Nucleoprotein Complexes Research Articles

Single-Molecule Fluorescence Imaging of DNA Replication Stalling at Sites of Nucleoprotein Complexes.

DNA replication in cells occurs on crowded and often damaged template DNA, forming potentially deleterious roadblocks to the progressing replication fork. Numerous tools have been developed to investigate the mechanisms of DNA replication and the fate of stalled replication forks. Here, we describe single-molecule fluorescence imaging methods to visualize processive DNA replication and replication fork stalling at site-specific nucleoprotein complexes. Using dCas9 as a protein barrier and rolling-circle DNA templates, we visualize effective, stable, and site-specific blocking of the Escherichia coli replisome. Additionally, we present a protocol to produce an 18-kb rolling-circle DNA template that provides increased spatial resolution in imaging the interplay between replisomes and roadblocks. These methods can be used to investigate encounters of the replisome with nucleoprotein complexes at the single-molecule level, providing important mechanistic details of replisome stalling and downstream rescue or restart pathways.

Telomere sequence variability in genotypes from natural plant populations: unusual block-organized double-monomer terminal telomeric arrays

BackgroundTelomeres are the nucleoprotein complexes that physically cap the ends of eukaryotic chromosomes. Most plants possess Arabidopsis-type telomere sequences (TSs). In addition to terminal TSs, more diverse interstitial TSs exists in plants. Although telomeres have been sufficiently studied, the actual diversity of TSs in land plants is underestimated.ResultsWe investigate genotypes from seven natural populations with contrasting environments of four Chenopodium species to reveal the variability in TSs by analyzing Oxford Nanopore reads. Fluorescent in situ hybridization was used to localize telomeric repeats on chromosomes. We identified a number of derivative monomers that arise in part of both terminal and interstitial telomeric arrays of a single genotype. The former presents a case of block-organized double-monomer telomers, where blocks of Arabidopsis-type TTTAGGG motifs were interspersed with blocks of derivative TTTAAAA motifs. The latter is an integral part of the satellitome with transformations specific to the inactive genome fraction.ConclusionsWe suggested two alternative models for the possible formation of derivative monomers from telomeric heptamer motifs of Arabidopsis-type. It was assumed that derivatization of TSs is a ubiquitous process in the plant genome but occurrence and frequencies of derivatives may be genotype-specific. We also propose that the formation of non-canonical arrays of TSs, especially at chromosomal termini, may be a source for genomic variability in nature.

Rep protein accommodates together dsDNA and ssDNA which enables a loop-back mechanism to plasmid DNA replication initiation.

For DNA replication initiation in Bacteria, replication initiation proteins bind to double-stranded DNA (dsDNA) and interact with single-stranded DNA (ssDNA) at the replication origin. The structural-functional relationship of the nucleoprotein complex involving initiator proteins is still elusive and different models are proposed. In this work, based on crosslinking combined with mass spectrometry (MS), the analysis of mutant proteins and crystal structures, we defined amino acid residues essential for the interaction between plasmid Rep proteins, TrfA and RepE, and ssDNA. This interaction and Rep binding to dsDNA could not be provided in trans, and both are important for dsDNA melting at DNA unwinding element (DUE). We solved two crystal structures of RepE: one in a complex with ssDNA DUE, and another with both ssDNA DUE and dsDNA containing RepE-specific binding sites (iterons). The amino acid residues involved in interaction with ssDNA are located in the WH1 domain in stand β1, helices α1 and α2 and in the WH2 domain in loops preceding strands β1' and β2' and in these strands. It is on the opposite side compared to RepE dsDNA-recognition interface. Our data provide evidence for a loop-back mechanism through which the plasmid replication initiator molecule accommodates together dsDNA and ssDNA.

Causes of DNA Damage and Genomic Instability: A Review

Genomic instability defines all genetic alterations resulting from excessive or high frequency of mutation; base pairs sequence alterations known as microsatellite instability (MSI) and aneuploidy also called chromosome instability (CIN), chromosomal re- arrangements within the genome or the susceptibility of genome to alterations that occur during cell division cycle. The genome of most cancer cells is highly unstable, mostly due to damage to tumour suppressor genes example tp53 which encodes p53 or other genes coordinating cell division. Telomere is a nucleoprotein complex which extends the physical ends of eukaryotic chromosomes, protecting it from degradation, counteracting sequence loss, protecting genes closer to the chromosomes end and inhibiting cell cycle arrest. Studies have implicated the dysfunction of telomere to the formation of sub-tetraploid aneuploid cells that exhibit tumourigenic capacity and formation of anueploid cells that can elude programmed cell death (apoptosis) and form tumour cells, thus initiating genomic instability. In this review, we presented an insight to the causes of DNA damage and its associated genomic instability. We found that many factors such as fragile sites, end replication problem, DNA double strand breaks, DNA replication defects, deletion, insertion and translocation all are responsible to the fragile nature of the genome.

Robust ParB Binding to Half-parS Sites in Pseudomonas aeruginosa-A Mechanism for Retaining ParB on the Nucleoid?

Chromosome segregation in Pseudomonas aeruginosa is assisted by the tripartite ParAB-parS system, composed of an ATPase (ParA), a DNA-binding protein (ParB) and its target parS sequence(s). ParB forms a nucleoprotein complex around four parSs (parS1-parS4) that overlaps oriC and facilitates relocation of newly synthesized ori domains inside the cells by ParA. Remarkably, ParB of P. aeruginosa also binds to numerous heptanucleotides (half-parSs) scattered in the genome. Here, using chromatin immunoprecipitation-sequencing (ChIP-seq), we analyzed patterns of ParB genome occupancy in cells growing under conditions of coupling or uncoupling between replication and cell division processes. Interestingly, a dissipation of ParB-parS complexes and a shift of ParB to half-parSs were observed during the transition from the exponential to stationary phase of growth on rich medium, suggesting the role of half-parSs in retaining ParB on the nucleoid within non-dividing P. aeruginosa cells. The ChIP-seq analysis of strains expressing ParB variants unable to dislocate from parSs showed that the ParB spreading ability is not required for ParB binding to half-parSs. Finally, a P. aeruginosa strain with mutated 25 half-parSs of the highest affinity towards ParB was constructed and analyzed. It showed altered ParB coverage of the oriC region and moderate changes in gene expression. Overall, this study characterizes a novel aspect of conserved bacterial chromosome segregation machinery.

Partition complex structure can arise from sliding and bridging of ParB dimers

In many bacteria, chromosome segregation requires the association of ParB to the parS-containing centromeric region to form the partition complex. However, the structure and formation of this complex have been unclear. Recently, studies have revealed that CTP binding enables ParB dimers to slide along DNA and condense the centromeric region through the formation of DNA bridges. Using semi-flexible polymer simulations, we demonstrate that these properties can explain partition complex formation. Transient ParB bridges organize DNA into globular states or hairpins and helical structures, depending on bridge lifetime, while separate simulations show that ParB sliding reproduces the multi-peaked binding profile observed in Caulobacter crescentus. Combining sliding and bridging into a unified model, we find that short-lived ParB bridges do not impede sliding and can reproduce both the binding profile and condensation of the nucleoprotein complex. Overall, our model elucidates the mechanism of partition complex formation and predicts its fine structure.

Isolation of CP-PVY-Specific siRNA from PVY-Infected Plants of Solanum tuberosum

Tools for activating crop resistance to viruses are now becoming part of a comprehensive plant protection
 strategy. Artificial resistance to viruses through expression of the viral envelope protein in transgenic
 plants is fairly well understood. An urgent issue is the study of small RNAs involved in the protective mechanisms
 of RNA interference against viruses. Understanding the role of short interfering RNA (siRNA) in the
 regulation and shutdown of genes is important. The proteinase accessory component (HC-Pro), a multifunctional
 suppressor protein synthesized by the potato virus Y, is able to neutralize S. tuberosum plant defenses
 by trapping siRNA and removing them from the RNA interference process, thereby causing systemic infection
 of the host plant. Protein liquid chromatography combined with high performance sequencing can help
 recognize the large number of small RNAs resulting from viral RNA degradation and identify 21–23 bp.
 siRNA from PVY-infected S. tuberosum plants. The HC-Pro/siRNA nucleoprotein complex was detected in
 chromatographic fractions using antibodies against HC-Pro, Southern-blot indicated the presence of small
 RNAs in the complex, and analysis of data from deep sequencing of the small RNA population determined a
 specificity of 21–23 bp. siRNA to the envelope protein of the PVY virus. The research results can be applied
 in the study of intracellular signaling molecules and stimulate new research on antiviral mechanisms to
 develop effective strategies for plant protection against viruses.

Modulation of the functional interfaces between retroviral intasomes and the human nucleosome.

Infection by retroviruses as HIV-1 requires the stable integration of their genome into the host cells. This process needs the formation of integrase (IN)-viral DNA complexes, called intasomes, and their interaction with the target DNA wrapped around nucleosomes within cell chromatin. To provide new tools to analyze this association and select drugs, we applied the AlphaLISA technology to the complex formed between the prototype foamy virus (PFV) intasome and nucleosome reconstituted on 601 Widom sequence. This system allowed us to monitor the association between both partners and select small molecules that could modulate the intasome/nucleosome association. Using this approach, drugs acting either on the DNA topology within the nucleosome or on the IN/histone tail interactions have been selected. Within these compounds, doxorubicin and histone binders calixarenes were characterized using biochemical, in silico molecular simulations and cellular approaches. These drugs were shown to inhibit both PFV and HIV-1 integration in vitro. Treatment of HIV-1-infected PBMCs with the selected molecules induces a decrease in viral infectivity and blocks the integration process. Thus, in addition to providing new information about intasome-nucleosome interaction determinants, our work also paves the way for further unedited antiviral strategies that target the final step of intasome/chromatin anchoring. IMPORTANCE In this work, we report the first monitoring of retroviral intasome/nucleosome interaction by AlphaLISA. This is the first description of the AlphaLISA application for large nucleoprotein complexes (>200 kDa) proving that this technology is suitable for molecular characterization and bimolecular inhibitor screening assays using such large complexes. Using this system, we have identified new drugs disrupting or preventing the intasome/nucleosome complex and inhibiting HIV-1 integration both in vitro and in infected cells. This first monitoring of the retroviral/intasome complex should allow the development of multiple applications including the analyses of the influence of cellular partners, the study of additional retroviral intasomes, and the determination of specific interfaces. Our work also provides the technical bases for the screening of larger libraries of drugs targeting specifically these functional nucleoprotein complexes, or additional nucleosome-partner complexes, as well as for their characterization.

Thermotoga maritima oriC involves a DNA unwinding element with distinct modules and a DnaA-oligomerizing region with a novel directional binding mode

Initiation of chromosomal replication requires dynamic nucleoprotein complexes. In most eubacteria, the origin oriC contains multiple DnaA box sequences to which the ubiquitous DnaA initiators bind. In Escherichia coli oriC, DnaA boxes sustain construction of higher-order complexes via DnaA-DnaA interactions, promoting the unwinding of the DNA unwinding element (DUE) within oriC and concomitantly binding the single-stranded (ss) DUE to install replication machinery. Despite the significant sequence homologies among DnaA proteins, oriC sequences are highly diverse. The present study investigated the design of oriC (tma-oriC) from Thermotoga maritima, an evolutionarily ancient eubacterium. The minimal tma-oriC sequence includes a DUE and a flanking region containing five DnaA boxes recognized by the cognate DnaA (tmaDnaA). This DUE was comprised of two distinct functional modules, an unwinding module and a tmaDnaA-binding module. Three direct repeats of the trinucleotide TAG within DUE were essential for both unwinding and ssDUE binding by tmaDnaA complexes constructed on the DnaA boxes. Its surrounding AT-rich sequences stimulated only duplex unwinding. Moreover, head-to-tail oligomers of ATP-bound tmaDnaA were constructed within tma-oriC, irrespective of the directions of the DnaA boxes. This binding mode was considered to be induced by flexible swiveling of DnaA domains III and IV, which were responsible for DnaA-DnaA interactions and DnaA box binding, respectively. Phasing of specific tmaDnaA boxes in tma-oriC was also responsible for unwinding. These findings indicate that a ssDUE recruitment mechanism was responsible for unwinding and would enhance understanding of the fundamental molecular nature of the origin sequences present in evolutionarily divergent bacteria.

Nanopore sensing reveals a preferential pathway for the co-translocational unfolding of a conjugative relaxase-DNA complex.

Bacterial conjugation is the main mechanism for the dissemination of antibiotic resistance genes. A single DNA strand of the conjugative plasmid is transferred across bacterial membranes covalently bound to a large multi-domain protein, named relaxase, which must be unfolded to traverse the secretion channel. Two tyrosine residues of the relaxase (Y18 and Y26 in relaxase TrwC) play an important role in the processing of conjugative DNA. We have used nanopore technology to uncover the unfolding states that take place during translocation of the relaxase-DNA complex. We observed that the relaxase unfolding pathway depends on the tyrosine residue involved in conjugative DNA binding. Transfer of the nucleoprotein complex is faster when DNA is bound to residue Y18. This is the first time in which a protein-DNA complex that is naturally translocated through bacterial membranes has been analyzed by nanopore sensing, opening new horizons to apply this technology to study protein secretion.

High-yield ligation-free assembly of DNA constructs with nucleosome positioning sequence repeats for single-molecule manipulation assays

Force and torque spectroscopy have provided unprecedented insights into the mechanical properties, conformational transitions, and dynamics of DNA and DNA-protein complexes, notably nucleosomes. Reliable single-molecule manipulation measurements require, however, specific and stable attachment chemistries to tether the molecules of interest. Here, we present a functionalization strategy for DNA that enables high-yield production of constructs for torsionally constrained and very stable attachment. The method is based on two subsequent PCRs: first ∼380bp long DNA strands are generated that contain multiple labels, which are used as "megaprimers" in a second PCR to generate ∼kbp long double-stranded DNA constructs with multiple labels at the respective ends. To achieve high-force stability, we use dibenzocyclooctyne-based click chemistry for covalent attachment to the surface and biotin-streptavidin coupling to the bead. The resulting tethers are torsionally constrained and extremely stable under load, with an average lifetime of 70± 3h at 45 pN. The high yield of the approach enables nucleosome reconstitution by salt dialysis on the functionalized DNA, and we demonstrate proof-of-concept measurements on nucleosome assembly statistics and inner turn unwrapping under force. We anticipate that our approach will facilitate a range of studies of DNA interactions and nucleoprotein complexes under forces and torques.

Telomere loss is accompanied by decreased pool of shelterin proteins TRF2 and RAP1, elevated levels of TERRA and enhanced glycolysis in imatinib-resistant CML cells

Telomere length may be maintained by telomerase nucleoprotein complex and shelterin complex, namely TRF1, TRF2, TIN2, TPP1, POT1 and RAP1 proteins and modulated by TERRA expression. Telomere loss is observed during progression of chronic myeloid leukemia (CML) from the chronic phase (CML-CP) to the blastic phase (CML-BP). The introduction of tyrosine kinase inhibitors (TKIs), such as imatinib (IM), has changed outcome for majority of patients, however, a number of patients treated with TKIs may develop drug resistance. The molecular mechanisms underlying this phenomenon are not fully understood and require further investigation. In the present study, we demonstrate that IM-resistant BCR::ABL1 gene-positive CML K-562 and MEG-A2 cells are characterized by decreased telomere length, lowered protein levels of TRF2 and RAP1 and increased expression of TERRA in comparison to corresponding IM-sensitive CML cells and BCR::ABL1 gene-negative HL-60 cells. Furthermore, enhanced activity of glycolytic pathway was observed in IM-resistant CML cells. A negative correlation between a telomere length and advanced glycation end products (AGE) was also revealed in CD34+ cells isolated from CML patients. In conclusion, we suggest that affected expression of shelterin complex proteins, namely TRF2 and RAP1, TERRA levels, and glucose consumption rate may promote telomere dysfunction in IM-resistant CML cells.

Comparative Analysis of Molecular Functions and Biological Role of Proteins from Cell-Free DNA-Protein Complexes Circulating in Plasma of Healthy Females and Breast Cancer Patients.

Cell-free DNA (cfDNA) circulates in the bloodstream packed in membrane-coated structures (such as apoptotic bodies) or bound to proteins. To identify proteins involved in the formation of deoxyribonucleoprotein complexes circulating in the blood, native complexes were isolated using affinity chromatography with immobilized polyclonal anti-histone antibodies from plasma of healthy females (HFs) and breast cancer patients (BCPs). It was found that the nucleoprotein complexes (NPCs) from HF plasma samples contained shorter DNA fragments (~180 bp) than BCP NPCs. However, the share of DNA in the NPCs from cfDNA in blood plasma in HFs and BCPs did not differ significantly, as well as the share of NPC protein from blood plasma total protein. Proteins were separated by SDS-PAGE and identified by MALDI-TOF mass spectrometry. Bioinformatic analysis showed that in the presence of a malignant tumor, the proportion of proteins involved in ion channels, protein binding, transport, and signal transduction increased in the composition of blood-circulating NPCs. Moreover, 58 (35%) proteins are differentially expressed in a number of malignant neoplasms in the NPCs of BCPs. Identified NPC proteins from BCP blood can be recommended for further testing as breast cancer diagnostic/prognostic biomarkers or as being useful in developing gene-targeted therapy approaches.

Recruitment of heterologous substrates by bacterial secretion systems for transkingdom translocation.

Bacterial secretion systems mediate the selective exchange of macromolecules between bacteria and their environment, playing a pivotal role in processes such as horizontal gene transfer or virulence. Among the different families of secretion systems, Type III, IV and VI (T3SS, T4SS and T6SS) share the ability to inject their substrates into human cells, opening up the possibility of using them as customized injectors. For this to happen, it is necessary to understand how substrates are recruited and to be able to engineer secretion signals, so that the transmembrane machineries can recognize and translocate the desired substrates in place of their own. Other factors, such as recruiting proteins, chaperones, and the degree of unfolding required to cross through the secretion channel, may also affect transport. Advances in the knowledge of the secretion mechanism have allowed heterologous substrate engineering to accomplish translocation by T3SS, and to a lesser extent, T4SS and T6SS into human cells. In the case of T4SS, transport of nucleoprotein complexes adds a bonus to its biotechnological potential. Here, we review the current knowledge on substrate recognition by these secretion systems, the many examples of heterologous substrate translocation by engineering of secretion signals, and the current and future biotechnological and biomedical applications derived from this approach.

The CTP-binding domain is disengaged from the DNA-binding domain in a cocrystal structure of Bacillus subtilis Noc–DNA complex

In Bacillus subtilis, a ParB-like nucleoid occlusion protein (Noc) binds specifically to Noc-binding sites (NBSs) on the chromosome to help coordinate chromosome segregation and cell division. Noc does so by binding to CTP to form large membrane-associated nucleoprotein complexes to physically inhibit the assembly of the cell division machinery. The site-specific binding of Noc to NBS DNA is a prerequisite for CTP-binding and the subsequent formation of a membrane-active DNA-entrapped protein complex. Here, we solve the structure of a C-terminally truncated B.subtilis Noc bound to NBS DNA to reveal the conformation of Noc at this crucial step. Our structure reveals the disengagement between the N-terminal CTP-binding domain and the NBS-binding domain of each DNA-bound Noc subunit; this is driven, in part, by the swapping of helices 4 and 5 at the interface of the two domains. Site-specific crosslinking data suggest that this conformation of Noc-NBS exists in solution. Overall, our results lend support to the recent proposal that parS/NBS binding catalyzes CTP binding and DNA entrapment by preventing the reengagement of the CTP-binding domain and the DNA-binding domain from the same ParB/Noc subunit.

Molecular Modeling of the Anti-HIV Activity Mechanism of Iodine-Containing Drugs Armenicum and FS-1.

Drugs Armenicum and FS-1 are a solution of ionic nanostructured complexes of α-dextrin. In the active centers of these drugs, located inside the dextrin helix, molecular iodine has such an electronic form that minimizes toxic effects in the human body, so these drugs can be used for parenteral and oral administration. On the human lymphoblastoid cell line MT-2, the effect of the antiviral action of FS-1 against HIV-1 was established. Literature data on the results of treatment of people with HIV infection with Armenicum are presented. The mechanism of anti-HIV action of drugs Armenicum and FS-1 was proposed by the molecular modeling method. Using the DFT/B3PW91/6-31G** approach, it was shown that LiI(Cl)I2 active center drugs of Armenicum and FS-1 can be segregated from the dextrin helix and can form a complex with the ACT nucleotide triplet, which is part of a specific fragment of viral DNA that binds to the active center of integrase. The formation of this complex is a key moment in the mechanism of anti-HIV drug action. Molecular iodine and lithium halide, which are part of the active complexes, inhibit the active center of the catalytic domain of the integrase. A new nucleoprotein complex is created that destroys the nucleoprotein preintegration complex (PIC) and inhibits the HIV DNA and the active center of the catalytic domain, while a new N-I bond appears in the viral DNA in the cytosine pyrimidine cycle.

Association of alcohol consumption with telomere length in humans: a systematic review

Telomeres are complex nucleoprotein structures with specific proteins of noncoding terminal regions of linear chromosomes of eukaryotic cells. Telomere DNA consists of a large number of short sequence repeats (TTAGGG in vertebrates). Telomeres protect chromosomes from their fusion and degradation, limit the proliferative potential of the cell, participate in the segregation of chromosomes during cell division, etc. Reduction of telomeres length is an important factor with significant impact on cell viability and function, aging, and leads to the development of various diseases including cancer.
 Alcohol abuse has a significant impact on a person's health. Ethanol consumption by a human potentially affects the length of chromosome telomeres on the cellular level.
 Current review represents systematic analysis of studies on the effect of alcohol consumption on telomere length in humans. PubMed and eLIBRARY.RU databases were explored for the combinations of the terms ("Ethanol" OR "Alcohol") AND "Telomer" with a limitation on the publication date until 01 January 2011. The search resulted in 269 studies. In accordance with the preset criteria, total 238 studies were excluded from the analysis, and 3 publications were excluded due to unavailability of full text. A total of 28 epidemiological and clinical studies were included for this study review.
 The association of alcohol consumption with shortening of telomeres was reported in 16 of the studies conducted with various populations and cohorts including individuals with alcohol abuse, alcohol dependence, and some genetic variants of alcohol metabolism enzymes. 12 studies reported alcohol consumption was not associated with change in telomere length.
 The analysis of reviewed studies allows to conclude that they are ambiguous and that there is further urgency to study the effect of alcohol on telomere length by engaging modern methods for its determination.

Oct4 interprets and enhances nucleosome dynamics.

Transcription factors are proteins that bind to DNA to regulate gene expression. Historically, DNA accessibility has been considered a prerequisite indispensable for transcription factor function. Nevertheless, in recent years certain transcription factors have been shown to have “pioneering activity”, meaning that they can bind their specific binding sites in packed regions of the genome, where the DNA is wrapped around a nucleoprotein complex called the nucleosome. Oct4 is a pioneer transcription factor, involved in pluripotency maintenance and that can be used for reprogramming of adult cells into stem-cell like cells. Using experimental data, we built models of Oct4 bound to two different native nucleosomes. We then performed over 50 µs of atomistic simulations of the Oct4-nucleosome complexes, which we compared to our already existing library of 25 µs of nucleosome alone simulations. We found that nucleosome flexibility is a requirement for a multidomain transcription factor like Oct4 to bind. Furthermore, we described two different mechanisms in which Oct4 can alter nucleosome dynamics, either by stabilizing naturally occurring nucleosome opening events or inducing large opening events not seen in the nucleosome alone simulations. The atomistic resolution allowed us to observe an interplay between the histone tails and the two DNA binding domains of Oct4 for the opening events to occur. Overall, our findings provide a mechanistic description of the effects of a pioneer transcription factor bound to a nucleosome, at an unprecedented resolution. This work allows us to begin understanding the changes of genome architecture that happen upon expression and binding of certain transcription factors, and ultimately gives us the tools to understand cell-fate transitions.

Thermodynamics of E. coli SSB phase separation. Effects of monovalent salt concentration and type and intrinsically disordered linker modifications.

E. coli single strand (ss) DNA binding protein (SSB) is an essential protein that binds ssDNA intermediates formed during genome maintenance. Depending on solution conditions SSB can bind to ssDNA in different binding modes characterized by different extent of DNA wrapping around the SSB tetramer and different cooperativities. We and others showed recently that SSB can undergo salt dependent phase separation (PS) which is facilitated by increases in concentration of the physiological salt, KGlu, while inhibited by [KCl] and/or deletion of the intrinsically disordered linker ( IDL) within the SSB C-terminal tail. These same factors influence non-nearest neighbor (NNN) cooperative binding of SSB to long ssDNA resulting in collapse of the nucleoprotein complexes. Here we report a thermodynamic study of EcSSB PS over a broad range of [KCl] and [KGlu] and find that PS is driven by favorable enthalpy with unfavorable entropy changes. While the effects of [KGlu] and [KCl] are similar at low salt concentrations, dramatic differences are observed at [salt] above 0.1 M. At low [salt] PS is driven mostly by electrostatic (coulombic) interactions, whereas at high [salt] Hofmeister effects dominate involving weak preferential interactions relative to water. We also have examined the effects on PS of amino acid sequence and composition of the IDL. The results are discussed in relation with SSB function in E. coli (supported by NIH GM136632 to TML and GM118100 to MTR).

Identification of Proximity Interactors of Mammalian Nucleoid Proteins by BioID.

Mitochondrial nucleoids are compact nucleoprotein complexes, in which mtDNA is located, replicated, and transcribed. Several proteomic approaches have been previously employed to identify nucleoid proteins; however, a consensus list of nucleoid-associated proteins has not been generated. Here we describe a proximity-biotinylation assay, BioID, which allows identification of proximity interactors of mitochondrial nucleoid proteins. It uses a promiscuous biotin ligase fused to a protein of interest which covalently attaches biotin to lysine residues of its proximal neighbors. Biotinylated proteins can be further enriched by a biotin-affinity purification and identified by mass-spectrometry. BioID can identify transient and weak interactions and can be used to identify changes in the interactions upon different cellular treatments, for different protein isoforms or for pathogenic variants.