Philip H. Bolton

Base excision repair of oxidative DNA damage activated by XPG protein.

Oxidized pyrimidines in DNA are removed by a distinct base excision repair pathway initiated by the DNA glycosylase--AP lyase hNth1 in human cells. We have reconstituted this single-residue replacement pathway with recombinant proteins, including the AP endonuclease HAP1/APE, DNA polymerase beta, and DNA ligase III-XRCC1 heterodimer. With these proteins, the nucleotide excision repair enzyme XPG serves as a cofactor for the efficient function of hNth1. XPG protein promotes binding of hNth1 to damaged DNA. The stimulation of hNth1 activity is retained in XPG catalytic site mutants inactive in nucleotide excision repair. The data support the model that development of Cockayne syndrome in XP-G patients is related to inefficient excision of endogenous oxidative DNA damage.

Vertebrate telomere repeat DNAs favor external loop propeller quadruplex structures in the presence of high concentrations of potassium

The circular dichroism, CD, spectra of the telomere repeats of vertebrates, d(TTAGGG), indicate that parallel type quadruplex structures or disordered single-stranded structures are formed in low salt. Anti-parallel quadruplex structures are favored in the presence of high concentrations, 140 mM, of sodium. External loop, also known as propeller, parallel type structures are favored in the presence of high concentrations, 100 mM, of potassium in the presence of either 5 or 140 mM sodium. The cation dependence of the CD spectra of the vertebrate telomere repeat DNAs is distinctly different from that of the telomere repeats of Tetrahymena and Oxytricha as well as that of the thrombin binding aptamer. These results indicate that the external loop structures may be present in vertebrate telomeres under the conditions of high potassium and low sodium concentration found in nuclei.

Relayed coherence transfer spectroscopy of heteronuclear systems: detection of remote nuclei in NMR

Abstract Heteronuclear two-dimensional NMR experiments are presented which allow the correlation of nuclei that are not directly coupled but belong to a common coupling network. The techniques involve two or more consecutive coherence transfer steps. The applications discussed include the indirect observation of remote proton signals through phosphorus-31 and carbon-13 detection.

Assignments and structural information via relayed coherence transfer spectroscopy

Investigations of molecular structures by nuclear magnetic resonance have, for the most part, relied on interpreting spectral parameters such as chemical shifts and spin couplings. A more direct approach can be based on the determination of extended coupling networks. When the coupling networks include nuclei which are in no way coupled to one another but do share a common coupling partner then new information is gained which can aid in both structural determination and in the assignment of resonances (1, 2). An example of this approach is the correlation of the chemical shifts of carbon13 nuclei with not only the directly coupled neighbor protons but to those remote protons coupled to the neighbor protons. Adjacent carbon sites in a molecule can be identified since their coupling networks overlap. The experiment might be thought of as an extension of two-dimensional chemical shift correlation spectroscopy (3, 4) which uses a heteronuclear spy to report on those protons which are directly coupled to the heteronucleus (5, 6). In the experiment presented below the heteronuclear spy reports not only on the directly coupled protons but those protons coupled to the directly coupled protons. The procedure will be referred to as relayed coherence transfer as it is based on relaying the spectral information about the remote nuclei through a neighbor to the heteronucleus. The basic idea which allows the observation of the remote protons via the heteronucleus is the merging of two, or more, distinct coherence transfer processes. In the example presented here the first coherence transfer process is between protons, which is then followed by a heteronuclear transfer. A two-coherence transfer experiment is most easily illustrated by considering the case of an AMX spin system with the heteronuclear coupling JAX vanishing and with JMx being much larger than JAM as is typically the case for proton-proton and proton-carbon13 couplings. The pulse sequence used to do the coherence transfers is shown in Fig. 1. The first two proton pulses, separated by a time t,, transfer coherence from A to M spins, among other pathways, as occurs in a homonuclear correlation experiment (7, 8). Once the magnetization has been passed from the remote A spin to the neighbor M spin it can then be relayed to the heteronucleus. The heteronuclear transfer occurs with the proton and carbon-13 pulses at the end of the time t, + fu. The time r, + tH is chosen so that the multiplets which are out of phase with

Functional and dysfunctional roles of quadruplex DNA in cells

A number of biological roles have been proposed for quadruplex, also referred to as G4 or tetraplex, DNA. The presence of quadruplex DNA may lead to errors in some biological processes and be required in others. Proteins that interact with quadruplex DNA have been identified including those that cause Blooms and Werners syndromes. There are small molecules that specifically bind to quadruplex DNA, inhibit telomerase, and are cytotoxic towards tumor cells indicating a role for quadruplex DNA in telomere function. It is now possible to make testable proposals for the possible biological implications of quadruplex DNA in replication, transcription, and recombination as well as possible routes to therapeutic intervention.

Protein φ and ψ dihedral restraints determined from multidimensional hypersurface correlations of backbone chemical shifts and their use in the determination of protein tertiary structures

The chemical shifts of the backbone atoms of proteins can be used to obtainrestraints that can be incorporated into structure determination methods. Eachchemical shift can be used to define a restraint and these restraints can besimultaneously used to define the local, secondary structure features. Theglobal fold can be determined by a combined use of the chemical shift basedrestraints along with the long-range information present in the NOEs ofpartially deuterated proteins or the amide–amide NOEs but not from suchlimited NOE data sets alone. This approach has been demonstrated to be capableof determining the overall folding pattern of four proteins. This suggeststhat solution-state NMR methods can be extended to the structure determinationof larger proteins by using the information present in the chemical shifts ofthe backbone atoms along with the data that can be obtained on a small numberof labeled forms.

Enhancement of signals from insensitive nuclei by magnetization transfer via J couplings

Abstract The use of a population transfer method to enhance the signals of insensitive nuclei has been evaluated for some molecules of typical interest. It is shown that the population transfer method is clearly superior to the nuclear Overhauser effect in enhancing the signals of all of the protonated carbons of an amino acid and nucleotides. The population transfer method enhances the signals of protonated nitrogen-15 by as much as a factor of 7 greater than the nuclear Overhauser effect. The main advantage of the population transfer method lies in its ability to exploit the relatively rapid relaxation of the protons. The application of the method to tRNA, a polynucleotide of molecular weight about 30,000, is a failure. The population transfer method will not, in general, be applicable to macromolecules due to T2 losses during the population transfer and since the carbon-13 T1s will typically be shorter than those of the protons, as is the case for tRNA.

Mix and measure fluorescence screening for selective quadruplex binders

The human genome contains thousands of regions, including that of the telomere, that have the potential to form quadruplex structures. Many of these regions are potential targets for therapeutic intervention. There are many different folding patterns for quadruplex DNAs and the loops exhibit much more variation than do the quartets. The successful targeting of a particular quadruplex structure requires distinguishing that structure from all of the other quadruplex structures that may be present. A mix and measure fluorescent screening method has been developed, that utilizes multiple reporter molecules that bind to different features of quadruplex DNA. The reporter molecules are used in combination with DNAs that have a variety of quadruplex structures. The screening is based on observing the increase or decrease in the fluorescence of the reporter molecules. The selectivity of a set of test molecules has been determined by this approach.

Structure determination and analysis of local bending in an A-tract DNA duplex: comparison of results from crystallography, nuclear magnetic resonance, and molecular dynamics simulation on d(CGCAAAAATGCG).

We have presented a detailed analysis for structure determinations for the DNA duplex d(CGCAAAAATGCG) obtained from X-ray crystallography, nuclear magnetic resonance, and molecular dynamics simulation. Each of the structures for the duplex deviates from the structure of the canonical form of B-DNA in a number of observable characteristics. Specifically, the three determinations all contain DNA axis deflections at the junctions of the A-tract with the flanking sequences. The analysis provided shows that the general characteristics of the structures obtained for d(CGCAAAAATGCG) from X-ray, NMR, and MD methods turn out to be quite similar. The extent to which this result can be generalized remains to be established by consideration of similar cross-comparisons on diverse oligonucleotide structures.

A method for the observation of selected proton NMR resonances of proteins

The determination of protein structures in aqueous solution represents a formidable problem in biochemistry and mo lecular biology. Although NMR spectroscopy has the potential for providing structural information through proton chemical shifts, proton-proton couplings, and proton-proton nuclear Overhauser effects, the extent of overlap in the proton spectra, whether one or two dimensional, is often too great to allow detailed analysis. A number of approaches have been used to allow selective detection of only a particular subset of protons to ease assignment and overlap problems (I-4); however, all of these methods have significant shortcomings. We have devised a solution to this problem which relies upon both the high sensitivity selective observation of the NMR spectral properties of protons directly bonded to 13C and the use of recombinant plasmids which facilitate the economical preparation of the necessary isotopically labeled protein mo lecules. We have chosen staphylococcal nuclease, Nase, as our experimental system since the structural gene was recently c loned and sequenced (5). The availability of this gene has permitted the construction of a recombinant plasmid that permits the facile preparation of Nase samples that are labeled with r3C in a specific site of a given am ino acid; in addition, site directed mutagenesis can be used to generate mutant versions of the enzyme that may prove useful in the assignment of the ‘H NMR resonances of selected am ino acid residues. Such isotopically labeled Nase samples can be studied using NMR techniques that allow observation of only those protons directly bonded to a heteronucleus (neighbor protons) (6, 7); a two-dimensional NMR experiment can be used to observe those protons which are coupled to the neighbor protons (remote protons) (6). Selective detection of the neighbors was performed using the pulse sequence ‘H: 90”-r/2-180”-~/2-r-acquisition