Single-stranded Binding Research Articles

P-448 Changes in gene transcription induced by cyclophosphamide treatment in an experimental ovarian culture model

Abstract Study question How does cyclophosphamide (CPA) treatment impact at transcriptional level on mouse ovarian tissue? Summary answer Cultured murine ovarian tissue with CPA versus control showed up-regulated intrinsic and extrinsic apoptotic signaling pathways, associated with DNA damage, DNA repair and oxidative response. What is known already Alkylating chemotherapeutic treatment depletes the ovarian pool and induces infertility in women. The suggested mechanisms behind these adverse effects include apoptosis and/or over-activation of the dormant primordial follicle pool. However, there is a lack of knowledge about the pathways that lead to these outcomes and previous researches have been inconclusive. The investigation of changes in the ovarian transcriptomic profiling following the alkylating drug CPA treatment can be useful to identify new potential targets for fertility preservation in women treated for cancer. Study design, size, duration Controlled experimental study using 20 female B6CBA/F1 4-day-old mice. Ovaries were collected and randomly assigned to CPA (4-hydroperoxycyclophosphamide) treated group (n = 20) or control group (n = 20). Five ovaries/group were collected at 8, 12, 24 and 36 h to investigate the dynamic of the changes. RNA extraction and RNA sequencing analysis were carried out. Participants/materials, setting, methods Ovaries were cultured on Millicell cell culture inserts floating on 0.25 mL culture medium in a 24-well plate. Freshly prepared 4-hydroperoxycyclophosphamide solution was added to the wells of CPA group (final concentration = 5 µM). Equal amount of solvent was added to the wells of control group. Culture medium was refreshed at 48 h with culture medium only. RNA sequencing data were processed for subsequent differentially expressed genes (DEGs) and gene set enrichment analysis (GSEA). Main results and the role of chance At 8 h, CPA treatment induced the up-regulation of biological processes related to hypoxia, cell growth and embryonic organ development. At 12 h, DNA damage and the ovarian cell responses were evidenced by an increased activity of DNA damage response, DNA damage checkpoint, DNA repair (double-strand break, mismatch, single strand binding), stress-activated MAPK cascade, antioxidant activity and intrinsic apoptotic signaling pathway. The representative genes of these processes there were Bbc3, Bax, Trp73, Cdkn1a, Trp53inp1 and Mdm2. A dramatic increase in the number of DEGs was found at 24 h (8 h, n = 209; 12 h, n = 239; 24 h, n = 2013). Also at 24 h DNA repair, intrinsic and extrinsic apoptotic signaling pathways were the most representative processes evidenced by the addition of Rad9a, H2afx, Casp3, Bak1 and Casp8 genes to the above mentioned. Whereas, germ cell related genes Ybx2, Nobox and Ddx4 were all down-regulated. At 36 h, the number of DEGs (n = 3804) still increased, the up-regulated pathways were similar to 24 h, while meiosis and microtubule-based movements pathways were observed in the down-regulated set too. Limitations, reasons for caution Although the age of the mice chosen for the experiment ensured a high and representative content of primordial follicles in the ovary, whole ovaries were used for RNA sequencing analysis containing a heterogeneous composition of cells other than follicles. Wider implications of the findings Our results provide evidence of dynamic sequential changes in transcriptional level where apoptosis was involved in CPA-induced ovarian follicle depletion. Our research indicates a time frame before the occurrence of DNA definitive damage following CPA-treatment, where application of possible treatments in order to prevent the following apoptosis would be possible. Trial registration number Not Applicable

Low background interference SERS aptasensor for highly sensitive multiplex mycotoxin detection based on polystyrene microspheres-mediated controlled release of Raman reporters

Mycotoxin contamination is a serious global issue in food safety. The accurate detection of mycotoxins in complex samples, particularly via a portable detection system that realizes multi-analyte detection, remains a great challenge. Here, a polystyrene (PS)-mediated surface-enhanced Raman scattering (SERS) aptasensor was designed for ultrasensitive and simultaneous detection of multiple mycotoxins in food samples. First, the novel Raman tags were prepared using PS as nanocontainer to encapsulate abundant Raman reporters. The PS nanocontainer with Raman reporters was then functionalized by aptamer as an effective target-recognition platform. Second, the single-strand binding protein modified magnetic nanoparticles (MNPs@SSB) were used as a magnetic capture substrate. When the target mycotoxin was present and recognized by its aptamer, the combination of PS-Apt and mycotoxins can hinder the binding between PS-Apt and MNPs@SSB. Consequently, after the magnetic separation and treatment by THF solution, fewer biological-silent reporter probes were released from PS, causing the reduction of SERS signal as a function of target mycotoxins concentration. Due to the high loading capacity of PS and the excellent signal conversion and amplification of aptamer-assisted SERS assay, the proposed strategy is reliable and ultrasensitive for the determination of multiple mycotoxins. The limit of detection was as low as 0.159 fg L−1, 2.015 fg L−1, and 1.561 fg L−1 for ZEN, OTA and AFB1, respectively. This strategy not only raises a new idea for designing novel Raman tag, but also broadens the application of SERS for multiple detection of trace target.

Single-stranded nucleic acid binding and coacervation by linker histone H1.

The H1 linker histone family is the most abundant group of eukaryotic chromatin-binding proteins. However, their contribution to chromosome structure and function remains incompletely understood. Here we use single-molecule fluorescence and force microscopy to directly visualize the behavior of H1 on various nucleic acid and nucleosome substrates. We observe that H1 coalesces around single-stranded DNA generated from tension-induced DNA duplex melting. Using a droplet fusion assay controlled by optical tweezers, we find that single-stranded nucleic acids mediate the formation of gel-like H1 droplets, whereas H1-double-stranded DNA and H1-nucleosome droplets are more liquid-like. Molecular dynamics simulations reveal that multivalent and transient engagement of H1 with unpaired DNA strands drives their enhanced phase separation. Using eGFP-tagged H1, we demonstrate that inducing single-stranded DNA accumulation in cells causes an increase in H1 puncta that are able to fuse. We further show that H1 and Replication Protein A occupy separate nuclear regions, but that H1 colocalizes with the replication factor Proliferating Cell Nuclear Antigen, particularly after DNA damage. Overall, our results provide a refined perspective on the diverse roles of H1 in genome organization and maintenance, and indicate its involvement at stalled replication forks.

Small Prokaryotic DNA-Binding Proteins Protect Genome Integrity throughout the Life Cycle.

Genomes of all organisms are persistently threatened by endogenous and exogenous assaults. Bacterial mechanisms of genome maintenance must provide protection throughout the physiologically distinct phases of the life cycle. Spore-forming bacteria must also maintain genome integrity within the dormant endospore. The nucleoid-associated proteins (NAPs) influence nucleoid organization and may alter DNA topology to protect DNA or to alter gene expression patterns. NAPs are characteristically multifunctional; nevertheless, Dps, HU and CbpA are most strongly associated with DNA protection. Archaea display great variety in genome organization and many inhabit extreme environments. As of yet, only MC1, an archaeal NAP, has been shown to protect DNA against thermal denaturation and radiolysis. ssDNA are intermediates in vital cellular processes, such as DNA replication and recombination. Single-stranded binding proteins (SSBs) prevent the formation of secondary structures but also protect the hypersensitive ssDNA against chemical and nuclease degradation. Ionizing radiation upregulates SSBs in the extremophile Deinococcus radiodurans.

An improved nucleic acid sequence-based amplification method mediated by T4 gene 32 protein.

The uptake of Nucleic Acid Sequence-Based Amplification (NASBA) for point of care testing may be hindered by a complexity in the workflow due the requirement of a thermal denaturation step to initiate the cyclic isothermal amplification before the addition of the amplification enzymes. Despite reports of successful enhancement of other DNA and RNA amplification methods using DNA and RNA binding proteins, this has not been reported for NASBA. Here, three single-stranded binding proteins, RecA, Extreme Thermostable Single-stranded binding protein (ET SSB) and T4 gene gp32 protein (gp32), were incorporated in NASBA protocol and used for single pot, one-step NASBA at 41 °C. Indeed, all SSBs showed significantly improved amplifications compared with the 2-step process, but only gp32 showed no non-specific aberrant amplification, and slightly improved the time-to-positivity in comparison with the conventional NASBA. For synthetic HIV-1 RNA, gp32 was found to improve the time-to-positivity (ttp) by average of 13.6% of one-step NASBA and 6.7% of conventional NASBA for the detection of HIV-1 RNA, showing its potential for simplifying the workflow as desirable for point of care applications of NASBA.

An Adenosine Triphosphate- Dependent 5'-3' DNA Helicase From sk1-Like Lactococcus lactis F13 Phage.

Here, we describe functional characterization of an early gene (gp46) product of a virulent Lactococcus lactis sk1-like phage, vB_Llc_bIBBF13 (abbr. F13). The GP46F13 protein carries a catalytically active RecA-like domain belonging to the P-loop NTPase superfamily. It also retains features characteristic for ATPases forming oligomers. In order to elucidate its detailed molecular function, we cloned and overexpressed the gp46 gene in Escherichia coli. Purified GP46F13 protein binds to DNA and exhibits DNA unwinding activity on branched substrates in the presence of adenosine triphosphate (ATP). Size exclusion chromatography with multi-angle light scattering (SEC-MALS) experiments demonstrate that GP46F13 forms oligomers, and further pull-down assays show that GP46F13 interacts with host proteins involved in replication (i.e., DnaK, DnaJ, topoisomerase I, and single-strand binding protein). Taking together the localization of the gene and the obtained results, GP46F13 is the first protein encoded in the early-expressed gene region with helicase activity that has been identified among lytic L. lactis phages up to date.

The RecD2 helicase balances RecA activities.

DNA helicases of the RecD2 family are ubiquitous. Bacillus subtilis RecD2 in association with the single-stranded binding protein SsbA may contribute to replication fork progression, but its detailed action remains unknown. In this work, we explore the role of RecD2 during DNA replication and its interaction with the RecA recombinase. RecD2 inhibits replication restart, but this effect is not observed in the absence of SsbA. RecD2 slightly affects replication elongation. RecA inhibits leading and lagging strand synthesis, and RecD2, which physically interacts with RecA, counteracts this negative effect. In vivo results show that recD2 inactivation promotes RecA–ssDNA accumulation at low mitomycin C levels, and that RecA threads persist for a longer time after induction of DNA damage. In vitro, RecD2 modulates RecA-mediated DNA strand-exchange and catalyzes branch migration. These findings contribute to our understanding of how RecD2 may contribute to overcome a replicative stress, removing RecA from the ssDNA and, thus, it may act as a negative modulator of RecA filament growth.

Performance improved fluorescence polarization for easy and accurate authentication of chicken adulteration

Rapid, accurate, and effective authentication of unlabeled meat components in commercial meat products is of great significance for guaranteeing food safety and human health. In this research, we have constructed a single-strand binding protein (SSB)-assisted performance improved fluorescence polarization (FP) protocol for authentication of chicken component in meat products. The SSB-assisted FP strategy involves the use of fluorescein isothiocyanate (FITC)-labeled primers to perform polymerase chain reaction (PCR). In addition, FP enhancement was accomplished by mixing the amplified product with SSB. Based on the designed mechanism, the free primers in the final amplified product can be tightly bound by SSB, which greatly restricts the rotation of FITC, thereby resulting a high FP signal. In the presence of chicken DNA, the transformation of single-stranded primers to double-stranded amplicons greatly hinders the combination of SSB and primers, leading to the decrease of FP signal. This method well couples the advantages of both PCR and FP technology. Through this new strategy, as low as 0.035% (wt. %) of chicken adulteration in meat samples can be well authenticated. Moreover, this method is also available for commercial meat products. We expect this SSB-assisted FP strategy will provide new insights into the construction of versatile analysis tools in the field of food safety detection. • Fluorescence polarization protocol for rapid sensing of chicken adulteration. • Fluorescence polarization adopted for analysis of PCR products. • Single strand binding protein adopted to improve detection limit of adulteration. • Successful authentication of commercial meat products with improved FP strategy.

The Finely Coordinated Action of SSB and NurA/HerA Complex Strictly Regulates the DNA End Resection Process in Saccharolobus solfataricus.

Generation of the 3′ overhang is a critical step during homologous recombination (HR) and replication fork rescue processes. This event is usually performed by a series of DNA nucleases and/or helicases. The nuclease NurA and the ATPase HerA, together with the highly conserved MRE11/RAD50 proteins, play an important role in generating 3′ single-stranded DNA during archaeal HR. Little is known, however, about HerA-NurA function and activation of this fundamental and complicated DNA repair process. Herein, we analyze the functional relationship among NurA, HerA and the single-strand binding protein SSB from Saccharolubus solfataricus. We demonstrate that SSB clearly inhibits NurA endonuclease activity and its exonuclease activities also when in combination with HerA. Moreover, we show that SSB binding to DNA is greatly stimulated by the presence of either NurA or NurA/HerA. In addition, if on the one hand NurA binding is not influenced, on the other hand, HerA binding is reduced when SSB is present in the reaction. In accordance with what has been observed, we have shown that HerA helicase activity is not stimulated by SSB. These data suggest that, in archaea, the DNA end resection process is governed by the strictly combined action of NurA, HerA and SSB.

Rapid isothermal amplification of several kilo base pair linear DNA

Polymerase Chain Reaction (PCR) is an essential method used to amplify or copy nucleic acids. PCR requires a thermal cycler to alternate between strand separation at high temperatures and replication/copying at low temperature. We eliminated thermal cycling and kept all desired PCR features in place through enabling a new SSB (Single-Stranded Binding protein) and helicase assisted isothermal amplification method named SHARP (SSB-Helicase Assisted Rapid PCR). SHARP uses the same starting set of primers and template DNA as PCR does, carries out the reaction at a constant temperature, and outputs the same amplicon as PCR with lengths of up to 6000 base pairs within 5 to 30 minutes, a feature no existing isothermal amplification method can match.

Cooperative kinetics of ligand binding to linear polymers

Ligands change the chemical and mechanical properties of polymers. In particular, single strand binding protein (SSB) non-specifically bounds to single-stranded DNA (ssDNA), modifying the ssDNA stiffness and the DNA replication rate, as recently measured with single-molecule techniques. SSB is a large ligand presenting cooperativity in some of its binding modes. We aim to develop an accurate kinetic model for the cooperative binding kinetics of large ligands. Cooperativity accounts for the changes in the affinity of a ligand to the polymer due to the presence of another bound ligand. Large ligands, attaching to several binding sites, require a detailed counting of the available binding possibilities. This counting has been done by McGhee and von Hippel to obtain the equilibrium state of the ligands-polymer complex. The same procedure allows to obtain the kinetic equations for the cooperative binding of ligands to long polymers, for all ligand sizes. Here, we also derive approximate cooperative kinetic equations in the large ligand limit, at the leading and next-to-leading orders. We found cooperativity is negligible at the leading-order, and appears at the next-to-leading order. Positive cooperativity (increased affinity) can be originated by increased binding affinity or by decreased release affinity, implying different kinetics. Nevertheless, the equilibrium state is independent of the origin of cooperativity and only depends on the overall increase in affinity. Next-to-leading approximation is found to be accurate, particularly for small cooperativity. These results allow to understand and characterize relevant ligand binding processes, as the binding kinetics of SSB to ssDNA, which has been reported to affect the DNA replication rate for several SSB-polymerase pairs.

Arabidopsis thaliana PrimPol is a primase and lesion bypass DNA polymerase with the biochemical characteristics to cope with DNA damage in the nucleus, mitochondria, and chloroplast

PrimPol is a novel Primase–Polymerase that synthesizes RNA and DNA primers de novo and extents from these primers as a DNA polymerase. Animal PrimPol is involved in nuclear and mitochondrial DNA replication by virtue of its translesion DNA synthesis (TLS) and repriming activities. Here we report that the plant model Arabidopsis thaliana encodes a functional PrimPol (AtPrimPol). AtPrimPol is a low fidelity and a TLS polymerase capable to bypass DNA lesions, like thymine glycol and abasic sites, by incorporating directly across these lesions or by skipping them. AtPrimPol is also an efficient primase that preferentially recognizes the single-stranded 3′-GTCG-5′ DNA sequence, where the 3′-G is cryptic. AtPrimPol is the first DNA polymerase that localizes in three cellular compartments: nucleus, mitochondria, and chloroplast. In vitro, AtPrimPol synthesizes primers that are extended by the plant organellar DNA polymerases and this reaction is regulated by organellar single-stranded binding proteins. Given the constant exposure of plants to endogenous and exogenous DNA-damaging agents and the enzymatic capabilities of lesion bypass and re-priming of AtPrimPol, we postulate a predominant role of this enzyme in avoiding replication fork collapse in all three plant genomes, both as a primase and as a TLS polymerase.

Bacterial YedK represses plasmid DNA replication and transformation through its DNA single-strand binding activity

The SOS response-associated peptidase (SRAP) is an ancient protein superfamily in all domains of life. The mammalian SRAP was recently reported to covalently bind to the abasic sites (AP) in single stranded (ss) DNA to shield the chromosome integrity. YedK, the Escherichia coli SRAP, is not functionally characterized. Here we report the fortuitous pull-down of YedK from bacterial cell lysates by short (<20 bp) double stranded (ds) DNAs, further enrichment of YedK was observed when single stranded (ss) DNA was added. YedK can bind multiple DNA substrates, particularly with a high affinity to DNA duplex with single strand segment. As a SRAP protein, the involvement of YedK in SOS response was extensively examined, however yedK mutant of Escherichia coli showed no difference from the wild type strain upon the treatments with UV and various DNA damaging reagents, indicating its non-essentiality or redundancy in E. coli. Surprisingly, yedK mutants derived from Escherichia coli and Samonella enterica both showed an increased plasmid DNA transformation efficiency compared to the wild types. In accordance with this, induction of YedK effectively decreased the copy number of plasmid DNA. Site-directed mutagenesis of YedK demonstrated that residues involved in single strand DNA binding and cysteine residue at position 2 from N-terminus can discharge the repression of the plasmid transformation efficiency.

The Simple Biology of Flipons and Condensates Enhances the Evolution of Complexity.

The classical genetic code maps nucleotide triplets to amino acids. The associated sequence composition is complex, representing many elaborations during evolution of form and function. Other genomic elements code for the expression and processing of RNA transcripts. However, over 50% of the human genome consists of widely dispersed repetitive sequences. Among these are simple sequence repeats (SSRs), representing a class of flipons, that under physiological conditions, form alternative nucleic acid conformations such as Z-DNA, G4 quartets, I-motifs, and triplexes. Proteins that bind in a structure-specific manner enable the seeding of condensates with the potential to regulate a wide range of biological processes. SSRs also encode the low complexity peptide repeats to patch condensates together, increasing the number of combinations possible. In situations where SSRs are transcribed, SSR-specific, single-stranded binding proteins may further impact condensate formation. Jointly, flipons and patches speed evolution by enhancing the functionality of condensates. Here, the focus is on the selection of SSR flipons and peptide patches that solve for survival under a wide range of environmental contexts, generating complexity with simple parts.

Plasmid replication based on the T7 origin of replication requires a T7 RNAP variant and inactivation of ribonuclease H.

T7 RNA polymerase (RNAP) is a valuable tool in biotechnology, basic research and synthetic biology due to its robust, efficient and selective transcription of genes. Here, we expand the scope of T7 RNAP to include plasmid replication. We present a novel type of plasmid, termed T7 ori plasmids that replicate, in an engineered Escherichia coli, with a T7 phage origin as the sole origin of replication. We find that while the T7 replication proteins; T7 DNA polymerase, T7 single-stranded binding proteins and T7 helicase-primase are dispensable for replication, T7 RNAP is required, although dependent on a T7 RNAP variant with reduced activity. We also find that T7 RNAP-dependent replication of T7 ori plasmids requires the inactivation of cellular ribonuclease H. We show that the system is portable among different plasmid architectures and ribonuclease H-inactivated E. coli strains. Finally, we find that the copy number of T7 ori plasmids can be tuned based on the induction level of RNAP. Altogether, this study assists in the choice of an optimal genetic tool by providing a novel plasmid that requires T7 RNAP for replication.

Efficient screening for 8-oxoguanine DNA glycosylase binding aptamers via capillary electrophoresis

8-氧代鸟嘌呤DNA糖基化酶(OGG1)是人体中重要的功能蛋白,在修复DNA氧化性损伤过程中起关键作用。氧化应激等引起的氧化损伤易导致炎症反应的发生,对OGG1的抑制可以一定程度上起到缓解作用;对癌细胞OGG1的抑制有望作为癌症治疗的新方法。目前的研究多集中于小分子对OGG1功能的影响和调控,而OGG1的适配体筛选尚未见报道。作为功能配体,适配体具有合成简单、高亲和力及高特异性等优点。该文筛选了OGG1的核酸适配体,结合毛细管电泳高效快速的优点建立了两种基于毛细管电泳-指数富集进化(CE-SELEX)技术的筛选方法:同步竞争法和多轮筛选法。同步竞争法利用单链结合蛋白(SSB)与核酸库中单链核酸的强结合能力,与目标蛋白OGG1组成竞争体系,并通过增加SSB浓度来增加竞争筛选压力,以去除与OGG1弱结合的核酸序列,一步筛选即可获得与OGG1强结合的核酸序列。多轮筛选法在相同孵育条件和电泳条件下,经3轮筛选获得OGG1的核酸适配体。比较两种筛选方法的筛选结果,筛选结果中频次最高的3条候选核酸适配体序列一致,其解离常数(KD)值在1.71~2.64 μmol/L之间。分子对接分析结果表明候选适配体1(Apt 1)可能与OGG1中具有修复氧化性损伤功能的活性口袋结合。通过对两种筛选方法的对比,证明同步竞争法更加快速高效,对其他蛋白核酸适配体筛选方法的选择具有一定的指导意义。得到的适配体有望用于OGG1功能调控,以抑制其修复功能。

Recombinant production of Thermus aquaticus single-strand binding protein for usage as PCR enhancer

Single-stranded DNA-binding (SSB) proteins play an important role in DNA metabolism involving DNA replication, recombination, and repair in all living beings. In molecular biology, SSB proteins are used as enhancers to increase the efficiency and specificity of PCR. Thermostable SSB protein eliminates secondary structure or dimer formation and significantly increase the effectiveness of amplification of DNA fragments. In this study, it was ensured that the SSB gene of thermophilic bacteria Thermus aquaticus (T. aquaticus) was cloned into the pET28b vector and expressed in E. coli BL21 (DE3) PLysE cells. Then, the purification of the SSB protein produced in E. coli BL21 (DE3) PLysE cells was performed. 20 mg SSB protein was obtained from 1L bacterial culture, and its purity was more than 90%. It was shown by the PCR experiment that the SSB protein produced in this study could increase the amplification efficiency.

Identification of gene signatures and potential therapeutic targets for acquired chemotherapy resistance in gastric cancer patients.

Gastric cancer (GC) is the most common type of gastrointestinal cancer, and has been studied extensively. However, resistance to chemotherapeutic agents has become a major problem, leading to treatment failure. This study aimed to investigate the molecular mechanisms mediating acquired resistance to cisplatin and fluorouracil (CF) combination-based chemotherapy in GC patients. The microarray datasets (GSE14209, GSE30070) were downloaded from the Gene Expression Omnibus (GEO) database to identify differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) using the limma package in R/Bioconductor. Possible targets of the DEMs were predicted using miRWalk, and the putative miRNA-mRNA regulatory network was constructed using Cytoscape software. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein-protein interaction (PPI) analyses were then conducted and visualized using the Search Tool for Retrieval of Interacting Genes (STRING) and Cytoscape. The prognostic value of hub genes was revealed by Kaplan-Meier Plotter. The causal relationships and interactions between proteins were displayed using DisNor. Finally, similarity analysis was conducted using the Connectivity Map (CMap) profiles to predict a group of small molecules in GC treatment. A total of 394 DEGs and 31 DEMs were identified after analysis of pre- and post-treatment samples of clinical responders to CF therapy. TM9SF4, hsa-miR-185-5p, and hsa-miR-145-5p were found to be critical in the miRNA-mRNA regulatory network. The DEGs were found to be mainly enriched in the processes of ribonucleoprotein complex assembly, catalytic activity acting on RNA, mitochondrial matrix, and thermogenesis. The DEMs were predominantly found to be involved in single-stranded RNA binding and endoplasmic reticulum lumen. HDAC5, DDX17, ILF3, and SDHC were identified as hub genes in the PPI network. Of these, HDAC5, DDX17, and ILF3 were found to be closely related to the overall survival of GC patients. DisNor identified the first neighbors of the key genes. Furthermore, CMap profiles predicted a group of small molecules, including several histone deacetylase inhibitors (HDACIs), menadione, and mibefradil, which could serve as promising therapeutic agents to reverse acquired resistance to CF therapy. Our findings reveal new targets and alternative therapies to overcome the acquired resistance of GC patients to CF treatment.

Elucidating Recombination Mediator Function Using Biophysical Tools.

Simple SummaryThis review recapitulates the initial knowledge acquired with genetics and biochemical experiments on Recombination mediator proteins in different domains of life. We further address how recent in vivo and in vitro biophysical tools were critical to deepen the understanding of RMPs molecular mechanisms in DNA and replication repair, and unveiled unexpected features. For instance, in bacteria, genetic and biochemical studies suggest a close proximity and coordination of action of the RecF, RecR and RecO proteins in order to ensure their RMP function, which is to overcome the single-strand binding protein (SSB) and facilitate the loading of the recombinase RecA onto ssDNA. In contrary to this expectation, using single-molecule fluorescent imaging in living cells, we showed recently that RecO and RecF do not colocalize and moreover harbor different spatiotemporal behavior relative to the replication machinery, suggesting distinct functions. Finally, we address how new biophysics tools could be used to answer outstanding questions about RMP function.The recombination mediator proteins (RMPs) are ubiquitous and play a crucial role in genome stability. RMPs facilitate the loading of recombinases like RecA onto single-stranded (ss) DNA coated by single-strand binding proteins like SSB. Despite sharing a common function, RMPs are the products of a convergent evolution and differ in (1) structure, (2) interaction partners and (3) molecular mechanisms. The RMP function is usually realized by a single protein in bacteriophages and eukaryotes, respectively UvsY or Orf, and RAD52 or BRCA2, while in bacteria three proteins RecF, RecO and RecR act cooperatively to displace SSB and load RecA onto a ssDNA region. Proteins working alongside to the RMPs in homologous recombination and DNA repair notably belongs to the RAD52 epistasis group in eukaryote and the RecF epistasis group in bacteria. Although RMPs have been studied for several decades, molecular mechanisms at the single-cell level are still not fully understood. Here, we summarize the current knowledge acquired on RMPs and review the crucial role of biophysical tools to investigate molecular mechanisms at the single-cell level in the physiological context.

Single molecule microscopy reveals key physical features of repair foci in living cells.

In response to double strand breaks (DSB), repair proteins accumulate at damaged sites, forming membrane-less sub-compartments or foci. Here we explored the physical nature of these foci, using single molecule microscopy in living cells. Rad52, the functional homolog of BRCA2 in yeast, accumulates at DSB sites and diffuses ~6 times faster within repair foci than the focus itself, exhibiting confined motion. The Rad52 confinement radius coincides with the focus size: foci resulting from 2 DSBs are twice larger in volume that the ones induced by a unique DSB and the Rad52 confinement radius scales accordingly. In contrast, molecules of the single strand binding protein Rfa1 follow anomalous diffusion similar to the focus itself or damaged chromatin. We conclude that while most Rfa1 molecules are bound to the ssDNA, Rad52 molecules are free to explore the entire focus reflecting the existence of a liquid droplet around damaged DNA.