Formation Of DNA-protein Complexes Research Articles

In Vitro Determination of Temperature-Dependent DNA Binding of the Evening Complex Using Electrophoretic Mobility Shift Assays.

Electrophoretic mobility shift assays (EMSAs) of DNA-binding proteins and labeled DNA allow the qualitative and quantitative characterization of protein-DNA complex formation using native (nondenaturing) polyacrylamide or agarose gel electrophoresis. By varying the incubation temperature of the protein-DNA binding reaction and maintaining this temperature during electrophoresis, temperature-dependent protein-DNA interactions can be investigated. Here, we provide examples of the binding of a transcriptional repressor complex called the Evening Complex, comprising the DNA-binding protein LUX ARRYTHMO (LUX), the scaffold protein EARLY FLOWERING 3 (ELF3), and the adapter protein ELF4, to its cognate DNA and demonstrate direct detection and visualization of thermoresponsive binding in vitro. As negative controls we use the LUX DNA-binding domain and LUX full length protein, which do not exhibit temperature-dependent DNA binding.

Gallic acid inhibits Staphylococcus aureus RecA protein functions: role in countering antibiotic resistance in bacteria.

Recombinase RecA and its homologs play a key role in homologous recombination DNA repair and revive stalled replication fork DNA synthesis. RecA plays an essential role in the evolution of antibiotic-resistant strains via stress-induced DNA repair mechanisms during the SOS response. Accordingly, RecA has become an attractive target to slow down antibiotic resistance rates and prevent mutations in pathogenic bacterial species. We employed RecA conserved activities: DNA binding, displacement loop formation, strand exchange, ATP hydrolysis, and LexA cleavage, to elucidate the inhibitory role of gallic acid on S. aureus RecA functions. Gallic acid inhibition of the SOS response by western blot analysis and its antibacterial activity were measured. The gallic acid inhibited all the canonical activities of S. aureus RecA protein. The natural phenolic compound gallic acid interferes with RecA protein DNA complex formation and inhibits activities such as displacement loop formation, strand exchange reaction, ATP hydrolysis, and coprotease activity of S. aureus. Additionally, gallic acid can obstruct ciprofloxacin-induced RecA expression and eventually confer the inhibitory role of gallic acid in the SOS survival mechanism in S. aureus.

Base Dynamics in the HhaI Protein Binding Site.

Protein-DNA interactions play an important role in numerous biological functions within the living cell. In many of these interactions, the DNA helix is significantly distorted upon protein-DNA complex formation. The HhaI restriction-modification system is one such system, where the methylation target is flipped out of the helix when bound to the methyltransferase. However, the base flipping mechanism is not well understood. The dynamics of the binding site of the HhaI methyltransferase and endonuclease (underlined) within the DNA oligomer [d(G1A2T3A4G5C6G7C8T9A10T11C12)]2 are studied using deuterium solid-state NMR (SSNMR). SSNMR spectra obtained from DNAs deuterated on the base of nucleotides within and flanking the [5'-GCGC-3']2 sequence indicate that all of these positions are structurally flexible. Previously, conformational flexibility within the phosphodiester backbone and furanose ring within the target sequence has been observed and hypothesized to play a role in the distortion mechanism. However, whether that distortion was occurring through an active or passive mechanism remained unclear. These NMR data demonstrate that although the [5'-GCGC-3']2 sequence is dynamic, the target cytosine is not passively flipping out of the double-helix on the millisecond-picosecond time scale. Additionally, although previous studies have shown that both the furanose ring and phosphodiester backbone experience a change in dynamics upon methylation, which may play a role in recognition and cleavage by the endonuclease, our observations here indicate that methylation has no effect on the dynamics of the base itself.

Sequence-Dependent Interaction of the Human Papillomavirus E2 Protein with the DNA Elements on Its DNA Replication Origin.

The human papillomavirus (HPV) E2 protein is essential for regulating the initiation of viral DNA replication as well as the regulation of transcription of certain HPV-encoded genes. Its ability to recognize and bind to its four recognition sequences in the viral origin is a key step in the initiation of HPV DNA replication. Thus, understanding the mechanism of DNA binding by E2 protein and the unique roles played by individual DNA sequence elements of the replication origin is essential. We have purified the recombinant full-length HPV type 11 E2 protein. Quantitative DNA binding analysis indicated E2 protein bound all four DNA binding sites with reasonably high affinities but with distinct preferences. It bound its cognate binding sites 1, 2, and 4 with higher affinities, but bound binding site 3 with lower affinity. Analysis of binding to these sites unraveled multiple sequence elements that appeared to influence E2 binding affinity and target discrimination, including the sequence of spacer region, flanking sequences, and proximity of E2 binding sites. Thermodynamic analysis indicated hydrophobic interaction in the protein-DNA complex formation. Our studies indicate a large multi-protein complex formation on the HPV-origin DNA, likely due to reasonably high binding affinities as well as intrinsic oligomerization propensity of E2 dimers.

Evolutionary Invariant of the Structure of DNA Double Helix in RNAP II Core Promoters.

Eukaryotic and archaeal RNA polymerase II (POL II) machinery is highly conserved, regardless of the extreme changes in promoter sequences in different organisms. The goal of our work is to find the cause of this conservatism. The representative sets of aligned promoter sequences of fifteen organisms belonging to different evolutional stages were studied. Their textual profiles, as well as profiles of the indexes that characterize the secondary structure and the mechanical and physicochemical properties, were analyzed. The evolutionarily stable, extremely heterogeneous special secondary structure of POL II core promoters was revealed, which includes two singular regions—hexanucleotide “INR” around TSS and octanucleotide “TATA element” of about −28 bp upstream. Such structures may have developed at some stage of evolution. It turned out to be so well matched for the pre-initiation complex formation and the subsequent initiation of transcription for POL II machinery that in the course of evolution there were selected only those nucleotide sequences that were able to reproduce these structural properties. The individual features of specific sequences representing the singular region of the promoter of each gene can affect the kinetics of DNA-protein complex formation and facilitate strand separation in double-stranded DNA at the TSS position.

WRKY transcription factors: evolution, regulation, and functional diversity in plants.

The recent advancements in sequencing technologies and informatic tools promoted a paradigm shift to decipher the hidden biological mysteries and transformed the biological issues into digital data to express both qualitative and quantitative forms. The transcriptomic approach, in particular, has added new dimensions to the versatile essence of plant genomics through the large and deep transcripts generated in the process. This has enabled the mining of super families from the sequenced plants, both model and non-model, understanding their ancestry, diversity, and evolution. The elucidation of the crystal structure of the WRKY proteins and recent advancement in computational prediction through homology modeling and molecular dynamic simulation has provided an insight into the DNA-protein complex formation, stability, and interaction, thereby giving a new dimension in understanding the WRKY regulation. The present review summarizes the functional aspects of the high volume of sequence data of WRKY transcription factors studied from different species, till date. The review focuses on the dynamics of structural classification and lineage in light of the recent information. Additionally, a comparative analysis approach was incorporated to understand the functions of the identified WRKY transcription factors subjected to abiotic (heat, cold, salinity, senescence, dark, wounding, UV, and carbon starvation) stresses as revealed through various sets of studies on different plant species. The review will be instrumental in understanding the events of evolution and the importance of WRKY TFs under the threat of climate change, considering the new scientific evidences to propose a fresh perspective.

Facilitated dissociation of nucleoid-associated proteins from DNA in the bacterial confinement

Transcription machinery depends on the temporal formation of protein-DNA complexes. Recent experiments demonstrated that not only the formation but also the lifetime of such complexes can affect the transcriptional machinery. In parallel, in vitro single-molecule studies showed that nucleoid-associated proteins (NAPs) leave the DNA rapidly as the bulk concentration of the protein increases via facilitated dissociation (FD). Nevertheless, whether such a concentration-dependent mechanism is functional in a bacterial cell, in which NAP levels and the 3d chromosomal structure are often coupled, is not clear a priori. Here, by using extensive coarse-grained molecular simulations, we model the unbinding of specific and nonspecific dimeric NAPs from a high-molecular-weight circular DNA molecule in a cylindrical structure mimicking the cellular confinement of a bacterial chromosome. Our simulations confirm that physiologically relevant peak protein levels (tens of micromolar) lead to highly compact chromosomal structures. This compaction results in rapid off rates (shorter DNA residence times) for specifically DNA-binding NAPs, such as the factor for inversion stimulation, which mostly dissociate via a segmental jump mechanism. Contrarily, for nonspecific NAPs, which are more prone to leave their binding sites via 1d sliding, the off rates decrease as the protein levels increase. The simulations with restrained chromosome models reveal that chromosome compaction is in favor of faster dissociation but only for specific proteins, and nonspecific proteins are not affected by the chromosome compaction. Overall, our results suggest that the cellular concentration level of a structural DNA-binding protein can be highly intermingled with its DNA residence time.

Tumuc1: A New Accurate DNA Force Field Consistent with High-Level Quantum Chemistry.

An accurate molecular mechanics force field forms the basis of Molecular Dynamics simulations to obtain a realistic view of the structure and dynamics of biomolecules such as DNA. Although frequently updated to improve agreement with available experimental data, DNA force fields still rely in part on parameters introduced more than 20 years ago. We have developed an entirely new DNA force field, Tumuc1, derived from quantum mechanical calculations to obtain a consistent set of bonded parameters and partial atomic charges. The performance of the force field was extensively tested on a variety of DNA molecules. It excels in accuracy of B-DNA simulations but also performs very well on other types of DNA structures and structure formation processes such as hairpin folding, duplex formation, and dynamics of DNA-protein complexes. It can complement existing force fields in order to provide an increasingly accurate description of the structure and dynamics of DNA during simulation studies.

Impact of Self-Association on the Architectural Properties of Bacterial Nucleoid Proteins

The chromosomal DNA of bacteria is folded into a compact body called the nucleoid, which is composed essentially of DNA (∼80%), RNA (∼10%), and a number of different proteins (∼10%). These nucleoid proteins act as regulators of gene expression and influence the organization of the nucleoid by bridging, bending, or wrapping the DNA. These so-called architectural properties of nucleoid proteins are still poorly understood. For example, the reason why certain proteins compact the DNA coil in certain environments but make the DNA more rigid instead in other environments is the subject of ongoing debates. Here, we address the question of the impact of the self-association of nucleoid proteins on their architectural properties and try to determine whether differences in self-association are sufficient to induce large changes in the organization of the DNA coil. More specifically, we developed two coarse-grained models of proteins, which interact identically with the DNA but self-associate differently by forming either clusters or filaments in the absence of the DNA. We showed through Brownian dynamics simulations that self-association of the proteins dramatically increases their ability to shape the DNA coil. Moreover, we observed that cluster-forming proteins significantly compact the DNA coil (similar to the DNA-bridging mode of H-NS proteins), whereas filament-forming proteins significantly increase the stiffness of the DNA chain instead (similar to the DNA-stiffening mode of H-NS proteins). This work consequently suggests that the knowledge of the DNA-binding properties of the proteins is in itself not sufficient to understand their architectural properties. Rather, their self-association properties must also be investigated in detail because they might actually drive the formation of different DNA-protein complexes.

A Protein in the Yeast Saccharomyces cerevisiae Presents DNA Binding Homology to the p53 Checkpoint Protein and Tumor Suppressor.

Saccharomyces cerevisiae does not contain a p53 homolog. Utilizing this yeast as an in vivo test tube model, our aim was to investigate if a yeast protein would show p53 DNA binding homology. Electrophoretic mobility shift analyses revealed the formation of specific DNA-protein complexes consisting of S. cerevisiae nuclear protein(s) and oligonucleotides containing p53 DNA binding sites. A S. cerevisiae p53 binding site factor (Scp53BSF) bound to a p53 synthetic DNA-consensus sequence (SCS) and a p53 binding-site sequence from the MDM2 oncogene. The complexes were of comparable size. Like mammalian p53, the affinity of Scp53BSF for the SCS oligonucleotide was higher than for the MDM2 oligonucleotide. Binding of Scp53BSF to the SCS and MDM2 oligonucleotides was strongly competed by unlabeled oligonucleotides containing mammalian p53 sites, but very little by a mutated site oligonucleotide. Importantly, Scp53BSF-DNA binding activity was significantly induced in extracts from cells with DNA damage. This resulted in dose-dependent coordinated activation of transcription when using p53-binding site reporter constructs. An ancient p53-like DNA binding protein may have been found, and activation of DNA-associated factors to p53 response elements may have functions not yet determined.

Abstract P3-11-12: Epigenetic modulation to deepen and prolong the response to PARP inhibitors

Abstract Background: BRCA1 and BRCA2 are key mediators of DNA damage repair response including the homologous recombination repair (HRR) pathway. Mutations in these genes predispose women to various cancers including breast, ovarian and pancreatic cancers. A new class of drugs, the poly (ADP-ribose) polymerase inhibitors (PARPi), were designed to leverage impaired DNA repair in BRCA-mutated tumors and are FDA approved for various cancers (breast, ovarian). Efforts to combine PARPi with cytotoxic agents show increased efficacy but overlapping toxicities hamper their tolerability. Further, the short duration of response to PARPi monotherapy in diseases like breast cancer has inspired the search for PARPi combinations with other agents including epigenetic modifiers. To overcome these hurdles, we explored synergistic interactions between PARPi and DNA methyltransferases inhibitors (DNMTi) such as decitabine, a cytidine analog. When incorporated in newly synthesized DNA strands during S phase, these agents will trigger a covalent protein-DNA complex formation causing cell cycle arrest and cell death. Material & Methods: BRCA wild-type (WT) and BRCA-mutated breast cancer cell lines were used for in vitro analyses: SUM149PT and HCC-3153 (BRCA1-mutated), MDA-MB-231 and BT-549 (BRCA-WT), UCRP231A (a CRISPR/Cas9 engineered BRCA2-mutated from parental MDA-MB-231), and MCF10A (a BRCA-WT non-tumorigenic breast cell line). The combined effect of talazoparib (PARPi) and decitabine (DNMTi) was analyzed using Combenefit® software to determine their synergistic interaction. Cell cycle analyses, dead/alive cells ratio analyses, as well as pH2AX levels were evaluated. In vivo analyses included xenograft models with SUM149PT as well as the parental BRCA-WT MDA-MB-231 and BRCA2-mutated MDA-MB-231 (UCRP231A) cells in immunodeficient NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ mice. Results: Our preclinical data in BRCA-deficient breast cancer cell lines, demonstrated a synergistic inhibition of cell growth and enhancement of cell death at concentrations of talazoparib and decitabine where each agent individually had minimal efficacy. In cells with intact HRR pathways, the drug combination showed an enhanced inhibition of cell growth but was not synergistic in inducing cell death compared to BRCA deficient cell lines. This difference was further manifested by different cell cycle profiles: non-BRCA mutated cells arrested in S-phase while BRCA-mutated cells progressed through S-phase without arrest and entered cell death from G2/M. In vivo mouse data showed a significant synergistic interaction between the two drugs without enhanced toxicity. Conclusion: The ability to induce a synergistic interaction between talazoparib and decitabine at low drug concentrations present a promising therapeutic strategy for patients with HRR-deficient tumors. Ongoing experiments are being conducted to generate additional engineered BRCA- and other HRR-deficient cell lines (ATM, CHEK2 and others) through CRISPR/Cas9 DNA editing system and further delineate the differential mechanism of interaction between the HRR-deficient and HRR-intact cell lines in preparation for clinical testing. Citation Format: Romain Pacaud, Mallika Dhawan, Scott Thomas, Elysia Roche, Jose Garcia, Nela Pawlowska, Pamela Munster. Epigenetic modulation to deepen and prolong the response to PARP inhibitors [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-12.

The effect of obstacles in multi-site protein target search with DNA looping

Many fundamental biological processes are regulated by protein-DNA complexes called synaptosomes, which possess multiple interaction sites. Despite the critical importance of synaptosomes, the mechanisms of their formation are not well understood. Because of the multisite nature of participating proteins, it is widely believed that their search for specific sites on DNA involves the formation and breaking of DNA loops and sliding in the looped configurations. In reality, DNA in live cells is densely covered by other biological molecules that might interfere with the formation of synaptosomes. In this work, we developed a theoretical approach to evaluate the role of obstacles in the target search of multisite proteins when the formation of DNA loops and the sliding in looped configurations are possible. Our theoretical method is based on analysis of a discrete-state stochastic model that uses a master equations approach and extensive computer simulations. It is found that the obstacle slows down the search dynamics in the system when DNA loops are long-lived, but the effect is minimal for short-lived DNA loops. In addition, the relative positions of the target and the obstacle strongly influence the target search kinetics. Furthermore, the presence of the obstacle might increase the noise in the system. These observations are discussed using physical-chemical arguments. Our theoretical approach clarifies the molecular mechanisms of formation of protein-DNA complexes with multiple interactions sites.

Type 2 diabetes-associated polymorphisms correlate with SIRT1 and TGF-β1 gene expression.

The polymorphisms rs3758391 and rs1800470 located in SIRT1 and TGF-β1 have been associated with type 2 diabetes in different populations but its functional effect is not clear. In this study, we evaluated their effect on the expression of SIRT1 and TGF-β1 in peripheral blood as well as their participation in the formation of DNA-protein complexes in a pancreas-derived cell line. It has been described that SIRT1 and TGF-β1 participate in cell growth and regulation of production and secretion of insulin in the pancreas. Anthropometric and biochemical profiles of 127 adults were measured. Genotypes for rs3758391 and rs1800470 were determined using TaqMan assays. Expression analysis of SIRT1 and TGF-β1 were performed using real-time PCR. Gene expression of these genes increased 1.8 ± 0.6- and 1.3 ± 0.6-fold in patients carrying the TT genotype of rs3758391 and rs1800470 when compared to carriers of the CC genotype. Then, we tested whether these single-nucleotide polymorphisms (SNPs) (and rs932658, which is in linkage disequilibrium with rs3758391) are located in regulatory DNA-protein binding sites by electrophoretic mobility shift assays using nuclear extract from the pancreas-derived cell line BxPC-3. The electrophoretic mobility shift assay showed no binding of nuclear proteins to DNA. In conclusion, the genotypes of rs3758391 and rs1800470 are associated with modifications in the expression of the genes SIRT1 and TGF-β1, respectively, but none of the tested SNPs are located in regulatory DNA-protein binding sites.

In Situ Analysis of DNA-Protein Complex Formation upon Radiation-Induced DNA Damage.

The importance of determining at the cellular level the formation of DNA–protein complexes after radiation-induced lesions to DNA is outlined by the evidence that such interactions represent one of the first steps of the cellular response to DNA damage. These complexes are formed through recruitment at the sites of the lesion, of proteins deputed to signal the presence of DNA damage, and of DNA repair factors necessary to remove it. Investigating the formation of such complexes has provided, and will probably continue to, relevant information about molecular mechanisms and spatiotemporal dynamics of the processes that constitute the first barrier of cell defense against genome instability and related diseases. In this review, we will summarize and discuss the use of in situ procedures to detect the formation of DNA-protein complexes after radiation-induced DNA damage. This type of analysis provides important information on the spatial localization and temporal resolution of the formation of such complexes, at the single-cell level, allowing the study of heterogeneous cell populations.

Signal-switchable lab-on-paper photoelectrochemical aptasensing system integrated triple-helix molecular switch with charge separation and recombination regime of type-II CdTe@CdSe core-shell quantum dots

Herein, a new “on-off-on” signal switch system combined triple helix molecular switch with efficient charge separation and transfer between different sensitization units was designed for the ultrasensitive photoelectrochemical (PEC) determination of prostate-specific antigen (PSA). Concretely, the initial “signal-on” state was obtained via the cascaded sensitization structure consisting of type-II CdTe@CdSe core-shell quantum dots (QDs), CdS QDs, and ZnO nanotubes, which were assembled on Au nanoparticles modified paper fibers with the aid of signal transduction probe (STP). Thereinto, the type-II CdTe@CdSe QDs with hole-localizing core and electron-localizing shell could enable the ultrafast charge transfer and retard the charge recombination, magnifying the initial photocurrent response and preserving the high efficiency of signal-switchable PEC aptasensing system. Subsequently, the PSA aptamer (PSA-Apt) modified with gold nanoparticles (GNPs) was introduced by the hybridization of PSA-Apt with STP and the hairpin configuration of STP changed from closed to open state, forming a triple-helix structure. Hence, the CdTe@CdSe QDs labeled on the terminal of STP moved away from the electrode surface while the GNPs kept attached close to it. The proposed aptasensor turned to “signal-off” state because of the dual inhibition of vanished cosensitization effect and signal quenching effect of GNPs. Upon the target recognition, the triple-helix structure was perturbed with the formation of DNA-protein complex and the recovery of STP hairpin structure, resulting in the second “switch-on” state. Based on the target-induced photocurrent enhancement, the proposed PEC aptasensor was utilized for the determination of PSA with high sensitivity, persuasive selectivity, and excellent stability.

Post-translational Modifications of Nucleotide Excision Repair Proteins and Their Role in the DNA Repair.

Nucleotide excision repair (NER) is one of the major DNA repair pathways aimed at maintaining genome stability. Correction of DNA damage by the NER system is a multistage process that proceeds with the formation of multiple DNA-protein and protein-protein intermediate complexes and requires precise coordination and regulation. NER proteins undergo post-translational modifications, such as ubiquitination, sumoylation, phosphorylation, acetylation, and poly(ADP-ribosyl)ation. These modifications affect the interaction of NER factors with DNA and other proteins and thus regulate either their recruitment into the complexes or dissociation from these complexes at certain stages of DNA repair, as well as modulate the functional activity of NER proteins and control the process of DNA repair in general. Here, we review the data on the post-translational modifications of NER factors and their effects on DNA repair. Protein poly(ADP-ribosyl)ation catalyzed by poly(ADP-ribose) polymerase 1 and its impact on NER are discussed in detail, since such analysis has not been done before.

Association of Genetic Variants of RANK, RANKL, and OPG with Ankylosing Spondylitis Clinical Features in Taiwanese.

Ankylosing spondylitis (AS) is a chronic inflammatory disease that leads to spinal ankylosis. The receptor activator of the nuclear factor-kappa (RANK), RANK ligand, and osteoprotegerin (OPG) (RANK/RANKL/OPG) pathway plays critical roles in bone metabolism and the immune system. The current study was aimed at investigating whether six single-nucleotide polymorphisms (SNPs) within the RANK, RANKL, and OPG genes essential for bone homeostasis are associated with AS. Genotype distributions, allele and haplotype frequencies, were compared between 1120 AS patients and 1435 healthy controls and among AS patients with stratification by syndesmophyte formation, onset age, and HLA-B27 positivity. We found that RANKL SNPs were associated with AS syndesmophyte formation. Notably, the RANKL SNP haplotype rs7984870C/rs9533155G/rs9525641C was negatively associated with AS susceptibility and appeared to protect against syndesmophyte formation in AS. Functionally, RANKL promoter SNPs (rs9525641 C/T and rs9533155 G/C) affected DNA-protein complex formation and promoter activity in promoter reporter analyses. The OPG SNP haplotype rs2073618G/rs3102735T was significantly associated with HLA-B27 negativity in AS patients. Furthermore, AS patients with syndesmophyte formation had significantly lower levels of soluble RANKL levels than those without syndesmophyte formation. Our data suggested a role for RANKL in AS susceptibility and severity.

Functional Analysis of Promoter Variants in Genes Involved in Sex Steroid Action, DNA Repair and Cell Cycle Control.

Genetic variants affecting the regulation of gene expression are among the main causes of human diversity. The potential importance of regulatory polymorphisms is underscored by results from Genome Wide Association Studies, which have already implicated such polymorphisms in the susceptibility to complex diseases such as breast cancer. In this study, we re-sequenced the promoter regions of 24 genes involved in pathways related to breast cancer including sex steroid action, DNA repair, and cell cycle control in 60 unrelated Caucasian individuals. We constructed haplotypes and assessed the functional impact of promoter variants using gene reporter assays and electrophoretic mobility shift assays. We identified putative functional variants within the promoter regions of estrogen receptor 1 (ESR1), ESR2, forkhead box A1 (FOXA1), ubiquitin interaction motif containing 1 (UIMC1) and cell division cycle 7 (CDC7). The functional polymorphism on CDC7, rs13447455, influences CDC7 transcriptional activity in an allele-specific manner and alters DNA–protein complex formation in breast cancer cell lines. Moreover, results from the Breast Cancer Association Consortium show a marginal association between rs13447455 and breast cancer risk (p = 9.3 × 10−5), thus warranting further investigation. Furthermore, our study has helped provide methodological solutions to some technical difficulties that were encountered with gene reporter assays, particularly regarding inter-clone variability and statistical consistency.

How proteins bind to DNA: target discrimination and dynamic sequence search by the telomeric protein TRF1.

Target search as performed by DNA-binding proteins is a complex process, in which multiple factors contribute to both thermodynamic discrimination of the target sequence from overwhelmingly abundant off-target sites and kinetic acceleration of dynamic sequence interrogation. TRF1, the protein that binds to telomeric tandem repeats, faces an intriguing variant of the search problem where target sites are clustered within short fragments of chromosomal DNA. In this study, we use extensive (>0.5 ms in total) MD simulations to study the dynamical aspects of sequence-specific binding of TRF1 at both telomeric and non-cognate DNA. For the first time, we describe the spontaneous formation of a sequence-specific native protein–DNA complex in atomistic detail, and study the mechanism by which proteins avoid off-target binding while retaining high affinity for target sites. Our calculated free energy landscapes reproduce the thermodynamics of sequence-specific binding, while statistical approaches allow for a comprehensive description of intermediate stages of complex formation.

Dual function of Ixr1 in transcriptional regulation and recognition of cisplatin-DNA adducts is caused by differential binding through its two HMG-boxes

Ixr1 is a transcriptional factor involved in the response to hypoxia, which is also related to DNA repair. It binds to DNA through its two in-tandem high mobility group box (HMG-box) domains. Each function depends on recognition of different DNA structures, B-form DNA at specific consensus sequences for transcriptional regulation, or distorted DNA, like cisplatin-DNA adducts, for DNA repair. However, the contribution of the HMG-box domains in the Ixr1 protein to the formation of different protein-DNA complexes is poorly understood. We have biophysically and biochemically characterized these interactions with specific DNA sequences from the promoters regulated by Ixr1, or with cisplatin-DNA adducts. Both HMG-boxes are necessary for transcriptional regulation, and they are not functionally interchangeable. The in-tandem arrangement of their HMG-boxes is necessary for functional folding and causes sequential cooperative binding to specific DNA sequences, with HMG-box A showing a higher contribution to DNA binding and bending than the HMG-box B. Binding of Ixr1 HMG boxes to specific DNA sequences is entropy driven, whereas binding to platinated DNA is enthalpy driven for HMG-box A and entropy driven for HMG-box B. This is the first proof that HMG-box binding to different DNA structures is associated with predictable thermodynamic differences. Based on our study, we present a model to explain the dual function of Ixr1 in the regulation of gene expression and recognition of distorted DNA structures caused by cisplatin treatment.