Peter Schultze

Solution structure of the HMG protein NHP6A and its interaction with DNA reveals the structural determinants for non-sequence-specific binding.

NHP6A is a chromatin‐associated protein from Saccharomyces cerevisiae belonging to the HMG1/2 family of non‐specific DNA binding proteins. NHP6A has only one HMG DNA binding domain and forms relatively stable complexes with DNA. We have determined the solution structure of NHP6A and constructed an NMR‐based model structure of the DNA complex. The free NHP6A folds into an L‐shaped three α‐helix structure, and contains an unstructured 17 amino acid basic tail N‐terminal to the HMG box. Intermolecular NOEs assigned between NHP6A and a 15 bp 13C, 15N‐labeled DNA duplex containing the SRY recognition sequence have positioned the NHP6A HMG domain onto the minor groove of the DNA at a site that is shifted by 1 bp and in reverse orientation from that found in the SRY–DNA complex. In the model structure of the NHP6A–DNA complex, the N‐terminal basic tail is wrapped around the major groove in a manner mimicking the C‐terminal tail of LEF1. The DNA in the complex is severely distorted and contains two adjacent kinks where side chains of methionine and phenylalanine that are important for bending are inserted. The NHP6A–DNA model structure provides insight into how this class of architectural DNA binding proteins may select preferential binding sites.

Solution structures of unimolecular quadruplexes formed by oligonucleotides containing Oxytricha telomere repeats

BACKGROUND Oligonucleotides containing the guanine-rich telomeric sequence of Oxytricha chromosomes (dT4G4) have previously been shown to form DNA quadruplexes comprising guanine quartets stabilized by cations. Two different structures have been reported for both d(G4T4G4) (Oxy1.5) and d(G4T4G4T4G4T4G4) (Oxy3.5). RESULTS Here we present the solution structure of a uracil- and inosine-containing derivative of Oxy3.5, d(G4TUTUG4T4G4UUTTG3I) (Oxy3.5-U4128), determined using two-dimensional 1H and 31P NMR techniques. This oligonucleotide forms a unimolecular quadruplex that is very similar to the dimeric Oxy1.5 solution structure, in that it contains a loop spanning the diagonal of an end quartet. The groove widths, strand polarities, and positions of the syn bases along the G4 tracts and within the quartets are all as reported for Oxy1.5. The first and third pyrimidine tracts form parallel loops spanning a wide groove and a narrow groove respectively. CONCLUSIONS Both Oxy3.5 and Oxy3.5-U(4)128 form unimolecular quadruplexes in solution with a diagonal central T4 loop. These results conflict with those reported for d(G4TUTUG4TTUUG4UUTTG4) in solution, in which the central loop spans a wide groove.

Polymorphism, Packing, Resolution, and Reliability in Single-Crystal DNA Oligomer Analyses

Abstract Synthetic double-helical B-DNA oligonucleotides crystallize in orthorhombic, monoclinic, and trigonal patterns that are determined by fine details of intermolecular contacts. Crystal packing apparently has relatively little influence on local helix structure, and noncrystallographic symmetry differences can be used to evaluate the quality of analyses.

Chirality errors in nucleic acid structures

The prevalence of errors as opposed to true anomalies in protein structures was discussed in Correspondence last year. We have analysed the nucleic acid structures in the Brookhaven Protein Data Bank using a simple automatic procedure (program available at http://www.mbi.ucla.edu/people/peter/chiral.html) to determine the configurations at tetrahedral chiral centres. It appears that a number of structures containing significant “chirality errors that no-one would argue with” have made their way into the data bank.

1H NMR spectroscopy of DNA

Publisher Summary This chapter focuses on the methods used to obtain, process, assign, and analyze the nuclear magnetic resonance (NMR) spectra of DNA oligonucleotides. The utility of these studies for qualitative analysis of DNA structures is also discussed in the chapter. Advances in NMR technology and instrumentation since the mid-1980s have led to a revolution in the use of NMR spectroscopy for the determination of macromolecular structures. Protein structures determined from data obtained by NMR methods are now accepted by both NMR spectroscopists and crystallographers. NMR spectroscopy of DNA oligonucleotides was largely made possible by the advent of convenient DNA synthesis methods at about the same time that two-dimensional NMR was beginning to be applied to the study of proteins. Prior to that, most 1 H NMR spectroscopy of nucleic acids was done on transfer RNA and synthetic RNA polymers. DNA in the milligram quantities needed for 1 H NMR spectroscopy can be conveniently synthesized on commercial DNA synthesizers. The most difficult part of obtaining DNA for NMR samples is purification. Many laboratories use high-performance liquid chromatography with adequate results for purification of DNA samples.