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Results for “DNA replication fork stabilization”

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PubMed1995

The phage T4-coded DNA replication helicase (gp41) forms a hexamer upon activation by nucleoside triphosphate.

Sedimentation and high performance liquid chromatography studies show that the functional DNA replication helicase of bacteriophage T4 (gp41) exists primarily as a dimer at physiological protein concentrations, assembling from gp41 monomers with an association constant of approximately 10(6) M-1. Cryoelectron microscopy, analytical ultracentrifugation, and protein-protein cross-linking studies demonstrate that the binding of ATP or GTP drives the assembly of these dimers into monodisperse hexameric complexes, which redissociate following depletion of the purine nucleotide triphosphatase (PuTP) substrates by the DNA-stimulated PuTPase activity of the helicase. The hexameric state of gp41 can be stabilized for detailed study by the addition of the nonhydrolyzable PuTP analogs ATP gamma S and GTP gamma S and is not significantly affected by the presence of ADP, GDP, or single-stranded or forked DNA template constructs, although some structural details of the hexameric complex may be altered by DNA binding. Our results also indicate that the active gp41 helicase exists as a hexagonal trimer of asymmetric dimers, and that the hexamer is probably characterized by D3 symmetry. The assembly pathway of the gp41 helicase has been analyzed, and its structure and properties compared with those of other helicases involved in a variety of cellular processes. Functional implications of such structural organization are also considered.

Adenosine Triphosphate↗
PubMed2026

R-loops and D-loops: a delicate balance in genomic stability and instability.

R-loops and D-loops are three-stranded nucleic acid structures that have emerged as central regulators of genome stability, gene expression, and DNA metabolism. R-loops form co-transcriptionally or post-transcriptionally when nascent RNA re-anneals with the template DNA strand, generating an RNA: DNA hybrid that displaces the non-template strand into a single-stranded state. These structures are enriched at CpG island promoters, transcription termination sites, and immunoglobulin class-switch regions, where they coordinate transcription regulation, chromatin remodeling, and DNA damage signaling. D-loops are formed when a single-stranded DNA segment pairs with one strand of a duplex and displaces the other, arising through context-dependent mechanisms that include RAD51- or DMC1-mediated strand invasion in homologous recombination, shelterin-assisted invasion at telomeres, and replication-coupled strand displacement at the mitochondrial DNA origin. They serve as indispensable intermediates in double-strand break repair, telomere maintenance, and mitochondrial DNA replication. Recent cryo-electron microscopy studies have resolved the stepwise RAD51-mediated strand exchange mechanism at near-atomic resolution, substantially advancing structural understanding of D-loop biogenesis. Despite their differences in molecular composition, both structures remodel Watson-Crick base pairing and, when dysregulated, are associated with replication fork stalling, transcription-replication conflicts, and aberrant recombination. This review systematically compares the structural features, formation mechanisms, regulatory networks, and biological functions of R-loops and D-loops, with emphasis on their convergent roles in safeguarding genome integrity. We further discuss rapidly evolving detection technologies and emerging therapeutic strategies targeting these structures in cancer and neurodegeneration, identifying key unresolved questions for future investigation.

Genomic Instability↗
PubMed1996

The stability of nucleosomes at the replication fork.

Purified simian virus (SV40) minichromosomes were photoreacted with psoralen under various conditions that moderately destabilize nucleosomes. This assay allows indirect distinction between stable nucleosomes, partially unravelled nucleosomes and nucleosomes containing (or lacking) histone H1. In replicating molecules the passage of the replication machinery destabilizes the nucleosomal organization of the chromatin fiber over a distance of 650 to 1100 bp. In front of the fork, an average of two nucleosomes are destabilized presumably by the dissociation of histone H1 and the advancing replication machinery. On daughter strands, the first nucleosome is detected at a distance of about 260 nucleotides from the elongation point. This nucleosome is interpreted to contain no histone H1, while no stepwise association of (H3-H4)2 tetramers with H2A/H2B dimers on nascent DNA can be detected in vivo. The second nucleosome after the replication fork appears to contain histone H1. The prolonged nuclease sensitivity of newly replicated chromatin described in the literature therefore may not be due to a slow reassociation of histone H1.

Animals↗
PubMed1995

Differential DNA secondary structure-mediated deletion mutation in the leading and lagging strands.

The frequencies of deletion of short sequences (mutation inserts) inserted into the chloramphenicol acetyl-transferase (CAT) gene were measured for pBR325 and pBR523, in which the orientation of the CAT gene was reversed, in Escherichia coli. Reversal of the CAT gene changes the relationship between the transcribed strand and the leading and lagging strands of the DNA replication fork in pBR325-based plasmids. Deletion of these mutation inserts may be mediated by slipped misalignment during DNA replication. Symmetrical sequences, in which the same potential DNA structural misalignment can form in both the leading and lagging strands, exhibited an approximately twofold difference in the deletion frequencies upon reversal of the CAT gene. Sequences that contained an inverted repeat that was asymmetric with respect to flanking direct repeats were designed. With asymmetric mutation inserts, different misaligned structural intermediates could form in the leading and lagging strands, depending on the orientation of the insert and/or of the CAT gene. When slippage could be stabilized by a hairpin in the lagging strand, thereby forming a three-way junction, deletion occurred by up to 50-fold more frequently than when this structure formed in the leading strand. These results support the model that slipped misalignment involving DNA secondary structure occurs preferentially in the lagging strand during DNA replication.

Base Sequence↗
PubMed1995

Purification and properties of human DNA helicase VI.

A novel ATP-dependent DNA unwinding enzyme, called human DNA helicase VI (HDH VI), was purified to apparent homogeneity from HeLa cells and characterized. From 327 g of cultured cells, 0.44 mg of pure enzyme was recovered, free of DNA polymerase, ligase, topoisomerase, nicking and nuclease activities. The enzyme behaves as a monomer having an M(r) of 128 kDa, whether determined with SDS-PAGE, or in native conditions. Photoaffinity labelling with [alpha-32P]ATP labelled the 128 kDa protein. Only ATP or dATP hydrolysis supports the unwinding activity for which a divalent cation (Mg2+ > Mn2+) is required. HDH VI unwinds exclusively DNA duplexes with an annealed portion < 32 bp and prefers a replication fork-like structure of the substrate. It cannot unwind blunt-end duplexes and is inactive also on DNA-RNA or RNA-RNA hybrids. HDH VI unwinds DNA unidirectionally by moving in the 3' to 5' direction along the bound strand.

Adenosine Triphosphate↗
PubMed1990

Patterns of strongly protein-associated simian virus 40 DNA replication intermediates resulting from exposures to specific topoisomerase poisons.

Exposure of infected CV-1 cells to specific type I and type II topoisomerase poisons caused strong protein association with distinct subsets of simian virus 40 (SV40) DNA replication intermediates. On the basis of the known specificity and mechanisms of action of these drugs, the proteins involved are assumed to be the respective topoisomerases. Camptothecin, a topoisomerase I poison, caused strong protein association with form II (relaxed circular) and form III (linear) viral genomes and replication intermediates having broken DNA replication forks but not with form I (superhelical) viral DNA or normal late replication intermediates which were present. In contrast, type II topoisomerase poisons caused completely replicated forms and late viral replication forms to be tightly bound to protein--some to a greater extent than others. Different type II topoisomerase inhibitors caused distinctive patterns of protein association with the replication intermediates present. Both intercalating and nonintercalating type II topoisomerase poisons caused a small amount of form I (superhelical) SV40 DNA to be protein-associated in vivo. The protein complex with form I viral DNA was entirely drug-dependent and strong, but apparently noncovalent. The protein associated with form I DNA may represent a drug-stabilized "topological complex" between type II topoisomerase and SV40 DNA.

Animals↗
PubMed1975

The deoxyribonucleic acid unwinding protein of Escherichia coli. Properties and functions in replication.

The DNA unwinding protein of Escherichia coli (Sigal, N., Delius, H., Kornberg, T., Gefter, M., and Alberts, B. (1972) Proc. Nat. Acad. Sci. U.S.A. 69, 3537-3541) has been purified to homogeneity by a simple procedure which utilizes its stability to heating. The protein is an asymmetric tetramer of 18,500 dalton subunits which binds preferentially to single-stranded DNA at a ratio of one protein molecule per 32 nucleotides. Binding to DNA is complete in less than 10 s at 0 degrees while release of the protein from single-stranded DNA is relatively slow even at 37 degrees. A simple functional assay for unwinding protein depends on its essential role in the conversion of phage G4 single-stranded DNA to the replicative form. Unwinding protein stimulates initiation of replication of all single-stranded phage DNAs. Approximately 300 copies of unwinding protein are present per cell, as estimated by antibody titration, an amount sufficient to cover substantial lengths of DNA in several replicating forks.

Bacterial Proteins↗
PubMed1994

Mitochondrial topoisomerase II activity is essential for kinetoplast DNA minicircle segregation.

Etoposide, a nonintercalating antitumor drug, is a potent inhibitor of topoisomerase II activity. When Trypanosoma equiperdum is treated with etoposide, cleavable complexes are stabilized between topoisomerase II and kinetoplast DNA minicircles, a component of trypanosome mitochondrial DNA (T. A. Shapiro, V. A. Klein, and P. T. Englund, J. Biol. Chem. 264:4173-4178, 1989). Etoposide also promotes the time-dependent accumulation of small minicircle catenanes. These catenanes are radiolabeled in vivo with [3H]thymidine. Dimers are most abundant, but novel structures containing up to five noncovalently closed minicircles are detectable. Analysis by two-dimensional gel electrophoresis and electron microscopy indicates that dimers joined by up to six interlocks are late replication intermediates that accumulate when topoisomerase II activity is blocked. The requirement for topoisomerase II is particularly interesting because minicircles do not share the features postulated to make this enzyme essential in other systems: for minicircles, the replication fork is unidirectional, access to the DNA is not blocked by nucleosomes, and daughter circles are extensively nicked and (or) gapped.

Animals↗
PubMed2026

Cohesin promotes genomic stability by suppressing unequal sister chromatid exchange.

The protein complex cohesin plays critical roles in genomic stability by tethering together sister chromatids at their pericentric regions and along their arms from S phase until anaphase. Cohesin-mediated pericentric cohesion prevents aneuploidy by ensuring bipolar attachment of sister kinetochores. Arm cohesion prevents loss of heterozygosity by biasing DNA repair via recombination between sister chromatids rather than between homologs. Here, we investigate in yeast whether cohesin also enhances genomic stability by suppressing unequal sister chromatid exchange (USCE) between repetitive sequences. In wild-type cells, the USCE rate between repeats 4kb apart (proximal) was 15X higher than repeats 68kb apart (distal). The USCE between distal repeats but not proximal repeats increased 4 to 7-fold in mutants with altered cohesin subunits or auxiliary factors. The level of increased distal USCE corresponded with reduced arm cohesion, reduced density of cohesion arm sites, and higher sister loci mobility. Our results suggest that high density of arm cohesion sites confines repair of DNA damage to local sequences. When the density of cohesion sites decreases, sister chromatid sequences are less confined, thereby enhancing distal repeat interactions and USCE. Another set of mutations disrupted both DNA replication and cohesin loading at the replication fork during S phase. Remarkably, distal USCE in these mutants increased approximately 100-fold and was 6-fold more likely than proximal USCE. This preferential hyperdistal USCE can be explained by an aberrant sister-chromatid structure that is normally prevented by proper coupling of cohesin function and replication.

Journal Article↗
PubMed1989

Lack of positional requirements for autonomously replicating sequence elements on artificial yeast chromosomes.

In the yeast Saccharomyces cerevisiae, origins of replication (autonomously replicating sequences; ARSs), centromeres, and telomeres have been isolated and characterized. The identification of these structures allows the construction of artificial chromosomes in which the architecture of eukaryotic chromosomes may be studied. A common feature of most, and possibly all, natural yeast chromosomes is that they have an ARS within 2 kilobases of their physical ends. To study the effects of such telomeric ARSs on chromosome maintenance, we introduced artificial chromosomes of approximately 15 and 60 kilobases into yeast cells and analyzed the requirements for telomeric ARSs and the effects of ARS-free chromosomal arms on the stability of these molecules. We find that terminal blocks of telomeric repeats are sufficient to be recognized as telomeres. Moreover, artificial chromosomes containing telomere-associated Y' sequences and telomeric ARSs were no more stable during both mitosis and meiosis than artificial chromosomes lacking terminal ARSs, indicating that yeast-specific blocks of telomeric sequences are the only cis-acting requirement for a functional telomere during both mitotic growth and meiosis. The results also show that there is no requirement for an origin of replication on each arm of the artificial chromosomes, indicating that a replication fork may efficiently move through a functional centromere region.

Blotting, Southern↗
PubMed1988

Deposition of newly synthesized histones: hybrid nucleosomes are not tandemly arranged on daughter DNA strands.

Density labeling procedures have been utilized to study the dynamics of histone-histone interactions in vivo. Cells were labeled for 60 min with dense amino acids, and the label was chased for up to 22 h (two replication events for these cells). Nuclei were isolated and treated with formaldehyde to stabilize the histone-histone interactions with a covalent cross-link that produces an octameric complex of two each of H3, H2B, H2A, and H4. This complex was then extracted from the DNA and analyzed on density gradients. The results indicate that new H3,H4 deposits as a tetramer and does not dissociate in the subsequent chases. New H2A,H2B deposited as a dimer and also does not dissociate in subsequent chases. These new histones form hybrid octamers with old histones. On the basis of the new:old ratio in the hybrid octamers, we propose that additional old H2A,H2B from elsewhere in the genome interacts with tetramers of new H3,H4 to form the newly synthesized nucleosomes. It is also observed that 5% of the cross-linked complexes produced by formaldehyde are octamer-octamer (dioctamer). Upon analysis of the density of the dioctamer, the hybrid octamers were found adjacent to octamers that were homogeneous with respect to containing normal density histones. Control experiments are presented to demonstrate that the octamer-octamer cross-links are a product of intrastrand and not interstrand interactions between nucleosomes. These same control experiments also indicate that these procedures do not induce histone exchange during the preparative procedure prior to density gradient analysis. The significance of these results with regard to the dynamics of histone-histone interactions at the replication fork and the potential role in the maintenance of differentiation is discussed.

Animals↗