Action Of DNA-binding Proteins Research Articles

Single-Molecule Microscopy Meets Molecular Dynamics Simulations for Characterizing the Molecular Action of Proteins on DNA and in Liquid Condensates.

DNA-binding proteins trigger various cellular functions and determine cellular fate. Before performing functions such as transcription, DNA repair, and DNA recombination, DNA-binding proteins need to search for and bind to their target sites in genomic DNA. Under evolutionary pressure, DNA-binding proteins have gained accurate and rapid target search and binding strategies that combine three-dimensional search in solution, one-dimensional sliding along DNA, hopping and jumping on DNA, and intersegmental transfer between two DNA molecules. These mechanisms can be achieved by the unique structural and dynamic properties of these proteins. Single-molecule fluorescence microscopy and molecular dynamics simulations have characterized the molecular actions of DNA-binding proteins in detail. Furthermore, these methodologies have begun to characterize liquid condensates induced by liquid-liquid phase separation, e.g., molecular principles of uptake and dynamics in droplets. This review discusses the molecular action of DNA-binding proteins on DNA and in liquid condensate based on the latest studies that mainly focused on the model protein p53.

Abstract 4607: Novel role of PARP4 in hormone-dependent breast cancer progression and metastasis

Abstract Estrogen/estrogen receptor-mediated actions play an important role in regulating a variety of biological process including development, homeostasis and breast cancer progression. Even though systemic hormone therapies that either block local estrogen production by aromatase inhibitors (AI) or block actions of estrogen/estrogen receptor (ER) by antiestrogens (AE) is well tolerated, the development of resistance to these agents is still a major concern. Since multiple signaling pathways involved in ER activation, combination of endocrine and non endocrine agents that block these signaling pathways may delay the onset of resistance to hormonal therapy. Recent studies from our laboratory have identified Poly(ADP-ribose) polymerase 4(PARP4) as a novel ER co-regulator. We have investigated with hormone resistant breast cancer cells to demonstrate whether blocking of PARP4 results in resensitization to hormone therapies. PARP comprise of a family of enzymes which catalyses poly(ADPribosyl) action of DNA-binding proteins. PARP-1, the best characterized member, however PARP4 is an understudied protein with numerous capabilities. PARP4 has both N-terminal PXXP and PPXXP motifs which are known to interact with SH3 domain of c-Src as well as LXXLL motif which interacts with estrogen receptor (ER). We show that PARP4 is widely expressed in breast cancer cells (MCF-7, MDA-MB-231, ZR-75, and SKBR3) and its expression is significantly higher in these cells compared to non malignant MCF-10A cells. To understand the important role of PARP4-ER axis in hormonal resistance, metastasis and to test whether blocking of PARP4 mediated actions provide a therapeutic advantage to treat endocrine resistant breast tumors, we have used hormone resistant breast cancer cells that over-express PARP4 or and treated with PARP4 siRNA nanoparticles. The PLGA-siRNA nanoparticles were designed multifunctionally using chemical conjugation. The nanoparticles were characterized physiochemically. Our studies show that PARP4 functionally interacts with ER. As demonstrated by immunoprecipitation, PARP4 interacts with ER upon ligand (E2)-mediated stimulation. PARP4 induces cell cycle progression, local aromatase activity and promotes in vitro tumorigenic potential of breast cancer cells. siRNA nanoparticles of PARP4 inhibit the metastatic incidence in vivo. Our study also shows that PARP4-ER axis enhances multiple signaling cross-talk leading to hormonal independence/resistance and metastasis. Our results suggests that targeting PARP4 may provide a therapeutic advantage to treat endocrine resistant tumors and blocking the development of resistance to hormonal therapeutic agents and as well as decrease the metastatic potential in therapy resistant breast tumors. (Supported by NIH/NCI P30 CA 54174, RRT) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4607.

Maintenance of open DNA base pairs through histone acetylated lysine-purine interaction leading to transcriptional activation: A proposed mechanism

Post-translational acetylation of lysines of the histone N-terminal tails is known to induce transcriptional activation, and thus plays a major role in gene regulation. A mechanism for this effect is suggested by our recent finding that the initial 'solvation' network, which is formed around the purines of base pairs immediately following their opening, has the tendency to be preserved. The experiments involved studying the solvation of nucleosides in water-alcohol mixtures; these systems model the hydrophobic/hydrophilic effects that participate in the interaction between histone-tail amino acid residues and nucleosomal DNA base pairs following their opening by the action of DNA-binding proteins in conjunction with remodeling complexes. A highly amphiphilic molecular environment, which has a four-carbon aliphatic chain and hydrogen bond accepting and donating capabilities, was found to promote this phenomenon. Acetylated lysines, unlike lysines, possess these properties and thus constitute such a molecular environment. Opened base pairs are maintained by direct interactions of their purines with acetylated lysines that are selectively recognized by the bromodomains of the recruited remodeling complexes. This maintenance is in accord with the reported large enhancements of i) transcription factor binding to nucleosomal DNA and (ii) the degree of DNA flexibility, which are associated with histone-tail acetylation. This mechanism suggests a way to possibly establish an orderly sequence of transcriptional events despite the reported prevailing stochasticity. It also suggests an approach for the artificial control of transcriptional activation through the use of highly amphiphilic small molecules that would mimic the action of acetylated lysines; in view of the known association of aberrant transcription with cancer, this approach holds promise for advances in cancer etiology and treatment and represents an alternative to the approach that currently uses histone deacetylase inhibitors (some of which are in clinical trials).

The combination of DNA methylation and H1 histone binding inhibits the action of a restriction nuclease on plasmid DNA

To investigate the potentials of DNA methylation and H1 histone in regulating the action of DNA binding proteins, well ordered complexes were formed by slow salt gradient dialysis of mixtures of H1 histone with either methylated or nonmethylated DNA. The sites methylated in the plasmids were CCGG. Methylation of cytosine in this site protects the DNA against HpaII endonuclease but not against MspI. However, when the methylated DNA was complexed to H1, it was protected against MspI. The protection was only effective for a subset of the MspI restriction sites. The protection of DNA afforded by the combination of H1 binding and DNA methylation did not apply to EcoRI, PstI, or BamHI sites and so did not seem to be due to aggregation of the DNA by H1 histone. Gel retardation assays indicated that the affinity of H1 for methylated DNA was not detectably different from its affinity for nonmethylated DNA. Probably methylated DNA when bound to H1 is in a conformation that is resistant to MspI endonuclease. Such conformational changes induced by DNA methylation and H1 binding might affect the action of other DNA binding proteins, perhaps in chromatin as well as in H1.DNA complexes.