the Alul sequence of A. franciscana could influence the sequence-dependent tridimensional conformation. The obtained data underline that, in spite of the high sequence mutation frequency (10%) of the repetitive DNA, the general structure of the heterochromatic DNA is not greatly influenced, but rather there is a substantial variation of the copy number of the repetitive AluI fragment. This variation could be responsible for the hypothetical function of the constitutive heterochromatin.Offprint requests to: N. Landsberger     

Sequence-directed curvature of repetitive AluI DNA in constitutive heterochromatin of Artemia franciscana.

An Alu I family of repeated DNA sequence 113 bp in length was found to be the major component of the heterochromatin in Artemia franciscana. On the basis of the analysis of cloned oligomeric (monomer to examer) heterchromatic fragments we predicted that the sequence could produce a stable curvature in chromosomal DNA. This prediction was confirmed by polyacrylamide gel electrophoresis analysis and by electron microscope observations. The anomalous mobility of these fragments is reversed when the DNA samples are electrophoresed in the presence of distamycin A. Moreover treatment of living Artemia with this drug produces visible decondensation of heterochromatic masses in the interphase nuclei.     

Induced bending of plasmid pLS1 DNA by the plasmid-encoded protein RepA

The broad host range streptococcal plasmid pLS1 encodes for a 5.1-kDa repressor protein, RepA. This protein has affinity for DNA (linear or supercoiled) and is translated from the same mRNA as the replication initiator protein RepB. By gel retardation assays, we observed that RepA shows specificity for binding to the plasmid HinfID fragment, which includes the target of the protein. The target of RepA within the plasmid DNA molecule has been located around the plasmid single site ApaLI. This site is included in a region that contains the promoter for the repA and repB genes and is contiguous to the plasmid ori(+). A complex sequence-directed DNA curvature is observed in this region of pLS1. Upon addition of RepA to plasmid linear DNA or to circularly permuted restriction fragments, this intrinsic curvature was greatly enhanced. Thus, a strong RepA-induced bending could be located in the vicinity of the ApaLI site. Visualization of the bent DNA was achieved by electron microscopy of complexes between RepA and plasmid DNA fragments containing the RepA target.     

A versatile primer for DNA sequencing in the M13mp2 cloning system

A primer for DNA sequencing by the chain-termination method in the M13mp2 cloning system was constructed and amplified. The primer was isolated as an EcoRI/AluI restriction fragment. After conversion of the AluI end into an EcoRI end the fragment was cloned in pBR325 from which it can be recovered by cleavage with EcoRI. The primer hybridizes to the single-stranded DNA of the mature M13mp2 phage next to the site of insertion thereby directing DNA synthesis along the inserted DNA.     

Human satellite 3 (HS3) binding protein from the nuclear matrix: isolation and binding properties

Satellite DNA (satDNA) is the main component of residual DNA in nuclear matrix (NM) preparations. Gel mobility shift assay (GMSA) revealed specific human satellite 3 (HS3) binding activity in NM extracts. An HS3 binding protein was purified using diethylaminoethyl (DEAE)-cellulose and preparative GMSA. The binding was specific, although other satDNA fragments compete to some extent for the binding. DNase I footprinting and methylation interference revealed multiple points of protection distributed throughout the HS3 fragment with periodicity of about 10 bp, mostly inside an AT island. Polyclonal antibodies (AB) were raised against HS3-protein complexes cut from the preparative GMSA gel. On immunoblots, AB recognise a protein, which is not lamin, with apparent molecular mass 70 kDa, the same as revealed by purification (p70). In in situ nuclear matrix preparations combined immunofluorescence (AB) and fluorescent in situ hybridisation (HS3) shows that HS3 and p70 areas correspond to each other. The localisation of this protein detected with AB in interphase nuclei coincides with the heterochromatic regions which surround nucleoli in correspondence with the known HS3 position in the nuclei.     

Molecular cloning and sequence of the gene for p9Ka. A cultured myoepithelial cell protein with strong homology to S-100, a calcium-binding protein

The gene for p9Ka, a protein of molecular weight 9000 that is expressed in cultured rat mammary myoepithelial cells, has been isolated from a normal rat genomic library in bacteriophage lambda, by its ability to hybridize to a cloned complementary DNA corresponding to p9Ka mRNA. The cloned rat genomic DNA fragment hybridized to translatable p9Ka mRNA. A nucleotide sequence of 2340 base-pairs of genomic DNA surrounding the p9Ka cDNA sequence has been obtained; the gene contains one intervening sequence of 675 nucleotides. The 3' end of the p9Ka mRNA has been identified on the gene sequence to be 13 nucleotides downstream from a poly(A) addition signal AATAAA. The gene contains an open reading frame of 101 amino acid residues, which is the only open reading frame in the entire gene long enough to encode a protein of molecular weight at least 9000. This hypothetical protein sequence shows greater than 40% homology to rat or bovine S-100 protein and over 30% homology to bovine intestinal calcium-binding protein. The results suggest that p9Ka may be related to a family of low molecular weight calcium-binding proteins.     

Specific interaction of mouse major satellite with MAR-binding protein SAF-A

A DNA-binding activity specific to the major mouse satellite (satMa) has been detected in a nuclear matrix protein extract by electrophoretic mobility shift assays (EMSA) after fractionation by ion exchange chromatography. An antibody raised against the satMa-protein complexes recovered from preparative EMSA recognizes on Western blots one major polypeptide with an apparent molecular mass of 120 kDa. The protein also has a similar affinity for a matrix-associated region (MAR) fragment. We demonstrate that the protein is a murine homologue of SAF-A which has been shown to bind selectively to MARs and is responsible for the satMa-binding activity in the chromatographic fractions. SatMa has significant homology to the mouse minor satellite fragments, but its binding of SAF-A shows much less affinity. No protected regions of significant length were found by footprinting, but multiple T residues scattered within the satMa sequence are protected, indicating that the whole fragment is involved in the binding to SAF-A. Combined immunofluorescence (SAF-A) and FISH (satMa) with in situ nuclear matrix procedures reveal that SAF-A and satMa colocalize. SAF-A appears as bright dots in interphase nuclei, presumably associated with MARs, predominantly surrounding and covering heterochromatic areas. A scheme based on morphological observations and biochemical data of SAF-A double satMa/MAR specificity is discussed.     

Purification and properties of a protein from the nuclear fraction of HeLa cells binding to a cloned fragment of human satellite DNA III

In nuclear extract of HeLa cells two proteins were identified having the specific binding activity to cloned 1.8kb fragment of human satellite DNA III (HS3). One of the satellite binding proteins (SBP1) purified by column chromatography using DEAE-, phospho- and DNA-cellulose steps interacted also with adenovirus 5 replication enhancer (ARE), another protein (SBP2) was separated during phosphocellulose chromatography from ARE-binding protein. It is suggested that SBP1 is possibly identical to the nuclear factor I purified earlier from the nuclear extract of HeLa cells by other authors.     

Further characterization of the cathepsin L-associated protein and its gene in two species of the brine shrimp, Artemia

The major cysteine protease in embryos and larvae of the brine shrimp Artemia franciscana is a heterodimer composed of a cathepsin L-like polypeptide of 28.5 kDa and a 31.5 kDa polypeptide called the cathepsin L-associated protein or CLAP. In a previous study, CLAP was shown to be a cell adhesion protein containing two Fas I domains and two GTP/ATP binding sites known as Walker A and B motifs. Here, we have characterized CLAP and its genes to better understand the role of this protein in Artemia development. The polymerase chain reaction was used to investigate the structure of the CLAP gene in two species of Artemia, the New World bisexual diploid A. franciscana and the Old World parthenogenetic tetraploid Artemia parthenogenetica. The protein coding region of the CLAP gene from each species was 99.5% identical for a protein of 332 amino acids, while the 3' non-coding region, representing nearly 45% of the gene, was only 86% identical between the two related species. However, while the CLAP gene is intronless in A. franciscana, in A. parthenogenetica the gene contained a mini-intron of 30 base pairs in the 3' non-coding region. The sequences representing the CLAP gene in A. franciscana and A. parthenogenetica have been entered into the NCBI database as AY757920 and DQ100385, respectively. Northern blot analysis showed that while the cathepsin L gene is expressed constitutively in Artemia franciscana embryos and young larvae, the CLAP gene is not expressed in late embryos and young larvae. In contrast, Western blots indicated that CLAP is present in developing embryos and young larvae, at least to the first larval molt, supporting results obtained previously showing CLAP's resistance to degradation by its dimeric partner, cathepsin L. At the protein level we showed that the GTP/ATP binding sites in CLAP are functional with rate constants of 0.024 and 0.022 for GTP and ATP hydrolase activity, respectively. GTP but not ATP also had a slight stimulatory effect on cathepsin L activity of the heterodimeric protease containing CLAP. Our results support the hypothesis that CLAP plays an important role in targeting and expression regulation of cathepsin L activity during early development of Artemia.     

Searching for target sequences by p53 protein is influenced by DNA length

One of the most important functions of the tumor suppressor p53 protein is its sequence-specific binding to DNA. Using a competition assay on agarose gels we found that the p53 consensus sequences in longer DNA fragments are better targets than the same sequences in shorter DNAs. Semi-quantitative evaluation of the competition experiments showed a correlation between the relative p53-DNA binding and the DNA lengths. Our results are consistent with a model of the p53-DNA interactions involving one-dimensional migration of the p53 protein along the DNA for distances of about 1000 bp while searching for its target sites. Positioning of the p53 target in the DNA fragment did not substantially affect the apparent p53-DNA binding, suggesting that p53 can slide along the DNA in a bi-directional manner. In contrast to full-length p53, the isolated core domain did not show any significant correlation between sequence-specific DNA binding and fragment length.     

DNA binding protein from ovaries of the frog, Xenopus laevis which promotes concatenation of linear DNA

A soluble extract of Xenopus laevis ovaries catalyzed ATP-dependent concatenation of linear duplex DNA molecules. DNA ligase and a unique X. laevis DNA binding protein were required for the formation of concatemers. A linear DNA concatenation system was reconstituted using T4 DNA ligase and homogeneous X. laevis DNA binding protein. This system catalyzed intermolecular ligation of DNA molecules into linear concatemers of up to ten or more times monomer length.     

A new protein domain for binding to DNA through the minor groove.

Protein p6 of the Bacillus subtilis phage phi 29 binds with low sequence specificity to DNA through the minor groove, forming a multimeric nucleoprotein complex that activates the initiation of phi 29 DNA replication. Deletion analysis suggested that the N-terminal part of protein p6, predicted to form an amphipathic alpha-helix, is involved in DNA binding. We have constructed site-directed mutants at the polar side of the putative alpha-helix. DNA binding and activation of initiation of phi 29 DNA replication were impaired in most of the mutant proteins obtained. A 19 amino acid peptide comprising the N-terminus of protein p6 interacted with a DNA fragment containing high-affinity signals for protein p6 binding with approximately 50-fold higher affinity than the peptide corresponding to an inactive mutant. Both wild-type peptide and protein p6 recognized the same sequences in this DNA fragment. This result, together with distamycin competition experiments, suggested that the wild-type peptide also binds to DNA through the minor groove. In addition, CD spectra of the wild-type peptide showed an increase in the alpha-helical content when bound to DNA. All these results indicate that an alpha-helical structure located in the N-terminal region of protein p6 is involved in DNA binding through the minor groove.     

DNA phosphate crowding correlates with protein cationic side chain density and helical curvature in protein/DNA crystal structures

Sequence-specific binding of proteins to their DNA targets involves a complex spectrum of processes that often induce DNA conformational variation in the bound complex. The forces imposed by protein binding that cause the helical deformations are intimately interrelated and difficult to parse or rank in importance. To investigate the role of electrostatics in helical deformation, we quantified the relationship between protein cationic residue density (Cpc) and DNA phosphate crowding (Cpp). The correlation between Cpc and Cpp was then calculated for a subset of 58 high resolution protein–DNA crystal structures. Those structures containing strong Cpc/Cpp correlation (>±0.25) were likely to contain DNA helical curvature. Further, the correlation factor sign predicted the direction of helical curvature with positive (16 structures) and negative (seven structures) correlation containing concave (DNA curved toward protein) and convex (DNA curved away from protein) curvature, respectively. Protein–DNA complexes without significant Cpc/Cpp (36 structures) correlation (-0.25<0<0.25) tended to contain DNA without significant curvature. Interestingly, concave and convex complexes also include more arginine and lysine phosphate contacts, respectively, whereas linear complexes included essentially equivalent numbers of Lys/Arg phosphate contacts. Together, these findings suggest an important role for electrostatic interactions in protein–DNA complexes involving helical curvature.     

Increase of the DNA strand assimilation activity of recA protein by removal of the C terminus and structure-function studies of the resulting protein fragment

A proteolytic fragment of recA protein, missing about 15% of the protein at the C terminus, was found to promote assimilation of homologous single-stranded DNA into duplex DNA more efficiently than intact recA protein. This difference was not found if Escherichia coli single-stranded DNA binding protein was present. The ATPase activity of both intact recA protein and the fragment was identical. The difference in strand assimilation activity cannot be due to differences in single-stranded DNA affinity, since both the fragment and intact proteins bind to single-stranded DNA with nearly identical affinities. However, the fragment was found to bind double-stranded DNA more tightly and to aggregate more extensively than recA protein; both of these properties may be important in strand assimilation. Aggregation of the fragment was extensive in the presence of duplex DNA under the same condition where recA protein did not aggregate. The double-stranded DNA binding of both recA protein and the fragment responds to nucleotide cofactors in the same manner as single-stranded DNA binding, i.e. ADP weakens and ATP gamma S strengthens the association. The missing C-terminal region of recA protein includes a very acidic region that is homologous to other single-stranded DNA binding proteins and which has been implicated in DNA binding modulation. This C-terminal region may serve a similar function in recA protein, possibly inhibiting double-stranded DNA invasion. The possible role of the enhanced double-stranded DNA affinity of the fragment protein in the mechanism of strand assimilation is discussed.     

Evidence of a ter specific binding protein essential for the termination reaction of DNA replication in Escherichia coli.

Activity binding specifically to the 22 bp of the DNA replication terminus (ter) sequence on plasmid R6K and the Escherichia coli genome was detected in the crude extract of E. coli cells. This activity was inactivated by heat or by protease but not by RNase treatments. Overproduction of the ter binding activity was observed when the extract was prepared from the cell carrying a plasmid with a chromosomal-derived 5.0 kb EcoRI fragment, on which one of the four terC sites, terC2, was also located. By mutagenesis of the 5.0 kb fragment on the plasmid with transposon Tn3 and subsequent replacement of the corresponding chromosomal region with the resulting mutant alleles, we isolated tau- mutants completely defective in ter binding activity. These mutants simultaneously lost the activity to block the progress of the DNA replication fork at any ter site, on the genome or the plasmid. It would thus appear that the ter binding protein plays an essential role in the termination reaction, at the ter sites.