Exploiting replication gaps for cancer therapy.

Defects in DNA double-strand break repair are thought to render BRCA1 or BRCA2 (BRCA) mutant tumors selectively sensitive to PARP inhibitors (PARPis). Challenging this framework, BRCA and PARP1 share functions in DNA synthesis on the lagging strand. Thus, BRCA deficiency or "BRCAness" could reflect an inherent lagging strand problem that is vulnerable to drugs such as PARPi that also target the lagging strand, a combination that generates a toxic accumulation of replication gaps.

Strand-specific ChIP-seq at DNA breaks distinguishes ssDNA versus dsDNA binding and refutes single-stranded nucleosomes.

In a first step of DNA double-strand break (DSB) repair by homologous recombination, DNA ends are resected such that single-stranded DNA (ssDNA) overhangs are generated. ssDNA is specifically bound by RPA and other factors, which constitutes a ssDNA-domain on damaged chromatin. The molecular organization of this ssDNA and the adjacent dsDNA domain is crucial during DSB signaling and repair. However, data regarding the presence of nucleosomes, the most basic chromatin components, in the ssDNA domain have been contradictory. Here, we use site-specific induction of DSBs and chromatin immunoprecipitation followed by strand-specific sequencing to analyze in vivo binding of key DSB repair and signaling proteins to either the ssDNA or dsDNA domain. In the case of nucleosomes, we show that recently proposed ssDNA nucleosomes are not a major, persistent species, but that nucleosome eviction and DNA end resection are intrinsically coupled. These results support a model of separated dsDNA-nucleosome and ssDNA-RPA domains during DSB repair.

(Single-stranded DNA) gaps in understanding BRCAness.

The tumour-suppressive roles of BRCA1 and 2 have been attributed to three seemingly distinct functions - homologous recombination, replication fork protection, and single-stranded (ss)DNA gap suppression - and their relative importance is under debate. In this review, we examine the origin and resolution of ssDNA gaps and discuss the recent advances in understanding the role of BRCA1/2 in gap suppression. There are ample data showing that gap accumulation in BRCA1/2-deficient cells is linked to genomic instability and chemosensitivity. However, it remains unclear whether there is a causative role and the function of BRCA1/2 in gap suppression cannot unambiguously be dissected from their other functions. We therefore conclude that the three functions of BRCA1 and 2 are closely intertwined and not mutually exclusive.

Rad5 Recruits Error-Prone DNA Polymerases for Mutagenic Repair of ssDNA Gaps on Undamaged Templates.

Post-replication repair (PRR) allows tolerance of chemical- and UV-induced DNA base lesions in both an error-free and an error-prone manner. In classical PRR, PCNA monoubiquitination recruits translesion synthesis (TLS) DNA polymerases that can replicate through lesions. We find that PRR responds to DNA replication stress that does not cause base lesions. Rad5 forms nuclear foci during normal S phase and after exposure to types of replication stress where DNA base lesions are likely absent. Rad5 binds to the sites of stressed DNA replication forks, where it recruits TLS polymerases to repair single-stranded DNA (ssDNA) gaps, preventing mitotic defects and chromosome breaks. In contrast to the prevailing view of PRR, our data indicate that Rad5 promotes both mutagenic and error-free repair of undamaged ssDNA that arises during physiological and exogenous replication stress.

R-Loops Promote Antisense Transcription across the Mammalian Genome.

Widespread antisense long noncoding RNA (lncRNA) overlap with many protein-coding genes in mammals and emanate from gene promoter, enhancer, and termination regions. However, their origin and biological purpose remain unclear. We show that these antisense lncRNA can be generated by R-loops that form when nascent transcript invades the DNA duplex behind elongating RNA polymerase II (Pol II). Biochemically, R-loops act as intrinsic Pol II promoters to induce de novo RNA synthesis. Furthermore, their removal across the human genome by RNase H1 overexpression causes the selective reduction of antisense transcription. Consequently, we predict that R-loops act to facilitate the synthesis of many gene proximal antisense lncRNA. Not only are R-loops widely associated with DNA damage and repair, but we now show that they have the capacity to promote de novo transcript synthesis that may have aided the evolution of gene regulation.

Molecular insights into the prototypical single-stranded DNA-binding protein from E. coli.

The SSB protein of Escherichia coli functions to bind single-stranded DNA wherever it occurs during DNA metabolism. Depending upon conditions, SSB occurs in several different binding modes. In the course of its function, SSB diffuses on ssDNA and transfers rapidly between different segments of ssDNA. SSB interacts with many other proteins involved in DNA metabolism, with 22 such SSB-interacting proteins, or SIPs, defined to date. These interactions chiefly involve the disordered and conserved C-terminal residues of SSB. When not bound to ssDNA, SSB can aggregate to form a phase-separated biomolecular condensate. Current understanding of the properties of SSB and the functional significance of its many intermolecular interactions are summarized in this review.

Improved prediction of DNA and RNA binding proteins with deep learning models.

Nucleic acid-binding proteins (NABPs), including DNA-binding proteins (DBPs) and RNA-binding proteins (RBPs), play important roles in essential biological processes. To facilitate functional annotation and accurate prediction of different types of NABPs, many machine learning-based computational approaches have been developed. However, the datasets used for training and testing as well as the prediction scopes in these studies have limited their applications. In this paper, we developed new strategies to overcome these limitations by generating more accurate and robust datasets and developing deep learning-based methods including both hierarchical and multi-class approaches to predict the types of NABPs for any given protein. The deep learning models employ two layers of convolutional neural network and one layer of long short-term memory. Our approaches outperform existing DBP and RBP predictors with a balanced prediction between DBPs and RBPs, and are more practically useful in identifying novel NABPs. The multi-class approach greatly improves the prediction accuracy of DBPs and RBPs, especially for the DBPs with ~12% improvement. Moreover, we explored the prediction accuracy of single-stranded DNA binding proteins and their effect on the overall prediction accuracy of NABP predictions.

The Histone Chaperones ASF1 and CAF-1 Promote MMS22L-TONSL-Mediated Rad51 Loading onto ssDNA during Homologous Recombination in Human Cells.

The access-repair-restore model for the role of chromatin in DNA repair infers that chromatin is a mere obstacle to DNA repair. However, here we show that blocking chromatin assembly, via knockdown of the histone chaperones ASF1 or CAF-1 or a mutation that prevents ASF1A binding to histones, hinders Rad51 loading onto ssDNA during homologous recombination. This is a consequence of reduced recruitment of the Rad51 loader MMS22L-TONSL to ssDNA, resulting in persistent RPA foci, extensive DNA end resection, persistent activation of the ATR-Chk1 pathway, and cell cycle arrest. In agreement, histones occupy ssDNA during DNA repair in yeast. We also uncovered DNA-PKcs-dependent DNA damage-induced ASF1A phosphorylation, which enhances chromatin assembly, promoting MMS22L-TONSL recruitment and, hence, Rad51 loading. We propose that transient assembly of newly synthesized histones onto ssDNA serves to recruit MMS22L-TONSL to efficiently form the Rad51 nucleofilament for strand invasion, suggesting an active role of chromatin assembly in homologous recombination.

A Role for N6-Methyladenine in DNA Damage Repair.

The leading cause of mutation due to oxidative damage is 8-oxo-2'-deoxyguanosine (8-oxoG) mispairing with adenine (Ade), which can occur in two ways. First, guanine of a G:C DNA base pair can be oxidized. If not repaired in time, DNA polymerases can mispair Ade with 8-oxoG in the template. This 8-oxoG:A can be repaired by enzymes that remove Ade opposite to template 8-oxoG, or 8-oxoG opposite to Cyt. Second, free 8-oxo-dGTP can be misincorporated by DNA polymerases into DNA opposite template Ade. However, there is no known repair activity that removes 8-oxoG opposite to template Ade. We suggest that a major role of N6-methyladenine in mammalian DNA is minimizing incorporation of 8-oxoG opposite to Ade by DNA polymerases following adduct formation.

Disenfranchised DNA: biochemical analysis of mutant øX174 DNA-binding proteins may further elucidate the evolutionary significance of the unessential packaging protein A.

Most icosahedral DNA viruses package and condense their genomes into pre-formed, volumetrically constrained capsids. However, concurrent genome biosynthesis and packaging are specific to single-stranded (ss) DNA micro- and parvoviruses. Before packaging, ~120 copies of the øX174 DNA-binding protein J interact with double-stranded DNA. 60 J proteins enter the procapsid with the ssDNA genome, guiding it between 60 icosahedrally ordered DNA-binding pockets formed by the capsid proteins. Although J proteins are small, 28-37 residues in length, they have two domains. The basic, positively charged N-terminus guides the genome between binding pockets, whereas the C-terminus acts as an anchor to the capsid's inner surface. Three C-terminal aromatic residues, W30, Y31, and F37, interact most extensively with the coat protein. Their corresponding codons were mutated, and the resulting strains were biochemically and genetically characterized. Depending on the mutation, the substitutions produced unstable packaging complexes, unstable virions, infectious progeny, or particles packaged with smaller genomes, the latter being a novel phenomenon. The smaller genomes contained internal deletions. The juncture sequences suggest that the unessential A* (A star) protein mediates deletion formation.IMPORTANCEUnessential but strongly conserved gene products are understudied, especially when mutations do not confer discernable phenotypes or the protein's contribution to fitness is too small to reliably determine in laboratory-based assays. Consequently, their functions and evolutionary impact remain obscure. The data presented herein suggest that microvirus A* proteins, discovered over 40 years ago, may hasten the termination of non-productive packaging events. Thus, performing a salvage function by liberating the reusable components of the failed packaging complexes, such as DNA templates and replication enzymes.

Welded Gold Nanoparticle Assemblies Defined Plasmonic Coupling.

Nanoparticle assemblies with interparticle ohmic contacts are crucial for nanodevice fabrication. Despite tremendous progress in DNA-programmable nanoparticle assemblies, seamlessly welding discrete components into welded continuous three-dimensional (3D) configurations remains challenging. Here, we introduce a single-stranded DNA-encoded strategy to customize welded metal nanostructures with tunable morphologies and plasmonic properties. We demonstrate the precise welding of gold nanoparticle assemblies into continuous metal nanostructures with interparticle ohmic contacts through chemical welding in solution. We find that the welded gold nanoparticle assemblies show a consistent morphology with welded efficiency over 90%, such as the rod-like, triangular, and tetrahedral metal nanostructures. Next, we show the versatility of this strategy by welding gold nanoparticle assemblies of varied sizes and shapes. Furthermore, the experiment and simulation show that the welded gold nanoparticle assemblies exhibit defined plasmonic coupling. This single-stranded DNA encoded welding system may provide a new route for accurately building functional plasmonic nanomaterials and devices.

The mechanisms of human lymphoid chromosomal translocations and their medical relevance.

The most common human lymphoid chromosomal translocations involve concurrent failures of the recombination activating gene (RAG) complex and Activation-Induced Deaminase (AID). These are two enzymes that are normally expressed for purposes of the two site-specific DNA recombination processes: V(D)J recombination and class switch recombination (CSR). First, though it is rare, a low level of expression of AID can introduce long-lived T:G mismatch lesions at 20-600 bp fragile zones. Second, the V(D)J recombination process can occasionally fail to rejoin coding ends, and this failure may permit an opportunity for Artemis:DNA-dependent kinase catalytic subunit (DNA-PKcs) to convert the T:G mismatch sites at the fragile zones into double-strand breaks. The 20-600 bp fragile zones must be, at least transiently, in a single-stranded DNA (ssDNA) state for the first step to occur, because AID only acts on ssDNA. Here we discuss the key DNA sequence features that lead to AID action at a fragile zone, which are (a) the proximity and density of strings of cytosine nucleotides (C-strings) that cause a B/A-intermediate DNA conformation; (b) overlapping AID hotspots that contain a methyl CpG (WRCG), which AID converts to a long-lived T:G mismatch; and (c) transcription, which, though not essential, favors increased ssDNA in the fragile zone. We also summarize chromosomal features of the focal fragile zones in lymphoid malignancies and discuss the clinical relevance of understanding the translocation mechanisms. Many of the key principles covered here are also relevant to chromosomal translocations in non-lymphoid somatic cells as well.

Interaction with single-stranded DNA-binding protein modulates Escherichia coli RadD DNA repair activities.

The bacterial RadD enzyme is important for multiple genome maintenance pathways, including RecA DNA strand exchange and RecA-independent suppression of DNA crossover template switching. However, much remains unknown about the precise roles of RadD. One potential clue into RadD mechanisms is its direct interaction with the single-stranded DNA binding protein (SSB), which coats single-stranded DNA exposed during genome maintenance reactions in cells. Interaction with SSB stimulates the ATPase activity of RadD. To probe the mechanism and importance of RadD-SSB complex formation, we identified a pocket on RadD that is essential for binding SSB. In a mechanism shared with many other SSB-interacting proteins, RadD uses a hydrophobic pocket framed by basic residues to bind the C-terminal end of SSB. We found that RadD variants that substitute acidic residues for basic residues in the SSB binding site impair RadD:SSB complex formation and eliminate SSB stimulation of RadD ATPase activity in vitro. Additionally, mutant Escherichia coli strains carrying charge reversal radD changes display increased sensitivity to DNA damaging agents synergistically with deletions of radA and recG, although the phenotypes of the SSB-binding radD mutants are not as severe as a full radD deletion. This suggests that cellular RadD requires an intact interaction with SSB for full RadD function.

Molecular flexibility of DNA as a key determinant of RAD51 recruitment.

The timely activation of homologous recombination is essential for the maintenance of genome stability, in which the RAD51 recombinase plays a central role. Biochemically, human RAD51 polymerises faster on single-stranded DNA (ssDNA) compared to double-stranded DNA (dsDNA), raising a key conceptual question: how does it discriminate between them? In this study, we tackled this problem by systematically assessing RAD51 binding kinetics on ssDNA and dsDNA differing in length and flexibility using surface plasmon resonance. By directly fitting a mechanistic model to our experimental data, we demonstrate that the RAD51 polymerisation rate positively correlates with the flexibility of DNA. Once the RAD51-DNA complex is formed, however, RAD51 remains stably bound independent of DNA flexibility, but rapidly dissociates from flexible DNA when RAD51 self-association is perturbed. This model presents a new general framework suggesting that the flexibility of DNA, which may increase locally as a result of DNA damage, plays an important role in rapidly recruiting repair factors that multimerise at sites of DNA damage.

PhieDBEs: a DBD-containing, PAM-flexible, high-efficiency dual base editor toolbox with wide targeting scope for use in plants.

Dual base editors (DBEs) enable simultaneous A-to-G and C-to-T conversions, expanding mutation types. However, low editing efficiency and narrow targeting range limit the widespread use of DBEs in plants. The single-strand DNA binding domain of RAD51 DBD can be fused to base editors to improve their editing efficiency. However, it remains unclear how the DBD affects dual base editing performance in plants. In this study, we generated a series of novel plant DBE-SpGn tools consisting of nine constructs using the high-activity cytidine deaminase evoFERNY, adenosine deaminase TadA8e and DBD in various fusion modes with the PAM-flexible Streptococcus pyogenes Cas9 (SpCas9) nickase variant SpGn (with NG-PAM). By analysing their editing performance on 48 targets in rice, we found that DBE-SpGn constructs containing a single DBD and deaminases located at the N-terminus of SpGn exhibited the highest editing efficiencies. Meanwhile, constructs with deaminases located at the C-terminus and/or multiple DBDs failed to function normally and exhibited inhibited editing activity. We identified three particularly high-efficiency dual base editors (C-A-SpGn, C-A-D-SpGn and A-C-D-SpGn), named PhieDBEs (Plant high-efficiency dual base editors), capable of producing efficient dual base conversions within a narrow editing window (M5 ~ M9, M = A/C). The editing efficiency of C-A-D-SpGn was as high as 95.2% at certain target sites, with frequencies of simultaneous C-to-T and A-to-G conversions as high as 81.0%. In summary, PhieDBEs (especially C-A-D-SpGn) can produce diverse mutants and may prove useful in a wide variety of applications, including plant functional genomics, precise mutagenesis, directed evolution and crop genetic improvement, among others.

Assessment of DNA quality for whole genome library preparation.

In recent years, more sophisticated DNA technologies for genotyping have enabled considerable progress in various fields such as clinical genetics, archaeogenetics and forensic genetics. DNA samples previously rejected as too challenging to analyze due to low amounts of degraded DNA can now provide useful information. To increase the chances of success with the new methodologies, it is crucial to know the fragment size of the template DNA molecules, and whether the DNA in a sample is mostly single or double stranded. With this knowledge, an appropriate library preparation method can be chosen, and the DNA shearing parameters of the protocol can be adjusted to the DNA fragment size in the sample. In this study, we first developed and evaluated a user-friendly fluorometry-based protocol for estimation of DNA strandedness. We also evaluated different capillary electrophoresis methods for estimation of DNA fragmentation levels. Next, we applied the developed methodologies to a broad variety of DNA samples processed with different DNA extraction protocols. Our findings show that both the applied DNA extraction method and the sample type affect the DNA strandedness and fragmentation. The established protocols and the gained knowledge will be applicable for future sequencing-based high-density SNP genotyping in various fields.

MSC microvesicles loaded G-quadruplex-enhanced circular single-stranded DNA-9 inhibits tumor growth by targeting MDSCs.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) promote tumor growth, metastasis, and lead to immunotherapy resistance. Studies revealed that miRNAs are also expressed in MDSCs and promote the immunosuppressive function of MDSCs. Currently, few studies have been reported on inducible cellular microvesicle delivery of nucleic acid drugs targeting miRNA in MDSCs for the treatment of malignant tumors. RESULTS AND CONCLUSION: In this study, we designed an artificial DNA named G-quadruplex-enhanced circular single-stranded DNA-9 (G4-CSSD9), that specifically adsorbs the miR-9 sequence. Its advanced DNA folding structure, rich in tandem repeat guanine (G-quadruplex), also provides good stability. Mesenchymal stem cells (MSCs) were prepared into nanostructured vesicles by membrane extrusion. The MSC microvesicles-encapsulated G4-CSSD9 (MVs@G4-CSSD9) was delivered into MDSCs, which affected the downstream transcription and translation process, and reduced the immunosuppressive function of MDSCs, so as to achieve the purpose of treating melanoma. In particular, it provides an idea for the malignant tumor treatment.

Secondary structure of the human mitochondrial genome affects formation of deletions.

BACKGROUND: Aging in postmitotic tissues is associated with clonal expansion of somatic mitochondrial deletions, the origin of which is not well understood. Such deletions are often flanked by direct nucleotide repeats, but this alone does not fully explain their distribution. Here, we hypothesized that the close proximity of direct repeats on single-stranded mitochondrial DNA (mtDNA) might play a role in the formation of deletions. RESULTS: By analyzing human mtDNA deletions in the major arc of mtDNA, which is single-stranded during replication and is characterized by a high number of deletions, we found a non-uniform distribution with a "hot spot" where one deletion breakpoint occurred within the region of 6-9 kb and another within 13-16 kb of the mtDNA. This distribution was not explained by the presence of direct repeats, suggesting that other factors, such as the spatial proximity of these two regions, can be the cause. In silico analyses revealed that the single-stranded major arc may be organized as a large-scale hairpin-like loop with a center close to 11 kb and contacting regions between 6-9 kb and 13-16 kb, which would explain the high deletion activity in this contact zone. The direct repeats located within the contact zone, such as the well-known common repeat with a first arm at 8470-8482 bp (base pair) and a second arm at 13,447-13,459 bp, are three times more likely to cause deletions compared to direct repeats located outside of the contact zone. A comparison of age- and disease-associated deletions demonstrated that the contact zone plays a crucial role in explaining the age-associated deletions, emphasizing its importance in the rate of healthy aging. CONCLUSIONS: Overall, we provide topological insights into the mechanism of age-associated deletion formation in human mtDNA, which could be used to predict somatic deletion burden and maximum lifespan in different human haplogroups and mammalian species.

Reversed Regulation Effects of ssDNA on the Mimetic Oxidase and Peroxidase Activities of Covalent Organic Frameworks.

Nanomaterials with enzyme mimetic activity have attracted extensive attention, especially in the regulation of their catalytic activities by biomolecules or other polymers. Here, a covalent organic framework (Tph-BT COF) with excellent photocatalytic activity is constructed by Schiff base reaction, and its mimetic oxidase activity and peroxidase activity is inversely regulated via single-stranded DNA (ssDNA). Under light-emitting diode (LED) light irradiation, Tph-BT exhibited outstanding oxidase activity, which efficiently catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to produce blue oxTMB, and ssDNA, especially those with poly-thymidine (T) sequences, can significantly inhibit its oxidase activity. On the contrary, Tph-BT showed weak peroxidase activity, and the presence of ssDNA, particularly poly-cytosine (C) sequences, can remarkably enhance the peroxidase activity. The influence of base type, base length, and other factors on the activities of two enzymes is also studied, and the results reveal that the adsorption of ssDNA on the surface of Tph-BT prevented intersystem crossing (ISC) and energy transfer processes to reduce 1 O2 generation, while the electrostatic interaction between ssDNA and TMB enhanced Tph-BT's affinity for TMB to facilitate the electron transfer from TMB to • OH. This study investigates multitype mimetic enzyme activities of nonmetallic D-A conjugated COFs and demonstrates their feasibility of regulation by ssDNA.

Low-Temperature Adaptation Targets Genome Packing Reactions in an Icosahedral Single-Stranded DNA Virus.

øX174, G4, and α3 represent the three sister genera of a Microviridae subfamily. α3-like genomes are considerably larger than their sister genera genomes, yet they are packaged into capsids of similar internal volumes. They also contain multiple A* genes, which are nested within the larger A gene reading frame. Although unessential under most conditions, A* proteins mediate the fidelity of packaging reactions. Larger genomes and multiple A* genes may indicate that genome packaging is more problematic for α3-like viruses, especially at lower temperatures, where DNA persistence lengths would be longer. Unlike members of the other genera, which reliably form plaques at 20°C, α3-like phages are naturally cold sensitive below 28°C. To determine whether there was a connection between the uniquely α3-like genome characteristics and the cold-sensitive phenotype, the α3 assembly pathway was characterized at low temperature. Although virions were not detected, particles consistent with off-pathway packaging complexes were observed. In a complementary evolutionary approach, α3 was experimentally evolved to grow at progressively lower temperatures. The two major responses to cold adaptation were genome reduction and elevated A* gene expression. IMPORTANCE The production of enzymes, transcription factors, and viral receptors directly influences the niches viruses can inhabit. Some prokaryotic hosts can thrive in widely differing environments; thus, physical parameters, such as temperature, should also be considered. These variables may directly alter host physiology, preventing viral replication. Alternatively, they could negatively inhibit infection processes in a host-independent manner. The members of three sister Microviridae genera (canonical species øX174, G4 and α3) infect the same host, but α3-like viruses are naturally cold sensitive, which could effectively exclude them from low-temperature environments (<28°C). Exclusion appeared to be independent of host cell physiology. Instead, it could be largely attributed to low-temperature packaging defects. The results presented here demonstrate how physical parameters, such as temperature, can directly influence viral diversification and niche determination in a host-independent manner.