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At least 19 recordsLinked to original sources
PubMed2026

Single-molecule tracking of RNA-DNA hybrid removal enzymes important for lagging-strand replication.

The formation of RNA-DNA hybrid (RDH) primers by primase is an essential step in the recruitment of DNA polymerase during replication initiation and for the synthesis of each Okazaki fragment on the lagging strand. In addition to primers, RDHs form through misincorporation of ribonucleotides by DNA polymerase during elongation and by formation of R loops during transcription. R loops are three-stranded structures that form when the nascent mRNA anneals to the template DNA strand, displacing the complementary DNA strand. The persistence of RDHs is deleterious to genome stability in all cells because they increase susceptibility to mutations, impaired replication fork progression, DNA double-stranded breaks, and genomic rearrangements. In many bacteria, it is well established that components of the replicative DNA polymerase form a macromolecular complex that can be imaged using single-molecule or ensemble fluorescence approaches. The spatiotemporal regulation of proteins involved in RDH removal during lagging-strand maturation is less clear. Here, we study three proteins that are involved in the removal of RDHs from the lagging strand during DNA replication in the Gram-positive bacterium Bacillus subtilis: DNA polymerase I (Pol I), FenA, and RNase HIII. We characterized the behavior of each PAmCherry-tagged lagging-strand enzyme in living cells using single-particle tracking photoactivated localization microscopy. We find that all three proteins are highly mobile, suggesting residence times at their target substrates are below our temporal resolution. We also find evidence that Pol I activity is modulated through interaction with the replisome, whereas FenA and RNase HIII are regulated through access to the nucleoid. Our results provide new insight into how enzymes are recruited to resolve RDHs during lagging-strand replication in vivo.

DNA Replication↗
PubMed2025

RNA-DNA hybrid binding domain broadens the editing window of base editors.

Adenine base editors (ABEs) and cytosine base editors (CBEs) are prominent tools for precise genome editing but are hindered by limited editing activity at positions proximal to the protospacer adjacent motif (PAM). This study investigates the potential of enhancing base editors editing activity by fusing them with RNA-DNA hybrid binding domains (RHBDs). Specifically, fusing ABE8e with the RHBD of Homo sapiens RNaseH1 (RHBD1) significantly increased A-to-G editing efficiency in the PAM-proximal region (A9-A15) by up to 3.5-fold, while reducing off-target cytosine editing. Additionally, RHBD1 is compatible with ABEmax, BE4max, and dual base editor (eA&C-BEmax), enhancing their editing activity at the PAM-proximal bases. Notably, RHBD1-fused BE4max led to a 3.1-fold improvement in C-to-T editing efficiency at PAM-proximal region (C9-C12). Furthermore, we demonstrated that RHBD1-fused ABE8e could effectively edit disease-related single nucleotide variations (SNVs) in human cells and validated its efficacy in adult mouse liver. These findings highlight the significance of the RHBD in expanding editing window and the applicability of base editors for gene therapy and disease modeling.

Gene Editing↗
PubMed2025

Rad53 regulates RNase H1, which promotes DNA replication through sites of transcription-replication conflict.

RNA-DNA hybrids and R-loops can lead to extensive DNA damage and loss of genomic integrity if not regulated in a timely manner. Although RNase H1 overexpression is frequently used as a tool to resolve R-loops, the regulation of RNase H1, overexpressed or endogenous, remains poorly characterized. We reveal that in yeast, overexpressed RNase H1 (RNH1) has no effect on gene expression, cell growth, or RNA-DNA hybrid resolution in wild-type cells. Overexpressed RNase H1 does, however, remove RNA-DNA hybrids in mutants where hybrids have become dysregulated. Endogenous RNase H1 becomes up-regulated and chromatin-associated in the absence of Sen1 in a DNA replication checkpoint-dependent manner. Rnh1 gets recruited to genomic loci where RNA-DNA hybrids accumulate following the loss of Sen1. Rnh1, together with Sen1, promotes DNA replication at sites of transcription-replication conflict. Hence, RNase H1, overexpressed or endogenous, responds to unscheduled, stress-inducing RNA-DNA hybrids.

Ribonuclease H↗
PubMed1975

RNA-dependent DNA polymerase activity of RNA tumor viruses. V. Rous sarcoma virus single-stranded RNA-DNA covalent hybrids in infected chicken embryo fibroblast cells.

RNA-DNA covalent hybrids containing viral RNA have been isolated from nuclear fractions of Rous sarcoma virus-infected chicken embryo fibroblast cells shortly after virus infection. The formation of covalent hybrid structures depends upon a functional reverse transcriptase in vivo, since its appearance in cells is temperature dependent when infected with Rous sarcoma virus mutant LA335, which contains a temperature-sensitive reverse transcriptase.

Animals↗
PubMed2025

DDX37 and DDX50 Maintain Genome Stability by Preventing Transcription-dependent R-loop Formation.

R-loops consist of an RNA-DNA hybrid and a displaced single-stranded DNA strand that play a central role in several biological processes. However, as the presence of aberrant R-loops forms a significant threat to genome stability, R-loop formation and resolution is strictly controlled by RNAse H and helicases. In a screening for RNA helicases, previously described as RNA-DNA hybrid interactors, that control genome integrity, we identified for the first time DDX37 and DDX50. Depletion of DDX37 and DDX50 promotes DNA damage, as demonstrated by H2AX phosphorylation and increased comet tail length. In addition, knock down of these RNA helicases decreases the DNA replication track length and leads to RPA focus formation, results that are indicative of replication stress. Downregulation of DDX37 and DDX50 triggers an increase in RNA-DNA hybrids, that can be reverted by the overexpression of RNase H1. Interestingly, inhibition of transcription prevented the increased RNA-DNA hybrid formation and DNA damage upon DDX37 or DDX50 depletion. Together these results demonstrate that DDX37 and DDX50 are important for resolving RNA-DNA hybrids appearing during transcription and thereby preventing DNA damage by replication stress.

DEAD-box RNA Helicases↗
PubMed1976

Purification of adenovirus messenger ribonucleic acid by an aqueous polymer two-phase system.

An aqueous polymer phase system containing 6.3% (w/w) dextran and 3.5% (w/w) poly(ethylene glycol) in 10 mM phosphate buffer (pH 8.0) was developed to select RNA-DNA hybrids from unhybridized RNA. The top phase of this phase system, which contains DNA and the RNA-DNA hybrids, can be used to purify adenovirus messenger RNA both early and late in the infectious cycle. The hybrids can be melted by heat in the top phase and the messenger RNA selected by oligo(dT)cellulose chromatography whereupon the polymers and the DNA percolate and the polyadenylated messenger RNA absorb to the column. The isolated messenger RNA appears to be almost quantitatively recovered at a purity from 70 to 90% depending on the concentration of the specific messenger RNA in the starting material. Early and late viral messenger RNA were selected on the complementary strands of adenovirus DNA according to this procedure.

Adenoviruses, Human↗
PubMed1978

Ribonuclease H from rat liver. II. Partial purification and characterization of cytosol ribonuclease H1.

We have detected in rat liver cytosol three enzymes (termed C-1, C-2, and C-3) which cleaved the RNA moiety of RNA-DNA hybrid. These enzymes were separated from each other by DEAE-Sephadex and Sephadex G-200 chromatography. C-1 and C-2 specifically act on the RNA moiety of RNA-DNA hybrid, while C-3 degrades single-stranded RNA as well as the RNA of the hybrid. The molecular weights of C-1, C-2, and C-3 are about 110,000, 35,000 and 110,000 daltons, respectively, and their activities are absolutely dependent on divalent cations such as Mg2+ and Mn2+. Cleavage by C-1 and C-2 is endonucleolytic, producing mostly oligonucleotides and a small amount of mononucleotides which possess 3'-hydroxyl termini. It seems likely that C-2 is originally present in the nucleus and is released into cytosol because of its loose binding to the nuclear components. As for biochemical properties, C-1 is very similar to the cytosol ribonuclease H initially reported by Roewekamp and Sekeris, and C-2 is very similar to the nuclear ribonuclease H reported by us in the preceding paper.

Animals↗
PubMed1979

Posttranscriptional regulation of glucocorticoid-regulated functions.

Relying heavily on studies of TAT regulation in cultured rat hepatoma cell lines, we have attempted in this brief review to discuss possible mechanisms for posttranscriptional regulation of glucocorticoid-sensitive enzymes and to chronicle the evidence for and against posttranscriptional mechanisms for specific enzyme induction by glucocorticoids. Initially, mechanisms were considered that would reconcile results showing sensitivity of both induction and deinduction of TAT to inhibitors of RNA synthesis with studies demonstrating first that glucocorticoids regulate the rates of specific enzyme synthesis and, then, that glucocorticoids regulate levels of enzyme-specific mRNA. Such reconciliation proved unnecessary when it was demonstrated that inhibitors of RNA synthesis such as actinomycin D were not specific for RNA synthesis, but also had effects on mRNA turnover and protein metabolism. The bulk of evidence to date establishes that glucocorticoids promote the production of enzyme-specific mRNA for the proteins whose synthesis is regulated by thses steroids. Nevertheless, there is still very little direct evidence that steroids can modulate rates of specific gene transcription. The glucocorticoid stimulation of mouse mammary tumor virus RNA production in cultured cell lines is the only example to date where such a mechanism is supported by RNA-DNA hybridization studies. Posttranscriptional actions of steroids on the turnover, processing, or extranuclear transport of specific mRNA precursors remain potential steps at which glucocorticoids might function. The rapid turnover of some glucocorticoid-regulated enzymes and their mRNAs not only ensures a rapid response to steroid addition or withdrawal, but also subjects these proteins to relatively large fluctuations upon alterations in overall protein or mRNA metabolism. Thus many of the inductions and repressions of hepatic TAT and TO by mediators other than the glucocorticoids may be attributable entirely to nonspecific mechanisms.

Animals↗
PubMed2025

Spatial Mapping and Interactome Profiling of m6A-Modified R-Loops via Chemically Inducible Split-APEX2 Proximity Labeling.

m6A-Modified R-loops (m6A-R-loops) play crucial roles in epigenetic regulation and genome stability, yet resolving their spatial distribution and protein interactomes in live cells remains challenging. To address this, we developed m6A-R-loop proximity labeling (m6A-RLPL), a chemically inducible split-APEX2 proximity labeling technology integrating dual-target recognition using the RNA-DNA hybrid binding domain of RNase H1 for R-loop targeting and m6A reader protein's YTH domain for m6A recognition, coupled with an abscisic acid (ABA)-inducible dimerization system for signal amplification. This technology revealed host m6A-R-loops enriched with nucleoli under normal conditions. When applied to herpes simplex virus (HSV) infection, it further demonstrated viral m6A-R-loops undergoing dramatic accumulation within phase-separated granules in replication compartments during late-stage infection. Proximity proteomics identified ZC3H4 and CCDC124 as essential regulators maintaining these structures, which serve as transcription sites for HSV late genes, with disruption selectively impairing viral transcription. m6A-RLPL establishes a generalizable approach for spatially resolved profiling of m6A-R-loop interactomes and organizational dynamics in living systems.

Humans↗
PubMed2026

RAD54L coordinates the nucleolar DNA damage response to maintain rDNA stability.

The nucleolus is organized around actively transcribed ribosomal RNA genes (rDNA), where high RNA polymerase I (Pol I) activity creates intrinsic susceptibility to replication stress and DNA damage. Here, we identify the DNA translocase RAD54L as a critical regulator of the nucleolar DNA damage response (nDDR) to rDNA double-strand breaks (DSBs) and replication stress. We show that RAD54L localizes to the nucleolus under basal conditions and is recruited to nucleolar caps following CRISPR-Cas9-induced rDNA-DSBs to promote repair. RAD54L loss results in persistent RAD51 foci, increased nucleolar γH2AX, and micronuclei formation, indicating defective resolution of rDNA lesions and genome instability. Under baseline conditions and replication stress induced by the Pol I transcription inhibitor CX-5461, RAD54L limits the accumulation of ssDNA and coordinates nDDR signaling. We further show that rDNA-DSBs induce RNA polymerase II-dependent RNA-DNA hybrids (R-loops) at intergenic rDNA regions, which facilitate nucleolar reorganization and cap formation and repair factor recruitment. Together, these findings establish RAD54L as a key regulator that coordinates replication stress response and rDNA repair, maintaining rDNA stability and genome integrity.

DNA, Ribosomal↗
PubMed1975

Deoxyribonucleic acid polymerases of Euglena gracilis. Primer-template utilization of and enzyme activities associated with the two deoxyribonucleic acid polymerases of high molecular weight.

The two high-molecular-weight DNA polymerases from Euglena gracilis, pol A (mol. wt. 190 000) and pol B (mol. wt. 240 000), were differentiated on the basis of associated enzymic activities and primer-template utilization. Neither enzyme had endodeoxyribonuclease activity, but pol B, like pol B of yeast and the corresponding enzyme from Tetrahymena pyriformis, exhibited at least one other nuclease activity directed against denatured DNA and the RNA of an RNA-DNA hybrid. These nuclease functions preferred an alkaline pH and Mg2+. Pol B also exhibited nucleoside diphosphokinase activity. Both enzymes were active with 'activated' DNA and poly[d(A-T)] as primer-templates and were sensitive, especially pol B, to inhibition by excess of native or heat-denatured DNA. Pol B also utilized oligo[d(T)] and poly(A) templates under certain conditions, whereas pol A exhibited only slight activity with poly[d(A)]. (U)6 was not used as a primer by either enzyme.

Cell-Free System↗
PubMed1978

Ribonuclease H from rat liver. I. Partial purification and characterization of nuclear ribonuclease H1.

A ribonuclease H, an enzyme that specifically degrades the RNA moiety of RNA-DNA hybrid, has been partially purified from rat liver nuclei and characterized. Neither native or denatured DNA, nor single or double-stranded synthetic polyribonucleotides were degraded by the enzyme. The enzyme possesses a molecular weight of about 36,000 and requires alkaline pH, magnesium ions, and ammonium sulphate for maximum activity. The enzyme acts on the hybrid as an endonuclease, resulting in oligonucleotides with 3'-hydroxyl termini. The properties of this enzyme were distinct from those of the rat liver cytosol enzyme reported by Roewekamp and Sekeris in many respects, such as molecular weight, optimal pH and requirements for divalent cations. Preliminary experiments suggest that the nuclear enzyme is localized in the nucleoplasm and nucleoli. These results indicate that multiple forms of ribonuclease H exist in different regions of rat liver cells.

Animals↗
PubMed1979

Multiple forms of ribonuclease H from rat liver cytosol.

Three forms (termed I, II, and III) of ribonuclease H (RNase H) [EC 3.1.4.34] activity are present in rat liver cytosol. These enzymes degrade RNA specifically in RNA-DNA hybrid structures. They were eluted at 0 M, 0.25 M, and 0.5 M KCl in phosphocellulose chromatography, and were further purified by using blue Sepharose. They are further distinguished from one another by their ionic requirements, optimal pH, molecular weights, sedimentation coefficients, and sensitivity to the -SH reagent, p-chloromercuribenzoate, although I and III have similar characteristics. They liberate a mixture of oligonucleotides with 5'-phosphate and 3'-hydroxyl termini.

Animals↗
PubMed2024

Integration of Imaging-based and Sequencing-based Spatial Omics Mapping on the Same Tissue Section via DBiTplus.

Spatially mapping the transcriptome and proteome in the same tissue section can significantly advance our understanding of heterogeneous cellular processes and connect cell type to function. Here, we present Deterministic Barcoding in Tissue sequencing plus (DBiTplus), an integrative multi-modality spatial omics approach that combines sequencing-based spatial transcriptomics and image-based spatial protein profiling on the same tissue section to enable both single-cell resolution cell typing and genome-scale interrogation of biological pathways. DBiTplus begins with in situ reverse transcription for cDNA synthesis, microfluidic delivery of DNA oligos for spatial barcoding, retrieval of barcoded cDNA using RNaseH, an enzyme that selectively degrades RNA in an RNA-DNA hybrid, preserving the intact tissue section for high-plex protein imaging with CODEX. We developed computational pipelines to register data from two distinct modalities. Performing both DBiT-seq and CODEX on the same tissue slide enables accurate cell typing in each spatial transcriptome spot and subsequently image-guided decomposition to generate single-cell resolved spatial transcriptome atlases. DBiTplus was applied to mouse embryos with limited protein markers but still demonstrated excellent integration for single-cell transcriptome decomposition, to normal human lymph nodes with high-plex protein profiling to yield a single-cell spatial transcriptome map, and to human lymphoma FFPE tissue to explore the mechanisms of lymphomagenesis and progression. DBiTplusCODEX is a unified workflow including integrative experimental procedure and computational innovation for spatially resolved single-cell atlasing and exploration of biological pathways cell-by-cell at genome-scale.

Journal Article↗
PubMed2026

Peroxiredoxin 1 safeguards the nucleolar genome from oxidative damage.

Peroxiredoxin 1 (PRDX1) is a highly conserved, thiol-dependent peroxidase that rapidly scavenges reactive oxygen species to modulate redox signaling. PRDX1-null mice exhibited genomic instability, shortened life span, and accelerated tumorigenesis, including development of lymphomas, sarcomas, and carcinomas. Despite extensive characterization of these phenotypes, the molecular mechanism by which PRDX1 loss causes genomic instability remains poorly understood. Here, we show that PRDX1 deficiency alters nucleolar morphology, impairs RNA polymerase I (POL-I)-dependent transcription of pre-ribosomal RNAs, and triggers nucleolar genomic instability. This oxidative stress-induced nucleolar dysfunction promotes the stability of secondary DNA structures, such as RNA-DNA hybrids and G-quadruplex DNA, contributing to nucleolar genomic instability. We demonstrate that PRDX1 loss reduces nascent ribosomal RNA (rRNA) levels and impairs rRNA processing, further affecting ribosome biogenesis. Mechanistically, we established that PRDX1 loss triggers activation of the nucleolar DNA damage response characterized by activation of the DNA repair kinase ATM and elevated TCOF1 within the nucleolus. In addition, we observed recruitment of the MRE11-RAD50-NBS1 (MRN) complex subunit NBS1 to ribosomal DNA (rDNA) loci and this was further increased under oxidative stress. NBS1 accumulation correlates with the repression of rDNA transcription by POL-I, potentially delaying rRNA synthesis, and safeguarding the nucleolar genome from further oxidative damage. Collectively, these findings uncover a previously unrecognized, but critical role, for PRDX1 in maintaining nucleolar integrity and ribosomal biogenesis through redox-dependent regulation of rDNA transcription and processing machinery.

Oxidative Stress↗
PubMed2024

Integration of Imaging-based and Sequencing-based Spatial Omics Mapping on the Same Tissue Section via DBiTplus.

Spatially mapping the transcriptome and proteome in the same tissue section can significantly advance our understanding of heterogeneous cellular processes and connect cell type to function. Here, we present Deterministic Barcoding in Tissue sequencing plus (DBiTplus), an integrative multi-modality spatial omics approach that combines sequencing-based spatial transcriptomics and image-based spatial protein profiling on the same tissue section to enable both single-cell resolution cell typing and genome-scale interrogation of biological pathways. DBiTplus begins with in situ reverse transcription for cDNA synthesis, microfluidic delivery of DNA oligos for spatial barcoding, retrieval of barcoded cDNA using RNaseH, an enzyme that selectively degrades RNA in an RNA-DNA hybrid, preserving the intact tissue section for high-plex protein imaging with CODEX. We developed computational pipelines to register data from two distinct modalities. Performing both DBiT-seq and CODEX on the same tissue slide enables accurate cell typing in each spatial transcriptome spot and subsequently image-guided decomposition to generate single-cell resolved spatial transcriptome atlases. DBiTplus was applied to mouse embryos with limited protein markers but still demonstrated excellent integration for single-cell transcriptome decomposition, to normal human lymph nodes with high-plex protein profiling to yield a single-cell spatial transcriptome map, and to human lymphoma FFPE tissue to explore the mechanisms of lymphomagenesis and progression. DBiTplusCODEX is a unified workflow including integrative experimental procedure and computational innovation for spatially resolved single-cell atlasing and exploration of biological pathways cell-by-cell at genome-scale.

Journal Article↗
PubMed2026

Reduced R-loop abundance at proinflammatory loci: a shared epigenetic mechanism in inflammatory and metabolic diseases.

INTRODUCTION: R-loops, RNA-DNA hybrid structures with a displaced single-stranded DNA loop, are key regulators of transcriptional control, chromatin architecture, and genome stability and have emerging roles in inflammatory signaling. However, the relationship between R-loop abundance and strongly modulated inflammatory effector genes in metabolic inflammation and influenza virus infection remains underexplored. METHODS: We performed a locus-centric integrative analysis combining robust differentially expressed genes (DEGs) from multiple inflammatory and infection-related murine and human transcriptomic disease models with experimentally validated multi-cell R-loop annotations from the reference atlas RLoopBase. Our correlation framework evaluated the directional relationship between R-loop abundance and inflammatory gene expression rather than assuming disease-sample-matched R-loop measurements. We further analyzed R-loop regulatory proteins, NRF2-associated R-loop regulators, and overlaps between R-loop regulators and CRISPRi-identified mitochondrial and cellular reactive oxygen species (ROS) regulators. RESULTS: In angiotensin II-infused apolipoprotein E-deficient (ApoE-/-) mice, a model of abdominal aortic aneurysm (AAA), genomic regions encoding the top significantly upregulated genes exhibited significantly fewer R-loops than those encoding downregulated genes at days 14 and 28. Similarly, in atherosclerotic ApoE-/- mice fed a high-fat diet for 32 and 78 weeks, upregulated genes were associated with fewer R-loops than downregulated genes. Reduced R-loop abundance was also observed in genomic regions encoding the top significantly upregulated genes in liver tissues from patients with non-alcoholic steatohepatitis (NASH), as well as in monosodium urate (MSU)-stimulated lymphatic endothelial cells (LECs) and influenza virus-infected human umbilical vein endothelial cells (HUVECs). R-loop regulatory proteins upregulated during metabolic inflammation were enriched in immune and inflammatory pathways. NRF2 was identified as a regulator of 27 R-loop regulatory proteins, including 10 positively and 17 negatively regulated proteins. Furthermore, 54 R-loop regulatory proteins overlapped with CRISPRi-identified mitochondrial and cellular ROS regulators, suggesting potential reciprocal regulation between R-loop homeostasis and ROS signaling. Disease-associated changes in pro-ROS and anti-ROS R-loop regulatory proteins further linked R-loop regulation to inflammatory and oxidative stress pathways. DISCUSSION: These findings identify reduced R-loop abundance at genomic regions encoding strongly upregulated inflammatory genes as a shared feature across multiple models of metabolic inflammation and influenza virus infection. The results further suggest that immune-associated R-loop regulatory proteins and the NRF2-ROS axis may contribute to R-loop remodeling during inflammatory disease. This integrative framework provides new insight into the potential role of R-loops and ROS-sensitive R-loop regulators in inflammatory and metabolic diseases and identifies candidate pathways for future mechanistic investigation and therapeutic targeting.

R-loop regulatory proteins↗
PubMed1977

Endonuclease V of Escherichia coli.

A small endodeoxyribonuclease )2.3 S) that is active on single-stranded DNA has been extensively purified from Escherichia coli so as to be free of other known DNases. It has an alkaline pH optimum (9.5), requires Mg2+, and makes 3'-hydroxy and 5'-phosphate termini. The nuclease nicks duplex DNA, particularly if treated with OsO4, irradiated with ultraviolet light, or exposed to pH 5. The uracil-containing duplex DNA from the Bacillus subtilis phage PBS-2 is an especially good substrate; it is made acid-soluble by levels of the enzyme which fail to produce any acid-soluble material in other single-stranded or duplex DNAs. Neither RNA nor RNA-DNA hybrid are degraded by the enzyme. The enzyme specificity suggests that it might act at abnormal regions in DNA, so that its in vivo function could be to initiate an excision repair sequence. Its high activity on uracil-containing DNA could imply that the enzyme provides an alternative mechanism for excising uracil residues from DNA to the pathway utilizing uracil-DNA N-glycosidase. We suggest that this enzyme be designated as endonuclease V of E. coli.

Cations, Divalent↗