Quantitative analysis of chromatin structure by circular dichroism

Quantitative analysis of the circular dichroism of nucleohistones and protein-free DNA was carried out in order to determine the structure and the role of the linker region DNA in chromatin, in terms of the conformational change of chromatin as a function of the ionic strength. It is shown clearly that the circular dichroism of Hl-depleted chromatin isolated from calf thymus is determined only by the ratio of the core region to the linker region and demonstrated by the linear combination of the spectrum of protein-free DNA and that of the nucleosome core in 5 mm-Tris · HCl, 1 mm-EDTA (pH 7.8). The calculated spectrum for the linker region in the H1-depleted chromatin was in good agreement with that of protein-free DNA. From the difference spectra between nucleohistones and protein-free DNA, it is suggested that the chromatin has an additional winding of DNA other than 146 base-pairs of DNA around the histone core. By decreasing the ionic strength to values lower than 5 mm-Tris · HCl, 1 mm-EDTA, the ellipticity of H1-depleted chromatin increased greatly between 250 nm and 300 nm while the increase was small in the case of chromatin and the nucleosome core. Nucleosomes with linker region DNA but without histone H1 also show great increase in ellipticity in this range of wavelengths as the ionic strength is decreased. Therefore, the linker region in H1-depleted chromatin plays an important role in the conformational changes brought about by changes in the ionic strength, and the conformational changes caused in the DNA of chromatin by decreasing the ionic strength are suppressed by the presence of histone H1.     

Somatic DNA recombination yielding circular DNA and deletion of a genomic region in embryonic brain

In this study, a mouse genomic region is identified that undergoes DNA rearrangement and yields circular DNA in brain during embryogenesis. External region-directed inverse polymerase chain reaction on circular DNA extracted from late embryonic brain tissue repeatedly detected DNA of this region containing recombination joints. Wide-range genomic PCR and digestion-circularization PCR analysis showed this region underwent recombination accompanied with deletion of intervening sequences, including the circularized regions. This region was mapped by fluorescence in situ hybridization to C1 on mouse chromosome 16, where no gene and no physiological DNA rearrangement had been identified. DNA sequence in the region has segmental homology to an orthologous region on human chromosome 3q.13. These observations demonstrated somatic DNA recombination yielding genomic deletions in brain during embryogenesis.     

A complementary single-stranded docking site is required for enhancement of strand exchange by human immunodeficiency virus nucleocapsid protein on substrates that model viral recombination

Enhancement of strand exchange by nucleocapsid protein (NC) is proposed to occur during retroviral recombination. The mechanism was examined using an RNA (donor)-DNA hybrid that mimicked a retrovirus replication intermediate. This consisted of a 25 base pair hybrid region flanked on each side by single-stranded RNA or DNA. A second set of acceptor RNAs that could bind to the 25-base hybrid region and to various lengths of additional bases on the DNA was used to displace the donor by hybridizing with the DNA. Displacement required a complementary single-stranded DNA region outside the donor-DNA 25-nucleotide hybrid region. NC enhanced displacement slightly when the acceptor could bind 10 nucleotides and significantly when binding 22 or more nucleotides in the single-stranded region. Two mutated acceptors that bound over 47 total nucleotides on the DNA (22 in the single-stranded region plus 25 in the hybrid region) were constructed. One had three mismatches in the hybrid region; the other, three in the single-stranded region and one in the hybrid region. Each acceptor bound the DNA with approximately equal thermodynamic stability, yet NC stimulated exchange with the former and actually inhibited with the latter. This emphasized the importance of the single-stranded region in NC stimulation. The results support a mechanism where NC enhances the docking of the acceptor to the single-stranded region and then the acceptor "zippers" through the hybrid and displaces the donor. Results with the mutated acceptors indicate that NC may actually inhibit strand exchange between genomes in nonhomologous regions.     

Replication origin of the Bacillus subtilis chromosome determined by hybridization of the first-replicating DNA with cloned fragments from the replication origin region of the chromosome

The replication origin (ori) on the Bacillus subtilis genome was determined by the hybridization between the first-replicating DNA region and the cloned fragments from the ori region. The first-replicating DNA region was labeled specifically by [³H]thymidine in the presence of an inhibitor for DNA polymerase during a synchronous initiation of the chromosomal replication by germinating spores starved for thymine, and isolated by a sucrose density gradient centrifugation. Most of the labeled DNA molecules are small in size (up to 1000 bases long). The 45-kb ori region was cloned first in a λ Charon vector and then subcloned in pBR vectors. Restriction fragments from these cloned DNAs were purified by electrophoresis in agarose gels.

Only one region within the 45-kb ori region shows strong hybridization with the first-replicating DNA. Restriction fragments from this region were cloned in a phage M13 vector and separated into complementary strands. Hybridization of the labeled DNA with these cloned single-stranded fragments revealed that one site of the ori is located in each strand and they are some 2-kb apart from each other. Replication starts from these sites and proceeds inwards to pass each other.     

Cloning of immunoglobulin kappa light chain genes from mouse liver and myeloma MOPC 173

The organization of the kappa chain constant region gene was compared in DNA from an immunoglobulin-producing mouse myeloma (MOPC 173) and from liver. In situ hybridization using the Southern blotting technique revealed constant region gene-containing EcoRI-DNA fragments of 14 and 20 kb in the myeloma tissue whereas one EcoRI-DNA fragment with a length of 15 kb was found in liver DNA. After enrichment by RPC-5 chromatography and preparative electrophoresis the 14 kb fragment from MOPC 173 DNA and the 15 kb fragment from liver DNA were cloned in the bacteriophage lambda vector Charon 4A using in vitro packaging. Extensive characterization of the two fragments by restriction endonuclease mapping, in situ hybridization, and electron microscopy (R-loop and heteroduplex) showed that both fragments contain the constant region but no MOPC 173 variable region gene. Both fragments are homologous over a length of 12.5 kb including the constant region but differ from one another starting about 2.7 kb from the 5' end of the constant region gene. This indicates that the 14 kb EcoRI-DNA fragment from the myeloma tissue clearly resulted from somatic DNA rearrangement although it does not seem to carry the MOPC 173 variable region gene. These observations suggest that somatic DNA rearrangement of immunoglobulin light chain genes can involve both homologous chromosomes.Images     

Activation of mammalian DNA ligase I through phosphorylation by casein kinase II.

Mammalian DNA ligase I has been shown to be a phosphoprotein. Dephosphorylation of purified DNA ligase I causes inactivation, an effect dependent on the presence of the N-terminal region of the protein. Expression of full-length human DNA ligase I in Escherichia coli yielded soluble but catalytically inactive enzyme whereas an N-terminally truncated form expressed activity. Incubation of the full-length preparation from E. coli with purified casein kinase II (CKII) resulted in phosphorylation of the N-terminal region and was accompanied by activation of the DNA ligase. Of a variety of purified protein kinases tested, only CKII stimulated the activity of calf thymus DNA ligase I. Tryptic phosphopeptide analysis of DNA ligase I revealed that CKII specifically phosphorylated a major peptide also apparently phosphorylated in cells, implying that CKII is a protein kinase acting on DNA ligase I in the cell nucleus. These data suggest that DNA ligase I is negatively regulated by its N-terminal region and that this inhibition can be relieved by post-translational modification.     

Structural modifications induced by the mtDBP-C protein in the replication origin of Xenopus laevis mitochondrial DNA

The structure of the non-coding region of Xenopus laevis mitochondrial DNA has been studied by electron microscopy analysis of DNA molecules end-labelled with streptavidin-ferritin. We have shown that the effect of a protein modifying the shape of the DNA double-helix can be studied and precisely located by this method. It was found that the non-coding region contains curved segments and that the mitochondrial protein mtDBP-C preferentially enhances the curvature of the promoters-replication origin region.     

Identification of two functional regions in Fis: the N-terminus is required to promote Hin-mediated DNA inversion but not lambda excision.

The Fis protein of E. coli binds to a recombinational enhancer sequence that is required to stimulate Hin-mediated DNA inversion. Fis is also required for efficient lambda prophase excision in vivo. The properties of mutant Fis proteins were examined in vivo and in vitro with respect to their stimulatory effects on these two different site-specific DNA recombination reactions. Both recombination reactions are dramatically affected by mutations altering a helix-turn-helix DNA binding motif located near the Fis C-terminus (residues 74-93). These mutations invariably decrease DNA binding affinity and some cause reduced DNA bending. Mutations in the Fis N-terminal region reduce or abolish the stimulation of Hin-mediated DNA recombination by Fis, but have little or no effect on DNA binding or lambda excision. We conclude that there are at least two functionally distinct domains in Fis: a C-terminal DNA binding region that is required for promoting both DNA recombination reactions and an N-terminal region that is uniquely required for Hin-mediated inversion.     

Binding of the EcoRII methyltransferase to 5-fluorocytosine-containing DNA. Isolation of a bound peptide.

The properties of the interaction of 5-fluorocytosine-containing DNA with the EcoRII methyltransferase were studied. The DNA used was either a polymer synthesized in vitro, or a 20-mer containing one CCA/TGG sequence. The DNA could be methylated by the enzyme. In the process the enzyme formed a tight binding adduct with the DNA that could be identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Enzyme activity was inhibited by this interaction. The 20-mer could be used to titrate the active site of the enzyme. The DNA polymer formed a tight binding complex that could be identified following digestion of the DNA with pancreatic deoxyribonuclease or micrococcal nuclease. A peptide-DNA adduct could be isolated after digestion of the EcoRII-DNA adduct with staphylococcal protease V8 by high pressure liquid chromatography and polyacrylamide gel electrophoresis. Sequencing of the peptide indicated the DNA bound to a region of the protein that is conserved in all procaryotic DNA(cytosine-5)-methyltransferases. We have previously shown that this region contains a cysteine that can be photomethylated with adenosylmethionine. This region, in addition to forming part of, or being adjacent to, the AdoMet binding site, also forms part of the DNA binding site.     

Unique positioning of reconstituted nucleosomes occurs in one region of simian virus 40 DNA

A direct end label method was used to study the positioning of nucleosome arrays on several long (greater than 2200 base pairs) SV40 DNA fragments reconstituted in vitro with core histones. Comparison of micrococcal nuclease cutting sites in reconstituted and naked DNA fragments revealed substantial differences in one DNA region. When sufficient core histones were annealed with the DNA to form closely spaced nucleosomes over most of the molecule, a uniquely positioned array of four nucleosomes could be assigned, by strict criteria, to a 610-base pair portion of the SV40 "late region," with a precision of about +/- 20 base pairs. In some other DNA regions, a number of alternative nucleosome positions were indicated. The uniquely positioned four-nucleosome array spanned the same 610 nucleotides on two different DNA fragments that possessed different ends. Removal of a DNA region that had contained a terminal nucleosome of the array, by truncation of the fragment before reconstitution, did not affect the positioning of the other three nucleosomes. As the core histone to DNA ratio was lowered, evidence for specific positioning of nucleosomes diminished, except within the region where the four uniquely positioned nucleosomes formed. This region, however, does not appear to have a higher affinity for core histones than other regions of the DNA.