No abstract

High-mobility-group 1 protein (HMG1) is an abundant eukaryotic DNA-binding protein, the cellular role of which remains ill-dermed. To test the ability of HMG1 itself to mediate curvature in double-stranded DNA, we examined its effect on the phage T4 DNA ligase-dependent cyclization of short DNA fragments. HMG1 caused circle formation for fragments 2 87 bp. Fragments of 123, 100, 92, and 87 bp did not cyclize in the absence of protein but formed covalently closed circular monomers efficiently in the presence of HMG1, indicating that the protein is capable of introducing bends into the duplex. The bending activity was maintained by a 79-amino acid polypeptide corresponding to a single HMG-box domain of HMG1. The binding affinity for the DNA minicircle was greater than for the corresponding linear fragment. These findings indicate that the role of HMG1 could involve both structure-specific recognition of prebent DNA and distortion of the DNA helix by bending and that the HMG-box domain may actually be responsible for this activity.High-mobility-group protein 1 (HMG1) is a 24.5-kDa chromosomal protein found in higher eukaryotes, the cellular function of which remains unknown (1). In the two decades since HMG1 was first observed as a nonhistone protein that could be extracted from chromatin with 0.35 M NaCl (2), it has been extensively investigated. These studies have revealed that HMG1 binds preferentially to and unwinds negatively supercoiled DNA (3), has the ability to recognize cruciform DNA molecules (4), and exhibits single-stranded DNA binding activity (5). Other studies have indicated that HMG1 may function as a general class II transcription factor (6) and as a suppressor of nucleosome assembly (7) and that it can facilitate the binding of transcription factors (8).The DNA-binding activity of HMG1 is derived from two homologous repeats of an 80-amino acid sequence, the socalled HMG-box domain, that comprise two-thirds of the protein. Each of these domains by itself can bind cruciform structures in DNA (9). The HMG-box domain appears in a number of DNA-binding proteins (10) including the transcription factors LEF-1 (lymphoid enhancer factor) (11), SRY (sex determining factor) (12), and hUBF (human upstream binding factor) (13) as well as a protein that binds both V-(D)-J (variable-diversity-joining) junction sequences (14) terial DNA-bending protein in a recombination experiment (10, 39). The present study addresses the question ofwhether HMG1 can induce the bending of DNA independent of sequence or the existence of prebent regions.Protein-induced bending of DNA has been examined by x-ray crystallography (20), by circular permutation of a protein binding site within a DNA fragment followed by gel electrophoresis (21), and by electron microscopy (22). A method has been described that allows the study of DNA bending in solution by covalent ring closure in the presence of T4 ligase (23). In such an experiment, the cyclization efficiency directly correlates with the degree ofbending. This method was used...

In all kingdoms of life, RNAs undergo specific post-transcriptional modifications. More than 100 different analogues of the four standard RNA nucleosides have been identified. Modifications in ribosomal RNAs are highly prevalent and cluster in regions of the ribosome that have functional importance, a high level of nucleotide conservation, and that typically lack proteins. Modifications also play roles in determining antibiotic resistance or sensitivity. A wide spectrum of chemical diversity from the modifications provides the ribosome with a broader range of possible interactions between ribosomal RNA regions, transfer RNA, messenger RNA, proteins, or ligands by influencing local ribosomal RNA folds and fine-tuning the translation process. The collective importance of the modified nucleosides in ribosome function has been demonstrated for a number of organisms, and further studies may reveal how the individual players regulate these functions through synergistic or cooperative effects.In all kingdoms of life, ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nucleolar RNAs (snoRNAs), and other RNAs undergo specific post-transcriptional modification by a wide variety of enzymes (1). To date, >100 different modifications of the four standard RNA nucleosides, adenosine, cytidine, guanosine, and uridine, have been identified (2). These modifications can be organized into four main types (Figure 1) (1). The first involves isomerization of uridine to pseudouridine (5-ribosyluracil, Ψ), which contains a C-rather than the typical N-glycosidic linkage, as well as an additional imino group that is available for unique hydrogen-bonding interactions. The second includes alterations to the bases, such as methylation (typically on carbon, primary nitrogen, or tertiary nitrogen), deamination (e.g., inosine), reduction (e.g., dihydrouridine), thiolation, or alkylation (e.g., isopentenylation or threonylation). The third involves methylation of the ribose 2′ hydroxyl (Nm). The fourth type includes more complex modifications, such as multiple modifications (e.g., 5-methylaminomethyl-2-thiouridine; 3-(3-amino-3-carboxypropyl)uridine, acp 3 U; 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, m 1 acp 3 Ψ) or "hypermodifications" that can be incorporated by specific exchange mechanisms (e.g., queuosine). The possible electronic and steric effects of the nucleoside modifications on base pairing, base stacking, and sugar pucker in RNA have been discussed in detail by Davis (3) and Agris (4), among others (1). The effects of modifications such as Ψ on RNA hydration and dynamics have also been considered (4).Modified nucleotides in the ribosome are varied in their identity, but highly localized in their positions (5). If the sites of modification are mapped on the secondary structures of the small and large subunit (SSU and LSU) rRNAs, they might appear to be random; however, if the same modifications are located within the ribosome tertiary structures from high-resolution Xray crystal structures (6,7), they occur in the mo...

Nanopatterns of thiolated single-stranded DNA (ssDNA) are produced by using an atomic force microscopy (AFM)-based lithography technique known as nanografting. Under high shear force, AFM tips displace resist molecules within a self-assembled monolayer, while ssDNA molecules adsorb chemically onto the exposed gold area through the sulfur headgroup. Nanostructures of ssDNA are characterized directly and in situ by using the same tip. Lines as narrow as 10 nm have been produced. The ssDNA molecules stand up on the gold surfaces and adapt a stretched conformation. In situ and real-time imaging studies have revealed that DNA molecules within the nanostructures are accessible by enzyme molecules.Arrays of DNA patterns are important in gene mapping, drug discovery, DNA sequencing and disease diagnosis. 1 Various approaches have been taken to create DNA arrays on surfaces. One approach is to use light-directed oligonucleotide synthesis to attach DNA nucleotides at mask-defined areas and build subsequent DNA strands by coupling. 2 Another method is to attach presynthesized DNA strands onto designated sites of a solid support. 3,4 A broad range of solid supports have been used, such as gold, conductive polymer, SAMs, and carbon paste. 5-15 Typically, the size of the DNA patterns is tens to hundreds of micrometers. 16,17 Further miniaturization is essential for the development of ultrasmall biosensors and biochips. The performance of chips or sensors can be enhanced after miniaturization because of the higher density of receptor elements, higher detection sensitivity, and smaller amounts of reaction reagents. New generations of nanochips also offer the hope of faster analysis time, less waste of costly reagents, and massive parallelization. 18 Scanning probe microscopy (SPM) techniques are well known for their ability to visualize surfaces of materials with the highest spatial resolution. 19-21 Taking advantage of the sharpness of the tips and strong and local interactions between the tip and surface molecules, SPM has also been used to produce nanostructures on surfaces. 22-30 "Dip-pen" nanolithography (DPN) has been used to pattern nanostructures of DNA on a gold surface. The size of the DNA pattern depends on the substrate, humidity of the environment, and fabrication speed, thus making it difficult to reach high spatial precision. The tip coating process is relatively difficult, and a different tip needs to be used to characterize the produced pattern. 31 Recently, a meniscus force nanografting method was used to pattern DNA on surfaces, and the patterns have been coupled with complementary oligonucleotides tagged by gold particles. 32 The spatial precision and selectivity is not sufficiently high and the method used to characterize the DNA patterns involves tagging. We have developed three AFM-based lithography techniques for creating nanopatterns of self-assembled monolayers (SAMs) and biosensors: nanoshaving, nanografting, and nanopen reader and writer (NPRW). 25-29 Using these methods, nanostructures of thiols as s...

The structure of neamine bound to the A site of the bacterial ribosomal RNA was used in the design of novel aminoglycosides. The design took into account stereo and electronic contributions to interactions between RNA and aminoglycosides, as well as a random search of 273 000 compounds from the Cambridge structural database and the National Cancer Institute 3-D database that would fit in the ribosomal aminoglycoside-binding pocket. A total of seven compounds were designed and subsequently synthesized, with the expectation that they would bind to the A-site RNA. Indeed, all synthetic compounds were found to bind to the target RNA comparably to the parent antibiotic neamine, with dissociation constants in the lower micromolar range. The synthetic compounds were evaluated for antibacterial activity against a set of important pathogenic bacteria. These designer antibiotics showed considerably enhanced antibacterial activities against these pathogens, including organisms that hyperexpressed resistance enzymes to aminoglycosides. Furthermore, analyses of four of the synthetic compounds with two important purified resistance enzymes for aminoglycosides indicated that the compounds were very poor substrates; hence the activity of these synthetic antibiotics does not appear to be compromised by the existing resistance mechanisms, as supported by both in vivo and in vitro experiments. The design principles disclosed herein hold the promise of the generation of a large series of designer antibiotics uncompromised by the existing mechanisms of resistance.

Understanding the basic principles that govern RNA binding by aminoglycosides is important for the design of new generations of antibiotics that do not suffer from the known mechanisms of drug resistance. With this goal in mind, we examined the binding of kanamycin A and four derivatives (the products of enzymic turnovers of kanamycin A by aminoglycoside-modifying enzymes) to a 27 nucleotide RNA representing the bacterial ribosomal A site. Modification of kanamycin A functional groups that have been directly implicated in the maintenance of specific interactions with RNA led to a decrease in affinity for the target RNA. Overall, the products of reactions catalyzed by aminoglycoside resistance enzymes exhibit diminished binding to the A site of bacterial 16S rRNA, which correlates well with a loss of antibacterial ability in resistant organisms that harbor these enzymes.

No abstract

Circularly permuted linear DNAs of approximately 100 bp were constructed containing the major adduct of the anticancer drug cisplatin, a cis-[Pt(NH3)2[d(GpG)-N7(1),-N7(2)]] intrastrand cross-link, at a specific site. Gel electrophoresis mobility shift assays with these probes were used to investigate the effects of binding of HMG domain proteins to the platinated DNAs. The site-specifically platinated duplexes were recognized by six different HMG domain proteins--HMG1, mtTFA, Ixr1, and HMG domains from HMG1 (domain B), mSRY, and LEF-1--with comparable binding affinities (Kd approximately 10(-6) to 10(-7) M). In the presence of the HMG domain proteins, the platinated DNAs were bent significantly more than in their absence, the values being 86 +/- 2 degrees, 87-90 +/- 5 degrees, and 68 +/- 6 degrees, respectively, for the proteins and 65-74 +/- 4 degrees, approximately 50 degrees, and 72 +/- 6 degrees, respectively, for the domains. The variability in bend angles suggests that, although the HMG domain proteins share a common ability to bend platinated DNA, specific contacts between the proteins and the platinated duplex are different. The assay further revealed the bend loci to be centered quite near the platinum adduct. The methodology employed in the present study should be generally applicable for synthesizing other small, circularly permuted, covalently modified DNAs which cannot otherwise be readily obtained.