Inherited mutations in BRCA1 confer susceptibility to breast and ovarian neoplasms. However, the function of BRCA1 and the role of BRCA1 in noninherited cancer remain unknown. Characterization of alternately spliced forms of BRCA1 may identify functional regions; thus, we constructed expression vectors of BRCA1 and a splice variant lacking exon 11, designated BRCA1⌬672-4095. Immunofluorescence studies indicate nuclear localization of BRCA1 but cytoplasmic localization of BRCA1⌬672-4095. Two putative nuclear localization signals (designated NLS1 and NLS2) were identified in exon 11; immunofluorescence studies indicate that only NLS1 is required for nuclear localization. RNA analysis indicates the expression of multiple, tissue-specific forms of BRCA1 RNAs; protein analysis with multiple antibodies suggests that at least three BRCA1 isoforms are expressed, including those lacking exon 11. The results suggest that BRCA1 is a nuclear protein and raise the possibility that splicing is one form of regulation of BRCA1 function by alteration of the subcellular localization of expressed proteins.BRCA1 was isolated by positional cloning methods as a gene linked to breast cancer in families with a pattern of autosomal dominant inheritance of the disease (21). Single dominant susceptibility alleles are thought to account for 5 to 10% of all breast cancers, and BRCA1 germ line mutations are widely held to be responsible for approximately 50% of all inherited breast cancers. Inherited BRCA1 mutations are also thought to be responsible for the disease in 80 to 90% of all families with breast-ovarian cancer syndrome (9). Women inheriting mutations in BRCA1, most of which are truncating mutations that result in nonfunctional or unstable proteins (8), have an 85% chance of developing breast cancer in their lifetime (9). An on-line database now provides a listing of known BRCA1 mutations (http:// www. nchgr. nih. gov/ Intramural_research/ Lab_transfer/Bic/index.html), but little is known about the regulation of this gene or the function of its protein product. Unfortunately, mutations in BRCA1 are distributed evenly over the gene, providing little in the way of clues for localizing critical functional regions.BRCA1 is a large gene, with a coding region of 5.5 kb and a total mRNA of approximately 8.0 kb. There is little identifiable homology to known genes, with the exception of a short region in the 5Ј end (spanning exons 2 to 5) that encodes a RING finger with a typical Cys 3 -His-Cys 4 structure. This is a zincbinding motif that is found in a family of transcription factors and may be a protein-protein interaction site. Initial reports also provided evidence for a complex pattern of alternate splicing and the potential for translation of a number of BRCA1 protein isoforms (21). BRCA1 fits the model of a classic tumor suppressor gene, a hypothesis supported by recent work demonstrating that expression of BRCA1 inhibits growth of breast and ovarian cancer cell lines and MCF7-based tumor development in nude mice (16). Additional da...

G-quadruplexes, DNA tertiary structures highly localized to functionally important sites within the human genome, have emerged as important new drug targets. The putative G-quadruplex-forming sequence (Pu27) in the NHE-III1 promoter region of the c-Myc gene is of particular interest as stabilization of this G-quadruplex with TMPyP4 has been shown to repress c-Myc transcription. In this study, we examine the Pu27 G-quadruplex-forming sequence and its interaction with TMPyP4. We report that the Pu27 sequence exists as a heterogeneous mixture of monomeric and higher-order G-quadruplex species in vitro and that this mixture can be partially resolved by size exclusion chromatography (SEC) separation. Within this ensemble of configurations, the equilibrium can be altered by modifying the buffer composition, annealing procedure, and dialysis protocol thereby affecting the distribution of G-quadruplex species formed. TMPyP4 was found to bind preferentially to higher-order G-quadruplex species suggesting the possibility of stabilization of the junctions of the c-Myc G-quadruplex multimers by porphyrin end-stacking. We also examined four modified c-Myc sequences that have been previously reported and found a narrower distribution of quadruplex configurations compared to the parent Pu27 sequence. We could not definitively conclude whether these G-quadruplex structures were selected from the original ensemble or if they are new G-quadruplex structures. Since these sequences differ considerably from the wild-type promoter sequence, it is unclear whether their structures have any actual biological relevance. Additional studies are needed to examine how the polymorphic nature of G-quadruplexes affects the interpretation of in vitro data for c-Myc and other G-quadruplexes. The findings reported here demonstrate that experimental conditions contribute significantly to G-quadruplex formation and should be carefully considered, controlled, and reported in detail.

We report the separation of several quadruplex species formed by ten promoter sequences by Size Exclusion Chromatography (SEC). Modification at the 5’ or 3’ ends or in loop regions of quadruplex forming sequences has become the standard technique for dealing with quadruplex polymorphism. However, conformations produced employing this method or by other means of artificially shifting the equilibrium may not represent the species that are present in vivo. This method enables an unperturbed view of the structural polymorphism inherent to quadruplex formation. Separation via SEC facilitates studies on quadruplex structure and biophysical properties without the need for sequence modification.

G-quadruplexes are noncannonical four-stranded DNA or RNA structures formed by guanine-rich repeating sequences. Guanines nucleotides can hydrogen bond to form a planar tetrad structure. Such tetrads can stack to form quadruplexes of various molecularities with a variety of types of single-stranded loops joining the tetrads. High-resolution structures may be obtained by X-ray crystallography or NMR spectroscopy for quadruplexes formed by short (≈25 nt) sequences but these methods have yet to succeed in characterizing higher-order quadruplex structures formed by longer sequences. An integrated computational and experimental approach was implemented in our laboratory to obtain structural models for higher-order quadruplexes that might form in longer telomeric or promoter sequences. In our approach atomic-level models are built using folding principles gleaned from available high-resolution structures and then optimized by molecular dynamics. The program HYDROPRO is then used to construct bead models of these structures to predict experimentally testable hydrodynamic properties. Models are validated by comparison of these properties with measured experimental values obtained by analytical ultracentrifugation or other biophysical tools. This chapter describes our approach and practical procedures.