Assembly of an active chromatin structure during replication.

MSB cells were pulse labeled with 3H-thymidine and the isolated nuclei digested with either staphylococcal nuclease (to about 40% acid solubility) or DNase I (to 15% acid solubility). The purified, nuclease resistant single-copy DNA was then hybridized to nuclear RNA (nRNA). The results of these experiments show that actively transcribed genes are assembled into nucleosome-like structures within 5-10 nucleosomes of the replication fork and that they also acquire a conformation characteristic of actively transcribed nucleosomes (ie, a DNase I sensitive structure) within 20 nucleosomes of the fork. Assuming DNA sequence specific interactions are required for establishing a DNase I sensitive conformation on active genes during each round of replication, our results indicate that a specific recognition event can occur very rapidly and very specifically in eukaryotic cells. The results are discussed in terms of the possible mechanisms responsible for propagating active, chromosomal conformations from mother cells to daughter cells.     

Stability and asymmetric replication of the Bacillus subtilis 168 chromosome structure.

Chromosomal DNAs from a number of strains derived from Bacillus subtilis 168 were digested with restriction endonucleases NotI or SfiI, and the locations of chromosomal alterations were compared with the recently constructed standard NotI-SfiI restriction map (M. Itaya and T. Tanaka, J. Mol. Biol. 220:631-648, 1991). In general, the chromosome structure of B. subtilis 168 was found to be stable, as expected from the genetic stability of this species. DNA alterations, typically deletions, are formed in three limited loci on the chromosome. One of these alterations was characterized as a spontaneous deletion formed between rrn operons, and another occurred as a result of prophage SP beta excision. I found that oriC and terC are not located on precisely opposite sides of the chromosome. Replication in the counter clockwise direction was 196 kb longer than replication in the clockwise direction. The characteristic of length difference is not changed by deletion formation.     

DNA replication and chromatin structure of simian virus 40 insertion mutants.

Insertion of DNA segments into the nuclease-sensitive region of simian virus 40 alters both replication efficiency and chromatin structure. Mutants containing large insertions between the simian virus 40 origin of replication (ori site) and the 21-base-pair repeated sequences replicated poorly when assayed by transfection into COS-1 cells. Replication of mutants with shorter insertions was moderately reduced. This effect was cis-acting and independent of the nucleotide sequence of the insert. The nuclease-sensitive chromatin structure was retained in these mutants, but the pattern of cleavage sites was displaced in the late direction from the ori site. New cleavage sites appeared within the inserted sequences, suggesting that information specifying the nuclease-sensitive chromatin structure is located on the late side of the inserts. Accessibility to BglI (which cleaves within the ori site) was reduced in the larger insertion mutants. These results support the conclusion that efficient function of the viral origin of replication is correlated with its proximity to an altered chromatin structure.     

Review: nuclear structure and DNA replication

DNA replication is a highly conserved process among eukaryotes where it occurs within a unique organelle-the nucleus. The importance of this structure is indicated by the fact that assembly of prereplication complexes on cellular chromatin is delayed until mitosis is completed and a nuclear structure has formed. Although nuclear structure is dispensable for DNA replication in vitro, it does appear to play a role in vivo by regulating the concentration of proteins required to initiate DNA replication, by facilitating the assembly or activity of DNA replication forks, and by determining where in the genome initiation of DNA replication occurs.     

Domain structure of the simian virus 40 core origin of replication.

The simian virus 40 core origin of replication consists of nucleotides 5211 through 31. These 64 base pairs contain three functional domains with strict sequence requirements and two spacer regions with relaxed sequence specificity but precise positional constraints. The early domain extends for 10 contiguous base pairs between nucleotides 5211 and 5220. A 9-base pair spacer from sequences 5221 through 5229 separates the early domain from the 23-base pair central palindrome that directs the binding of T antigen. The late end of the core between nucleotides 12 and 31 also contains spacer and sequence-specific functions that are not yet completely mapped. We propose that the sequence-specific domains are interaction sites for viral and cellular proteins, determinants of DNA conformation, or both. The spacers would position these signals at required distances and rotations relative to one another.     

A DNA structure is required for geminivirus replication origin function.

The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two single-stranded circular DNAs, A and B, that replicate through a rolling-circle mechanism in nuclei of infected plant cells. The TGMV origin of replication is located in a conserved 5' intergenic region and includes at least two functional elements: the origin recognition site of the essential viral replication protein, AL1, and a sequence motif with the potential to form a hairpin or cruciform structure. To address the role of the hairpin motif during TGMV replication, we constructed a series of B-component mutants that resolved sequence changes from structural alterations of the motif. Only those mutant B DNAs that retained the capacity to form the hairpin structure replicated to wild-type levels in tobacco protoplasts when the viral replication proteins were provided in trans from a plant expression cassette. In contrast, the same B DNAs replicated to significantly lower levels in transient assays that included replicating, wild-type TGMV A DNA. These data established that the hairpin structure is essential for TGMV replication, whereas its sequence affects the efficiency of replication. We also showed that TGMV AL1 functions as a site-specific endonuclease in vitro and mapped the cleavage site to the loop of the hairpin. In vitro cleavage analysis of two TGMV B mutants with different replication phenotypes indicated that there is a correlation between the two assays for origin activity. These results suggest that the in vivo replication results may reflect structural and sequence requirements for DNA cleavage during initiation of rolling-circle replication.     

The relationship between DNA replication and chromosome structure

Summary The results obtained by acridine orange staining of chromosomes, after BrdU treatment, during one or two cell cycles, are described. The alterations of chromosome structure do not depend only on BrdU incorporation into DNA. Some other mechanisms are necessarily involved, and it is postulated that they are disturbances of protein-DNA association, occurring in G₁ and in S- or G₂-phase. The aspect of metaphase chromosomes then appears as the result of several metabolic steps, all occurring during interphase.Presented at the Fifth Meeting of the Cytogenetics Section of the Society for Anthropology and Human Genetics, Basel, Switzerland, June 17–19, 1976.     

Chromatin structure within sites of DNA replication

Conformational changes in chromatin structure are nowadays the object of intensive research due to its importance for proper regulation of intranuclear processes. The fine structure of chromatin within the DNA replication sites was studied in in situ fixed cells and cells permebilized by low ionic strength solutions in the presence of divalent cations. The latter method provides visualization of higher level chromatin structures such as globular chromomeres and chromonema fibres. Nascent DNA was detected immunochemically using anti-BrdU antibodies on the surface of ultrathin sections prepared from Epon-embedded material. It was shown that newly replicated DNA preferentially localized within the zones filled with globular and fibrillar elements with characteristic diameter of 30 nm, and not in chromonema fibres, while after replication had been completed DNA became embedded into as thick as 60-80 nm chromonema elements. The results obtained are discussed in the context of conception of hierarchical folding of chromatin fibers.     

Function, replication and structure of the mammalian telomere

Telomeres are specialized structures at the ends of linear chromosomes that were originally defined functionally based on observations first by Muller (1938) and subsequently by McClintock (1941) that naturally occurring chromosome ends do not behave as double-stranded DNA breaks, in spite of the fact that they are the physical end of a linear, duplex DNA molecule. Double-stranded DNA breaks are highly unstable entities, being susceptible to nucleolytic attack and giving rise to chromosome rearrangements through end-to-end fusions and recombination events. In contrast, telomeres confer stability upon chromosome termini, as evidenced by the fact that chromosomes are extraordinarily stable through multiple cell divisions and even across evolutionary time. This protective function of telomeres is due to the formation of a nucleoprotein complex that sequesters the end of the DNA molecule, rendering it inaccessible to nucleases and recombinases as well as preventing the telomere from activating the DNA damage checkpoint pathways. The capacity of a functional end-protective complex to form is dependent upon maintenance of sufficient telomeric DNA. We have learned a great deal about telomere structure and how this specialized nucleoprotein complex confers stability on chromosome ends since the original observations that defined telomeres were made. This review summarizes our current understanding of mammalian telomere replication, structure and function.