Holliday Junction Research Articles

Advances in holliday junction recognition protein (HJURP): Structure, molecular functions, and roles in cancer.

Oncogenes are increasingly recognized as important factors in the development and progression of cancer. Holliday Junction Recognition Protein (HJURP) is a highly specialized mitogenic protein that is a chaperone protein of histone H3. The HJURP gene is located on chromosome 2q37.1 and is involved in nucleosome composition in the mitotic region, forming a three-dimensional crystal structure with Centromere Protein A (CENP-A) and the histone 4 complex. HJURP is involved in the recruitment and assembly of centromere and kinetochore and plays a key role in stabilizing the chromosome structure of tumor cells, and its dysfunction may contribute to tumorigenesis. In the available studies HJURP is upregulated in a variety of cancer tissues and cancer cell lines and is involved in tumor proliferation, invasion, metastasis and immune response. In an in vivo model, overexpression of HJURP in most cancer cell lines promotes cell proliferation and invasiveness, reduces susceptibility to apoptosis, and promotes tumor growth. In addition, upregulation of HJURP was associated with poorer prognosis in a variety of cancers. These properties suggest that HJURP may be a possible target for the treatment of certain cancers. Various studies targeting HJURP as a prognostic and therapeutic target for cancer are gradually attracting interest and attention. This paper reviews the functional and molecular mechanisms of HJURP in a variety of tumor types with the aim of providing new targets for future cancer therapy.

Abstract P2-17-02: SMC6 down-regulation: marker of genetic instability and poor outcome in breast cancer

Abstract Genetic instability commonly found in tumors may be a consequence of imbalance in homologous recombination (HR), a mechanism of DNA double-strand break (DSB) repair dependent on the formation and resolution of Holliday junctions’ structures for genomic stability maintenance. The SMC6 is involved in DSB repair. The SMC6 gene is structurally located very close to the GEN1 gene in a head-to-head organization sharing a bi-directional promoter, and the encoded protein is also involved in HR. Herein, we explored the association between SMC6 expression, genomic instability, and prognosis of breast cancer (BC). SMC6 and GEN1 expression and Copy Number Variation (CNV) data measured by qRT-PCR and CGH array, respectively, were assessed in 33 BC of women non-carrier BRCA1/BRCA2-mutations. The SMC6 protein expression was evaluated on a Tissue Microarray containing 74 BC samples classified as low (negative/weak) or high (moderate/strong) according to SMC6 nuclear staining. SMC6 and GEN1 expressions were directly correlated (p=0.821). Moreover, SMC6-low tumors tend to show higher CNV. Yet, the SMC6-low group presented poorer disease-free survival (DFS) than the SMC6 high group (log-rank=0.008). Further, SMC6 expression broke the DFS ER+ cohort in groups of distinct prognoses (log-rank=0.029). By in silico data analysis, SMC6 downregulation was also associated with worse prognosis in some BC subtypes. Our findings point to a possible association between SMC6 down-regulation and BC poor outcomes, suggesting that SMC6 may serve as an indicator of tumor genetic instability for BC patients. Supported by Fapesp and CNPq. Citation Format: Flavia R. Rotea Mangone, Ana Cristina V. Krepischi, Dirce M. Carraro, Maria A. Nagai. SMC6 down-regulation: marker of genetic instability and poor outcome in breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-17-02.

Antigen footprint governs activation of the B cell receptor

Antigen binding by B cell receptors (BCR) on cognate B cells elicits a response that eventually leads to production of antibodies. However, it is unclear what the distribution of BCRs is on the naïve B cell and how antigen binding triggers the first step in BCR signaling. Using DNA-PAINT super-resolution microscopy, we find that most BCRs are present as monomers, dimers, or loosely associated clusters on resting B cells, with a nearest-neighbor inter-Fab distance of 20–30 nm. We leverage a Holliday junction nanoscaffold to engineer monodisperse model antigens with precision-controlled affinity and valency, and find that the antigen exerts agonistic effects on the BCR as a function of increasing affinity and avidity. Monovalent macromolecular antigens can activate the BCR at high concentrations, whereas micromolecular antigens cannot, demonstrating that antigen binding does not directly drive activation. Based on this, we propose a BCR activation model determined by the antigen footprint.

Molecular Dynamic Studies of Dye-Dye and Dye-DNA Interactions Governing Excitonic Coupling in Squaraine Aggregates Templated by DNA Holliday Junctions.

Dye molecules, arranged in an aggregate, can display excitonic delocalization. The use of DNA scaffolding to control aggregate configurations and delocalization is of research interest. Here, we applied Molecular Dynamics (MD) to gain an insight on how dye-DNA interactions affect excitonic coupling between two squaraine (SQ) dyes covalently attached to a DNA Holliday junction (HJ). We studied two types of dimer configurations, i.e., adjacent and transverse, which differed in points of dye covalent attachments to DNA. Three structurally different SQ dyes with similar hydrophobicity were chosen to investigate the sensitivity of excitonic coupling to dye placement. Each dimer configuration was initialized in parallel and antiparallel arrangements in the DNA HJ. The MD results, validated by experimental measurements, suggested that the adjacent dimer promotes stronger excitonic coupling and less dye-DNA interaction than the transverse dimer. Additionally, we found that SQ dyes with specific functional groups (i.e., substituents) facilitate a closer degree of aggregate packing via hydrophobic effects, leading to a stronger excitonic coupling. This work advances a fundamental understanding of the impacts of dye-DNA interactions on aggregate orientation and excitonic coupling.

The cryo‐EM structure of the CENP‐A nucleosome in complex with ggKNL2

Centromere protein A (CENP‐A) nucleosomes containing the centromere‐specific histone H3 variant CENP‐A represent an epigenetic mark that specifies centromere position. The Mis18 complex is a licensing factor for new CENP‐A deposition via the CENP‐A chaperone, Holliday junction recognition protein (HJURP), on the centromere chromatin. Chicken KINETOCHORE NULL2 (KNL2) (ggKNL2), a Mis18 complex component, has a CENP‐C‐like motif, and our previous study suggested that ggKNL2 directly binds to the CENP‐A nucleosome to recruit HJURP/CENP‐A to the centromere. However, the molecular basis for CENP‐A nucleosome recognition by ggKNL2 has remained unclear. Here, we present the cryo‐EM structure of the chicken CENP‐A nucleosome in complex with a ggKNL2 fragment containing the CENP‐C‐like motif. Chicken KNL2 distinguishes between CENP‐A and histone H3 in the nucleosome using the CENP‐C‐like motif and its downstream region. Both the C‐terminal tail and the RG‐loop of CENP‐A are simultaneously recognized as CENP‐A characteristics. The CENP‐A nucleosome–ggKNL2 interaction is thus essential for KNL2 functions. Furthermore, our structural, biochemical, and cell biology data indicate that ggKNL2 changes its binding partner at the centromere during chicken cell cycle progression.

Recognition of recombination intermediates by the mismatch repair protein Msh2-Msh6.

The mismatch repair (MMR) protein MutSα (Msh2-Msh6), known for improving the fidelity of DNA replication, also improves the fidelity of homologous recombination. Msh2-Msh6 and its associated proteins are responsible for suppressing homologous recombination when excessive base mispairing is present between the participating chromosomes. In MMR, Msh2-Msh6 recognizes small insertion/deletion loops and single base pair mismatches while scanning DNA post replication. Interestingly, Sc Msh2-Msh6 binds to Holliday Junctions (HJs) with an affinity comparable to that of mismatched duplex DNA (Kd =15 nM). We study the Msh2-Msh6-HJ binding interaction to gain insight into the mechanistic role of Msh2-Msh6 during the process of homologous recombination. In our previous study we examined the dynamics of single base mismatches and the changes associated with Msh2-Msh6 binding using the fluorescent DNA base analog 6-MI and time-resolved fluorescence methods. Here, we employ a similar strategy to study the dynamics of single bases within HJs alone and upon interaction with Msh2-Msh6. 6-MI probes incorporated into the center and arms of the junction only exhibit perturbed dynamics in the center location upon Msh2-Msh6 binding, suggesting protein binding takes place at the junction center. Successful UV-photo crosslinking of Msh2-Msh6 to HJs containing 5-bromouracil at the junction center further supports this finding. Base dynamics at the center of the junction resemble those of the mismatch, which possibly drives Msh2-Msh6 recognition. Interestingly, introduction of a mismatch in the arms of the HJ shifts the Msh2-Msh6 binding from the center of HJ to the mismatch location despite the similar binding affinities. Additionally, a molecular model of the Msh2-Msh6-HJ interaction has been created and subjected to molecular dynamics simulations to gain further structural insights with particular focus on how the DNA binding domain interacts with HJs compared to mismatched duplex DNA.

Elucidating the structure of the HU-junction complex.

Histone-like protein (HU) is one of the most abundant proteins in the bacterial nucleoid with a multi-functional role in DNA packaging, recombination, replication, and repair. HU binds with high affinity and in a non-sequence specific manner to Holliday or DNA 4-Way Junctions (HJ), a central intermediate in recombination and repair. Current understanding of how HU binds and recognizes junctions, and its role in recombination is limited.

Intramolecular Charge Transfer and Ultrafast Nonradiative Decay in DNA-Tethered Asymmetric Nitro- and Dimethylamino-Substituted Squaraines.

Molecular (dye) aggregates are a materials platform of interest in light harvesting, organic optoelectronics, and nanoscale computing, including quantum information science (QIS). Strong excitonic interactions between dyes are key to their use in QIS; critically, properties of the individual dyes govern the extent of these interactions. In this work, the electronic structure and excited-state dynamics of a series of indolenine-based squaraine dyes incorporating dimethylamino (electron donating) and/or nitro (electron withdrawing) substituents, so-called asymmetric dyes, were characterized. The dyes were covalently tethered to DNA Holliday junctions to suppress aggregation and permit characterization of their monomer photophysics. A combination of density functional theory and steady-state absorption spectroscopy shows that the difference static dipole moment (Δd) successively increases with the addition of these substituents while simultaneously maintaining a large transition dipole moment (μ). Steady-state fluorescence and time-resolved absorption and fluorescence spectroscopies uncover a significant nonradiative decay pathway in the asymmetrically substituted dyes that drastically reduces their excited-state lifetime (τ). This work indicates that Δd can indeed be increased by functionalizing dyes with electron donating and withdrawing substituents and that, in certain classes of dyes such as these asymmetric squaraines, strategies may be needed to ensure long τ, e.g., by rigidifying the π-conjugated network.

Probing DNA structural heterogeneity by identifying conformational subensembles of a bicovalently bound cyanine dye.

DNA is a re-configurable, biological information-storage unit, and much remains to be learned about its heterogeneous structural dynamics. For example, while it is known that molecular dyes templated onto DNA exhibit increased photostability, the mechanism by which the structural dynamics of DNA affect the dye photophysics remains unknown. Here, we use femtosecond, two-dimensional electronic spectroscopy measurements of a cyanine dye, Cy5, to probe local conformations in samples of single-stranded DNA (ssDNA-Cy5), double-stranded DNA (dsDNA-Cy5), and Holliday junction DNA (HJ-DNA-Cy5). A line shape analysis of the 2D spectra reveals a strong excitation-emission correlation present in only the dsDNA-Cy5 complex, which is a signature of inhomogeneous broadening. Molecular dynamics simulations support the conclusion that this inhomogeneous broadening arises from a nearly degenerate conformer found only in the dsDNA-Cy5 complex. These insights will support future studies on DNA's structural heterogeneity.

Towards control of excitonic coupling in DNA-templated Cy5 aggregates: the principal role of chemical substituent hydrophobicity and steric interactions.

Understanding and controlling exciton coupling in dye aggregates has become a greater focus as potential applications such as coherent exciton devices, nanophotonics, and biosensing have been proposed. DNA nanostructure templates allow for a powerful modular approach. Using DNA Holliday junction (HJ) templates variations of dye combinations and precision dye positions can be rapidly assayed, as well as creating aggregates of dyes that could not be prepared (either due to excess or lack of solubility) through alternative means. Indodicarbocyanines (Cy5) have been studied in coupled systems due to their large transition dipole moment, which contributes to strong coupling. Cy5-R dyes were recently prepared by chemically modifying the 5,5'-substituents of indole rings, resulting in varying dye hydrophobicity/hydrophilicity, steric considerations, and electron-donating/withdrawing character. We utilized Cy5-R dyes to examine the formation and properties of 30 unique DNA templated homodimers. We find that in our system the sterics of Cy5-R dyes play the determining factor in orientation and coupling strength of dimers, with coupling strengths ranging from 50-138 meV. The hydrophobic properties of the Cy5-R modify the percentage of dimers formed, and have a secondary role in determining the packing characteristics of the dimers when sterics are equivalent. Similar to other reports, we find that positioning of the Cy5-R within the HJ template can favor particular dimer interactions, specifically oblique or H-type dimers.

Emerging role of HJURP as a therapeutic target in cancers

Holliday junction recognition protein (HJURP) is a key molecular chaperone for centromere protein A (CENP-A), which is essential for chromosome separation during mitosis and cell cycle regulation. Recent studies have identified the essential role of HJURP in carcinogenesis. Abnormal upregulation of HJURP expression has been observed in various human cancers, such as non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), bladder cancer, and breast cancer, and is associated with poor pathologic development and prognosis. In vitro and in vivo studies have shown that HJURP mainly exerts oncogenic functions by regulating the cell cycle, cellular senescence, and epithelial-mesenchymal transition (EMT). The purpose of this review was to evaluate the prognostic significance of HJURP in human cancers and summarize anti-tumor studies targeting HJURP. The factors regulating HJURP in carcinogenesis and the corresponding effects are also discussed to provide new insight into targeting HJURP as a promising strategy for cancer treatment.

Single-molecular surface-induced fluorescence attenuation based on thermal reduced graphene oxide

Single-molecular surface-induced fluorescence attenuation (smSIFA) is a precise method of studying the vertical movement of biological macromolecules based on two-dimensional material receptors. This method is not affected by two-dimensional planar motion of membrane or proteins. However, the detection range and accuracy of vertical movement are determined by the properties of two-dimensional materials as receptors. In recent years, surface induced fluorescence attenuation based on graphene oxide and graphene has played an important role in studying biomacromolecules. However, the detection range of graphene and graphene oxide are limited owing to the fixed and limited characteristic quenching distance. Adjusting the detection range requires replacing the medium material, which poses difficulties in selecting and preparing materials. Therefore, it is urgently needed to develop controllable materials for single-molecular SIFA. In this study, the single-molecule SIFA with graphene oxide as the medium acceptor is improved by reducing graphene oxide through thermal reduction. By controlling the reduction temperature, reduced graphene oxides to different reduction degrees are prepared and the characteristic quenching distances are adjusted. The characteristic quenching distance is measured by fluorescent labeled DNA. Single-molecule SIFA based on reduced graphene oxide is used to observe the conformational changes of Holliday junction, and the detection range of reduced graphene oxide is demonstrated.

Conformational dynamics and mechanical properties of biomimetic RNA, DNA, and RNA-DNA hybrid nanotubes: an atomistic molecular dynamics study.

With the nanotechnology boom, artificially designed nucleic acid nanotubes have aroused interest due to their practical applications in nanorobotics, vaccine design, membrane channels, drug delivery, and force sensing. In this paper, computational study was performed to investigate the structural dynamics and mechanical properties of RNA nanotubes (RNTs), DNA nanotubes (DNTs), and RNA-DNA hybrid nanotubes (RDHNTs). So far, the structural and mechanical properties of RDHNTs have not been examined in experiments or theoretical calculations, and there is limited knowledge regarding these properties for RNTs. Here, the simulations were carried out using the equilibrium molecular dynamics (MD) and steered molecular dynamics (SMD) approaches. Using in-house scripting, we modeled hexagonal nanotubes composed of six double-stranded molecules connected by four-way Holliday junctions. Classical MD analyses were performed on the collected trajectory data to investigate structural properties. Analyses of the microscopic structural parameters of RDHNT indicated a structural transition from the A-form to a conformation between the A- and B-forms, which may be attributable to the increased rigidity of RNA scaffolds compared to DNA staples. Comprehensive research on the elastic mechanical properties was also conducted based on spontaneous thermal fluctuations of nanotubes and employing the equipartition theorem. The Young's modulus of RDHNT (E = 165 MPa) and RNT (E = 144 MPa) was found to be almost the same and nearly half of that found for DNT (E = 325 MPa). Furthermore, the results showed that RNT was more resistant to bending, torsional, and volumetric deformations than DNT and RDHNT. We also used non-equilibrium SMD simulations to acquire comprehensive knowledge of the mechanical response of nanotubes to tensile stress.

Abstract 1230: Molecular Mechanisms of Holliday Junction Branch Migration Catalyzed by an Asymmetric RuvB Hexamer

Abstract 1230: Molecular Mechanisms of Holliday Junction Branch Migration Catalyzed by an Asymmetric RuvB Hexamer

The mitosis-related gene OIP5 is a potential biomarker in pan-cancer.

OIP5 is found at the centromere and plays an important role in recruiting centromere protein-A (CENP-A) through interacting with Holliday junction recognition protein during cell mitosis. OIP5 is considered to be a cancer-testis specific gene, but its function in tumor development remains unclear. Increased expression of OIP5 has been reported in testis as well as in different cancers; however, the underlying mechanisms remain obscure. Data were collected from the Genotype-Tissue Expression project, the Cancer Cell Line Encyclopedia, and The Cancer Genome Atlas (TCGA) to analyze the effect of OIP5 in many common cancers. Analyses of the differential expression of OIP5 and its relationships with prognosis, the tumor microenvironment, immune infiltration, immune regulation, neoantigen production, and genomic stability in various cancers were performed using R software. Expression of OIP5 was significantly increased in 34 common tumor types compared with matched healthy samples; however, no significant increases were observed in pheochromocytoma and paraganglioma or kidney chromophobe. Elevated OIP5 expression predicted dismal overall survival in 14 tumors. The function of OIP5 in tumor-infiltrating immune cells (TIIC) was analyzed, and OIP5 might inhibit TIIC infiltration in the tumor microenvironment; a positive correlation was found in thymoma, while a negative correlation was observed in lung squamous cell carcinoma and lung adenocarcinoma. High OIP5 expression was related to immune regulation and neoantigen production, particularly in terms of the levels of immune regulatory molecules and the number of neoantigens produced in lung adenocarcinoma, uterine corpus endometrial carcinoma, breast cancer, stomach adenocarcinoma, low-grade glioma, and prostate adenocarcinoma. It was also associated with increased cell genome instability in lung adenocarcinoma. Gene set enrichment analysis revealed potential critical effects of OIP5 on the cell cycle, base excision repair, homologous recombination, DNA replication, the p53 signaling pathway, and mismatch repair pathways. High expression of OIP5 is found in many common tumors and predicts a dismal prognostic outcome. The gene is an important recruitment factor for CENP-A and may promote tumor progression by affecting the tumor immune microenvironment and genomic stability. Therefore, OIP5 can serve as a potential candidate factor to predict cancer prognosis and guide the use of therapeutics.

Integration Host Factor Binds DNA Holliday Junctions.

Integration host factor (IHF) is a nucleoid-associated protein involved in DNA packaging, integration of viral DNA and recombination. IHF binds with nanomolar affinity to duplex DNA containing a 13 bp consensus sequence, inducing a bend of ~160° upon binding. We determined that IHF binds to DNA Four-way or Holliday junctions (HJ) with high affinity regardless of the presence of the consensus sequence, signifying a structure-based mechanism of recognition. Junctions, important intermediates in DNA repair and homologous recombination, are dynamic and can adopt either an open or stacked conformation, where the open conformation facilitates branch migration and strand exchange. Using ensemble and single molecule Förster resonance energy transfer (FRET) methods, we investigated IHF-induced changes in the population distribution of junction conformations and determined that IHF binding shifts the population to the open conformation. Further analysis of smFRET dynamics revealed that even in the presence of protein, the junctions remain dynamic as fast transitions are observed for the protein-bound open state. Protein binding alters junction conformational dynamics, as cross correlation analyses reveal the protein slows the transition rate at 1 mM Mg2+ but accelerates the transition rate at 10 mM Mg2+. Stopped flow kinetic experiments provide evidence for two binding steps, a rapid, initial binding step followed by a slower step potentially associated with a conformational change. These measurements also confirm that the protein remains bound to the junction during the conformer transitions and further suggest that the protein forms a partially dissociated state that allows junction arms to be dynamic. These findings, which demonstrate that IHF binds HJs with high affinity and stabilizes junctions in the open conformation, suggest that IHF may play multiple roles in the processes of integration and recombination in addition to stabilizing bacterial biofilms.

Microbial biofilms are shaped by the constant dialogue between biological and physical forces in the extracellular matrix.

Microbial biofilms are shaped by the constant dialogue between biological and physical forces in the extracellular matrix.

Pan-cancer analysis based on epigenetic modification explains the value of HJURP in the tumor microenvironment

To analyze the expression levels, prognostic value and immune infiltration association of Holliday junction protein (HJURP) as well as its feasibility as a pan-cancer biomarker for different cancers. The Protter online tool was utilized to obtain the localization of HJURP, then the methylation of HJURP in tumors were further explored. Thereafter, the mRNA data and clinical characteristics of 33 tumor types from TCGA database were obtained to investigate the expression and prognostic relationship of HJURP in different tumor types. Finally, the composition pattern and immune infiltration of HJURP in different tumors were detected in Tumor Immune Estimation Resource. HJURP was abnormally expressed in most of the cancer types and subtypes in TCGA database. Also, it was associated with poor prognosis of different cohorts. At the same time, the results also showed that HJURP was related to tumor immune evasion through different mechanisms, including T cell rejection and methylation in different cancer types. Besides, the methylation of HJURP was inversely proportional to mRNA expression levels, which mediated the dysfunctional phenotypes of T cells and poor prognosis of different cancer types. Alternatively, our results indicated that HJURP expression was associated with immune cell infiltration in a variety of cancers. HJURP may serve as an oncogenic molecule, and its expression and immune infiltration characteristics can be used as a biomarker for cancer detection, prognosis, treatment design and follow-up.

Single-photon smFRET. III. Application to pulsed illumination

Förster resonance energy transfer (FRET) using pulsed illumination has been pivotal in leveraging lifetime information in FRET analysis. However, there remain major challenges in quantitative single-photon, single-molecule FRET (smFRET) data analysis under pulsed illumination including 1) simultaneously deducing kinetics and number of system states; 2) providing uncertainties over estimates, particularly uncertainty over the number of system states; and 3) taking into account detector noise sources such as cross talk and the instrument response function contributing to uncertainty; in addition to 4) other experimental noise sources such as background. Here, we implement the Bayesian nonparametric framework described in the first companion article that addresses all aforementioned issues in smFRET data analysis specialized for the case of pulsed illumination. Furthermore, we apply our method to both synthetic as well as experimental data acquired using Holliday junctions.

DNA Nanostructure-Guided Plasmon Coupling Architectures

DNA Nanostructure-Guided Plasmon Coupling Architectures