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PubMed1977

Shear degradation of DNA.

A concentric-cylinder flow-birefringence instrument is used to generate sufficient shear fields to break T2 DNA (M = 1.2 X 10(8)) and E. coli DNA (M = 2.5 X 10(9)) in dilute solution. Breakage is monitored in situ by measuring the change in birefringence relaxation after the flow has been stopped. The breakage of T2 DNA follows first-order kinetics. Rate constants are obtained as functions of shear rate and viscosity (varied by adding glycerol). The data are fitted by a modified Arrhenius equation, assuming that stess increases the rate by lowering the activation energy. The rate increases with temperature, pH, and water concentration, and appears to be a base-catalyzed hydrolysis of the phosphate-ester linkage. La3+ ions catalyze the reaction. E. coli DNA was reduced to half molecules at a shear stress of 0.4 dynes/cm2, which is about 2500 times less than that required for T2. The difference in rates is accounted for in part by the difference in size of the two, but may also reflect the presence of many single-strand nicks in the coli DNA.

Birefringence↗
PubMed1976

Repair of x-ray-induced single strand breaks in toluenized Escherichia coli cells.

We have used sedimentation in alkali to estimate the repair of X-ray-induced single strand breaks in the DNA of irradiated toluenized Escherichia coli cells. Extensive repair requires no exogenous cofactors except ATP although other individual NTPs (except U) or dNTPs can substitute for ATP. There is no repair in polA or resA cells and since nicotinamide mononucleotide (NMN) inhibits repair in wild type cells we interpret the results as indicating that both ligase and polymerase I are needed for repair but that the amount of any gap filling is small and extensive repair replication is not necessary.

Adenosine Triphosphate↗
PubMed1976

Mechanisms and selectivity of anthracycline aminoglycosides and other intercalating agents.

Many of the antiproliferative actions of daunorubicin and adriamycin are attributable to the results of their interaction with DNA: single and double strand breaks and inhibition of repair and of nucleic acid synthesis. Like other DNA-reactive agents, high cumulative doses produce pharmacological actions and species-specific cytotoxicities in non-proliferating myocardium and nerve cells. Evidence is reviewed suggesting that many, if not all, intercalating agents are membrane-reactive. Thus it seems likely that the selective nature of the responses by various cells and the discrete expressions of cytotoxicity may be determined by binding to specific receptors in membranes of target cells in susceptible organs and species.

Adenosine Triphosphatases↗
PubMed2025

Moss BRCA2 lacking the canonical DNA-binding domain promotes homologous recombination and binds to DNA.

BRCA2 is crucial for mediating homology-directed DNA repair (HDR) through its binding to single-stranded DNA (ssDNA) and the recombinases RAD51 and DMC1. Most BRCA2 orthologs have a canonical DNA-binding domain (DBD) with the exception of Drosophila melanogaster. It remains unclear whether such a noncanonical BRCA2 variant without DBD possesses a DNA-binding activity. Here, we identify a new noncanonical BRCA2 in the model plant Physcomitrium patens (PpBRCA2). We establish that PpBRCA2 is essential for genome integrity maintenance, somatic DNA double-strand break (DSB) repair, HDR-mediated gene targeting, and RAD51 foci recruitment at DNA break sites. PpBRCA2 is also critical for DSB repair during meiosis. Interestingly, PpBRCA2 interacts strongly with RAD51 but weakly with DMC1, suggesting a distinct meiotic function compared to other BRCA2 homologs. Despite lacking the canonical DBD, PpBRCA2 binds ssDNA through its disordered N-terminal region and efficiently promotes HDR. Our work highlights that the ssDNA binding capacity of BRCA2 homologs is conserved regardless of the presence of a canonical DBD and provides a deeper understanding of BRCA2's functional diversity across species.

BRCA2 Protein↗
PubMed1976

Naturally occurring cross-links in yeast chromosomal DNA.

Chromosome-size yeast DNA molecules with a number average molecular weight (Mn) of 3-4 X 10(8) were isolated from sucrose gradients after sedimentation of lysed yeast spheroplasts. Resedimentation showed that the molecules were isolated without introducing appreciable single-strand or double-strand breaks. The presence of cross-links in these molecules was suggested by the observation that the apparent Mn in alkali was greater than expected for separated single strands. Since cross-linked molecules would have strands which fail to separate upon denaturation, this was tested more directly. Neutralization of alkaline denaturing conditions resulted in up to 70% of the intact molecules rapidly reforming duplex structures, as shown by equilibrium banding in CsCI. Experiments with larger E. coli DNA molecules (Mn = 5.2 X 10(8)) indicated that the conditions used were sufficient to denature completely molecules of this size. Results of enzyme treatments suggest that the cross-links are not RNA or protein. Experiments with density-labeled yeast DNA molecules showed that the rapid reformation of duplex DNA is not the consequence either of a bimolecular reaction between separated DNA strands or of intrastrand renaturation. The data indicate that when the yeast DNA molecules are completely denatured, the strands fail to separate. Hence they must be cross-linked. Experiments with sheared DNA show that there are small number of cross-links, one to four, permolecule.

Centrifugation, Density Gradient↗
PubMed2026

Recurrent patterns of TOP1-mediated neuronal genomic damage shared by major neurodegenerative disorders.

Amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease (AD) represent two major categories of neurodegenerative disorders-TAR DNA-binding protein 43 (TDP-43) and tau proteinopathies-for which the mechanisms driving neuronal death remain unclear. Single-cell whole-genome sequencing of 469 neurons from C9ORF72 ALS, C9ORF72 FTD, AD, and control brains revealed increased somatic single-nucleotide variants (sSNVs) and insertions/deletions (sIndels) in all three diseases. Mutational signature analysis identified a disease-associated sSNV signature consistent with oxidative damage and an sIndel process affecting 22% of ALS, 76% of FTD, and 61% of AD neurons-but only 2% of control neurons-resembling signature ID4, previously linked to topoisomerase 1 (TOP1)-mediated mutagenesis. Rapid approach to DNA adduct recovery (RADAR) assays confirmed increased TOP1-DNA covalent complexes, and duplex sequencing confirmed the increased sIndels and identified single-strand events as likely precursor lesions. TOP1-associated sIndel mutagenesis and genome instability thus represent a mechanism shared by both TDP-43 and tau neurodegeneration.

Humans↗
PubMed2026

Linker Histone H1 Phosphorylation Promotes DNA Damage Repair during Replication Stress.

DNA replication fidelity depends on the integrity of the replication fork to prevent DNA damage and preserve genome stability. Disruptions to this process can trigger replication stress, leading to the accumulation of single-strand DNA (ssDNA) and double-strand breaks (DSBs), which drive mutagenesis and ultimately contribute to disease. While the roles of core histones and their post-translational modifications in this context have been more well-studied, far less is known about how linker histones regulate the replication stress response. Here, we demonstrate that the S-phase-phosphorylated form of the linker histone H1 (pH1) plays a key role in DNA damage repair at collapsed replication forks, both in vitro and in cells. Using phosphomimic and phosphonull H1 mutants, we show that phosphorylation enhances H1 assembly with ssDNA. Utilizing intein chemistry for the site-specific incorporation of a photocrosslinker to the C-terminus of H1, we map the direct interactors of H1. We identify phosphorylated H1 at replication forks, where it engages replication machinery and DNA damage response factors, including Histone PARylation Factor 1 (HPF1). We further demonstrate that ssDNA induces pH1-HPF1 interactions that promote liquid-like assemblies, correlating with reduced DNA damage and histone PARylation. Consistent with this role, reduction of total H1 increases cellular sensitivity to DNA damage, a phenotype that is partially rescued by reintroduction of H1.4. Together, these findings establish pH1 as a new regulator of DNA damage repair at collapsed replication forks through the controlled sequestration of repair factors.

Journal Article↗
PubMed1978

Bleomycin-specific fragmentation of double-stranded DNA.

Brief exposure of covalently closed circular duplex PM2 DNA to low concentrations of the clinical bleomycin mixture (Blenoxane) resulted in specific fragmentation of the genome that does not depend on the presence of superhelical turns. The double-strand breaks are in fact produced at several discrete sites on the PM2 genome but frequently occurring near the HpaII restriction endonuclease cleavage site. Initial rates of formation of nicked circular and linear duplex PM2 DNAs are reduced to different extents as the ionic strength of the reaction is increased. Increasing ionic strength is most effective in reducing the initial rate and overall yield of apparent double-strand scissions compared with single-strand scissions in the bleomycin-treated PM2 DNA.

Bacteriophages↗
PubMed1977

Characterization of DNA strand breakage in vitro by the antitumor protein neocarzinostatin.

The antitumor protein antibiotic neocarzinostatin causes strand scission of DNA in vitro in the presence of a sulfhydryl compound. The breaks are single stranded in nature and bear 5'-phosphoryl termini. All four deoxymononucleotides are recoverable at the 5'-ends of the cleavage sites although a higher proportion of dGMP and TMP are consistently found. The lesions are not repairable with polynucleotide ligase from Escherichia coli. A quantitative assay was developed to determine the pH profile and time course of the reaction. Data from protection experiments with synthetic and natural DNAs indicate the requirement for thymidylic acid and deoxyadenylic acid in the DNA for cutting. In DNA-RNA hybrids, riboadenylic acid can substitute for deoxyadenylic acid, whereas ribouridylic acid cannot substitute for thymidylic acid. Release of thymine is detected, and the amount of release correlates well with the number of strand scissions.

Antibiotics, Antineoplastic↗
PubMed2026

BCDX2-CX3 and DX2-CX3 complexes assemble and stabilize RAD51 filaments.

The repair of DNA double-strand breaks by homologous recombination is essential for genomic integrity, and its dysregulation is a hallmark of cancer1. Central to homologous recombination is the RAD51 recombinase, whose assembly into a nucleoprotein filament is governed by five RAD51 paralogues (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3)2. Mutations in any of these proteins predispose individuals to multiple cancers or genetic disorders3-6. These paralogues are thought to form two functionally separate complexes RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2) and RAD51C-XRCC3 (CX3), that act independently at different stages of homologous recombination7-11. Here we demonstrate that all five paralogues can assemble into a single, ATP-dependent BCDX2-CX3-RAD51 supercomplex. The architecture of this assembly bound to single-stranded DNA reveals a contiguous filament where the CX3 module stacks atop BCDX2, creating a protofilament template for RAD51 filament formation. We further identify a novel, RAD51B-independent DX2-CX3 complex (RAD51D-XRCC2-RAD51C-XRCC3) functioning as a stable RAD51 anchor on single-stranded DNA, and we capture it in multiple states, including capping RAD51 filament segment. These distinct assemblies are differentially regulated by ATPase activity, defining a dynamic BCDX2-CX3 'loader' and a stable DX2-CX3 'anchor' that provide functional modularity to the homologous recombination machinery. This work provides a unifying mechanism for human RAD51 paralogue function and delivers an atomic blueprint for interpreting disease-causing mutations.

Rad51 Recombinase↗
PubMed1977

Effect of camptothecin and adriamycin on bleomycin-induced tritiated thymidine triphosphate incorporation in a rat nuclear system.

We investigated the effect of camptothecin and adriamycin on [3H]TTP incorporation and bleomycin-stimulated [3H]TTP incorporation in host liver and hepatoma nuclei of rats. Camptothecin neither stimulated nor inhibited incorporation in the regular nuclear incorporating system. Bleomycin stimulated incorporation to a much greater extent in host liver nuclei and slow-growing hepatomas than it did in the fast-growing hepatoma 7777. Addition of camptothecin to bleomycin stimulated incorporation of [3H]TTP even further. This camptothecin stimulation was slightly greater in hepatoma nuclei than it was in host liver nuclei. Adriamycin inhibited [3H]TTP incorporation in the regular system as well as the bleomycin-induced incorporation. Hepatoma nuclei were more sensitive to this inhibition than were host liver nuclei. Sucrose density gradients indicated that camptothecin caused DNA strand scissions in addition to those produced by bleomycin. Camptothecin alone produced some single-strand but no double-strand scissions. The action of bleomycin was dependent on sulfhydryl-reducing agents. Camptothecin could partially substitute for this requirement. Adriamycin did not produce DNA breaks as determined by neutral or alkaline sucrose density gradients. Despite complete inhibition of bleomycin-induced [3H]TTP incorporation, adriamycin did not prevent bleomycin-induced DNA breaks. The inhibitory effect of adriamycin might have been on the repair system.

Animals↗
PubMed1975

Studies on deoxyribonucleases from Saccharomyces cerevisiae. Characterization of two endonuclease activities with a preference for double-stranded DNA.

Two new endonuclease activities, endonuclease B and endonuclease C, obtained from yeast nuclear preparations have been separated and partially characterized. Endonuclease B has a primary requirement for Mn2+ which cannot be replaced by Mg2+ or Ca2+, and makes single-strand scissions in double-stranded DNA. Endonculease C is activated by either Mn2+ or Mg2+, and makes single-strand scissions with Mg2+, while with Mn2+, scissions are made which result in double-strand breaks. Neither enzyme is active on denatured DNA, and both are inhibited by yeast RNA. Both enzymes exhibit pH optima at pH 5.0 and PH 7.2, and leave 5'-phosphoryl termini.

Cell Nucleus↗
PubMed2026

The dynamics of RAD51 foci formation and elongation in living human cells.

Homologous recombination is a DNA repair process that requires binding of RAD51 to ssDNA at the break site. This facilitates the search for a homologous repair template on the sister chromatid, or on the homologous chromosome. How broken DNA ends loaded with RAD51 filaments are brought toward their repair template in the crowded 3D genome is currently poorly understood. This is largely due to a lack of tools to visualize homology search in living human cells. Here, we show that RAD51 and MND1, two proteins operating in homology search, become visible in long, extended structures several hours after double-stranded break formation. Using GFP-MND1 we capture these elongated foci in living human cells and reveal their highly dynamic nature as they traverse the nuclear space and gradually disassemble. We show that resolution of these structures depends on RAD54L, known for its role in RAD51-driven homology search. In addition, we find that loss of cohesin inhibits their resolution, in accordance with a role for cohesin in homology search. Thus, our data suggest that these elongated foci are visible intermediates of an active DNA repair process, and that GFP-MND1 is a powerful tool to study the dynamics of homology search in living human cells.

Humans↗
PubMed2025

Targeting RAD52 overcomes PARP inhibitor resistance in preclinical Brca2-deficient ovarian cancer model.

BRCA-mutated ovarian cancer commonly develops resistance to poly (ADP-ribose) polymerase (PARP) inhibitors. Here, we investigated the DNA repair protein RAD52 as a potential target to overcome resistance. In analysis of The Cancer Genome Atlas datasets and immunohistochemistry of tissue microarrays, elevated RAD52 expression correlated with poor overall survival in patients with high-grade serous ovarian cancers. We tested two PARP inhibitor-resistant Brca2-deficient mouse ovarian cancer models, ID8-OR and HGS2-OR. HGS2-OR cells had higher RAD52 expression than parental lines. Rad52 knockout or knockdown restored PARP inhibitor sensitivity in both models. In syngeneic mice, ID8-OR cells in which Rad52 was knocked out yielded lower tumor burden and longer overall survival than control cells. Rad52 depletion impaired single-strand annealing and homologous recombination and led to accumulation of DNA double-strand breaks after PARP inhibitor treatment. RNA sequencing demonstrated that PARP inhibitor treatment induced Polq expression in Brca2- and Rad52-deficient cells, suggesting a switch to microhomology-mediated end joining. Finally, the RAD52 inhibitor D-I03 synergized with a PARP inhibitor to reduce cell viability and tumor burden and prolong survival. Collectively, our findings establish RAD52 as a promising therapeutic target to overcome PARP inhibitor resistance in BRCA2-mutated ovarian cancer and offer mechanistic insights to inform future clinical strategies.

Journal Article↗
PubMed1977

Possible in vitro repair of viral RNA by ligase-like enzyme(s) in poliovirus-infected cells.

A soluble polymerase-template complex prepared from poliovirus-infected cells was found to incorporate radioactive UTP into trichloroacetic acid-insoluble RNA linearly for 8 h in the presence of ATP and Mg2+. Radioactive CTP or GTP was not incorporated under identical conditions. Nearest-neighbor analysis of the in vitro product demonstrated that ATP was added to the viral RNA in the form of polyadenylic acid; UTP was added internally to the 3'-OH group of all four nucelotides. The data can best be explained by the addition of the UTP to the 3'-OH groups of single-stranded breaks in the double-stranded viral RNA and ligation to the adjacent 5'-phosphate groups. The enzymatic activity was also found in encephalomyocarditis virus- and rhinovirus type 1A-infected cells but not in uninfected cells.

Adenosine Triphosphate↗
PubMed1976

Fast neutron and x-ray induced single strand DNA breaks in cultured mammalian cells.

The relative biologic effectiveness (RBE) of fast neutrons in the production of single strand DNA breaks is 1.6 as compared to that of 250 kVp x rays. Monolayers of L-929 cells were treated with dinitrophenol during irradiation to prevent the DNA strands from rejoining; the extent of DNA damage was measured by the alkaline sucrose sedimentation method. The RBE for DNA damage is essentially the same as the RBE measured by cell survival methods.

Animals↗
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↗