Chromosome-wide distribution of haplotype blocks and the role of recombination hot spots.

Recent studies of human populations suggest that the genome consists of chromosome segments that are ancestrally conserved ('haplotype blocks'; refs. 1-3) and have discrete boundaries defined by recombination hot spots. Using publicly available genetic markers, we have constructed a first-generation haplotype map of chromosome 19. As expected for this marker density, approximately one-third of the chromosome is encompassed within haplotype blocks. Evolutionary modeling of the data indicates that recombination hot spots are not required to explain most of the observed blocks, providing that marker ascertainment and the observed marker spacing are considered. In contrast, several long blocks are inconsistent with our evolutionary models, and different mechanisms could explain their origins.

A quantitative method for evaluating the stabilities of nucleic acids.

We report a general method for screening, in solution, the impact of deviations from canonical Watson-Crick composition on the thermodynamic stability of nucleic acid duplexes. We demonstrate how fluorescence resonance energy transfer (FRET) can be used to detect directly free energy differences between an initially formed "reference" duplex (usually a Watson-Crick duplex) and a related "test" duplex containing a lesion/alteration of interest (e.g., a mismatch, a modified, a deleted, or a bulged base, etc.). In one application, one titrates into a solution containing a fluorescently labeled, FRET-active, reference duplex, an unlabeled, single-stranded nucleic acid (test strand), which may or may not compete successfully to form a new duplex. When a new duplex forms by strand displacement, it will not exhibit FRET. The resultant titration curve (normalized fluorescence intensity vs. logarithm of test strand concentration) yields a value for the difference in stability (free energy) between the newly formed, test strand-containing duplex and the initial reference duplex. The use of competitive equilibria in this assay allows the measurement of equilibrium association constants that far exceed the magnitudes accessible by conventional titrimetric techniques. Additionally, because of the sensitivity of fluorescence, the method requires several orders of magnitude less material than most other solution methods. We discuss the advantages of this method for detecting and characterizing any modification that alters duplex stability, including, but not limited to, mutagenic lesions. We underscore the wide range of accessible free energy values that can be defined by this method, the applicability of the method in probing for a myriad of nucleic acid variations, such as single nucleotide polymorphisms, and the potential of the method for high throughput screening.

Mammographic changes after hormone replacement therapy in patients who have undergone breast irradiation.

OBJECTIVE: We examined three patients who began hormone replacement therapy after lumpectomy and breast irradiation. In these women increased tissue density appeared on mammography only or disproportionately in the nonirradiated breast. To our knowledge, this observation has not been reported. CONCLUSION: Breast irradiation may induce tissue changes that prevent or diminish the proliferative response that can be induced in breast tissue by postmenopausal hormone replacement therapy. Because this treatment is now recommended for some women after breast conservation therapy, mammographers may recognize this finding with increasing frequency.

Effects of 3,N4-ethenodeoxycytidine on duplex stability and energetics.

The exocyclic cytosine adduct 3,N4-ethenocytosine is highly mutagenic in mammalian cells. We describe the impact of this adduct on DNA duplex stability. The adduct does not disrupt the overall B-form DNA structure; however, structural accommodation of the adduct is necessary at the lesion site. Despite the relatively small structural perturbation imparted by the adduct, there is a large adduct-induced destabilization of the DNA duplex. This destabilization is observed to be independent of the cross-strand partner base and neighbouring base pairs. The thermodynamic origins of the destabilization are, however, strongly dependent on the cross-strand partner base and neighbouring base pairs. Comparisons are made between the impact of the 3,N4-ethenocytosine adduct and other lesions on DNA thermodynamics. The lesions are similar in that all result in destabilization of the DNA duplex. The magnitudes and the thermodynamic origins of that destabilization vary widely, the 3,N4-ethenocytosine adduct being dramatically more destabilizing than other lesions. The impact of damaged sites on the stability of the DNA helix suggests that energetic differences between damaged and normal DNA may contribute to the recognition of damage by the cellular DNA repair machinery.

The impact of an exocyclic cytosine adduct on DNA duplex properties: significant thermodynamic consequences despite modest lesion-induced structural alterations.

The exocyclic base adduct 3,N4-deoxyethenocytosine (epsilonC) is a common DNA lesion that can arise from carcinogen exposure and/or as a biproduct of cellular processes. We have examined the thermal and thermodynamic impact of this lesion on DNA duplex properties, as well as the structural alterations imparted by the lesion. For these studies, we used calorimetric and spectroscopic techniques to investigate a family of 13-mer DNA duplexes of the form (5'CGCATGNGTACGC3')x(3'GCGTACNCATGCG5'), where the central NxN base pair represents the four standard Watson-Crick base pairs (corresponding to four control duplexes), and where either one of the N bases has been replaced by epsilonC, yielding eight test duplexes. Studies on these 12 duplexes permit us to assess the impact of the epsilonC lesion as a function of sequence context. Our spectroscopic and calorimetric data allow us to reach the following conclusions: (i) The epsilonC lesion imparts a large penalty on duplex stability, with sequence context only modestly modulating the extent of this lesion-induced destabilization. This result contrasts with our recent studies of duplexes with abasic sites, where sequence context was found to be the predominant determinant of thermodynamic damage. (ii) For the epsilonC-containing duplexes, sequence context effects are most often observed in the enthalpic contribution to lesion-induced duplex destabilization. However, due to compensating entropies, the free energy changes associated with this lesion-induced duplex destablization are nearly independent of sequence context. (iii) Despite significant lesion-induced changes in duplex energetics, our spectroscopic probes detect only modest lesion-induced changes in duplex structure. In fact, the overall duplex maintains a global B-form conformation, in agreement with NMR structural data. We discuss possible interpretations of the apparent disparity between the severe thermodynamic and relatively mild structural impacts of the epsilonC lesion on duplex properties. We also note and discuss the implications of empirical correlations between biophysical and biological properties of lesion-containing duplexes.

Thermodynamic consequences of an abasic lesion in duplex DNA are strongly dependent on base sequence.

The abasic site in DNA may arise spontaneously, as a result of nucleotide base damage, or as an intermediate in glycosylase-mediated DNA-repair pathways. It is the most common damage found in DNA. We have examined the consequences of this lesion and its sequence context on DNA duplex structure, as well as the thermal and thermodynamic stability of the duplex, including the energetic origins of that stability. To this end, we incorporated a tetrahydrofuran abasic site analogue into a family of 13-mer DNA duplexes, wherein the base opposite the lesion (A, C, G, or T) and the base pairs neighboring the lesion (C.G or G.C) were systematically varied and characterized by a combination of spectroscopic and calorimetric techniques. The resulting data allowed us to reach the following conclusions: (i) the presence of the lesion in all sequence contexts studied does not alter the global B-form conformation characteristic of the parent undamaged duplex; (ii) the presence of the lesion induces a significant enthalpic destabilization of the duplex, with the magnitude of this effect being dependent on the sequence context; (iii) the thermodynamic impact of the lesion is dominated by the identity of the neighboring base pairs, with the cross strand partner base exerting only a secondary thermodynamic effect on duplex properties. In the aggregate, our data reveal that even in the absence of lesion-induced alterations in global structure, the abasic lesion can significantly alter the thermodynamic properties of the host duplex, with the magnitude of this impact being strongly dependent on sequence context.

Binding of a hairpin polyamide in the minor groove of DNA: sequence-specific enthalpic discrimination.

Hairpin polyamides are synthetic ligands for sequence-specific recognition in the minor groove of double-helical DNA. A thermodynamic characterization of the DNA-binding properties exhibited by a six-ring hairpin polyamide, ImPyPy-gamma-PyPyPy-beta-Dp (where Im = imidazole, Py = pyrrole, gamma = gamma-aminobutyric acid, beta = beta-alanine, and Dp = dimethylaminopropylamide), reveals an approximately 1-2 kcal/mol greater affinity for the designated match site, 5'-TGTTA-3', relative to the single base pair mismatch sites, 5'-TGGTA-3' and 5'-TATTA-3'. The enthalpy and entropy data at 20 degrees C reveal this sequence specificity to be entirely enthalpic in origin. Correlations between the thermodynamic driving forces underlying the sequence specificity exhibited by ImPyPy-gamma-PyPyPy-beta-Dp and the structural properties of the heterodimeric complex of PyPyPy and ImPyPy bound to the minor groove of DNA provide insight into the molecular forces that govern the affinity and specificity of pyrrole-imidazole polyamides.

Role of cellular casein kinase II in the function of the phosphoprotein (P) subunit of RNA polymerase of vesicular stomatitis virus.

The phosphorylation of the P protein of vesicular stomatitis virus by cellular casein kinase II (CKII) is essential for its activity in viral transcription. Recent in vitro studies have demonstrated that CKII converts the inactive unphosphorylated form of P (P0) to an active phosphorylated form P1, after phosphorylation at two serine residues, Ser-59 and Ser-61. To gain insight into the role of CKII-mediated phosphorylation in the structure and function of the P protein, we have carried out circular dichroism (CD) and biochemical analyses of both P0 and P1. The results of CD analyses reveal that phosphorylation of P0 to P1 significantly increases the predicted alpha-helical structure of the P1 protein from 27 to 48%. The phosphorylation defective double serine mutant (P59/61), which is transcriptionally inactive, possesses a secondary structure similar to that of P0. P1, at a protein concentration of 50 micrograms/ml, elutes from a gel filtration column apparently as a dimer, whereas both P0 and the double serine mutant elute as a monomer at the same concentration. Interestingly, unlike wild-type P1 protein, the P mutants in which either Ser-59 or Ser-61 is altered to alanine required a high concentration of CKII for optimal phosphorylation. We demonstrate here that phosphorylation of either Ser-59 or Ser-61 is necessary and sufficient to transactivate L polymerase although alteration of one serine residue significantly decreases its affinity for CKII. We have also shown that P1 binds to the N-RNA template more efficiently than P0 and the formation of P1 is a prerequisite for the subsequent phosphorylation by L protein-associated kinase. In addition, mutant P59/61 acts as a transdominant negative mutant when used in a transcription reconstitution assay in the presence of wild-type P protein.

Spectral analysis and tryptic susceptibility as probes of HIV-1 capsid protein structure.

The structures of HIV-1 capsid protein (CA, p24) isolated from mature virions and CA protein autoprocessed from a recombinant Gag-Pol precursor expressed in Escherichia coli were compared using circular dichroic (CD) spectral analysis. The spectra obtained for the intact recombinant and viral proteins were indistinguishable, indicating that the backbone configurations directed by the primary amino acid sequences of the proteins were similar or identical. The structure predictions derived from CD were, in general, inconsistent with a model proposing the eight-stranded beta barrel motif found in several RNA viruses. However, aspects of the model were supported by experiments that identified surface-exposed regions. Biochemical analysis indicated that the recombinant CA protein formed nonrandom higher-ordered structures in vitro. Under physiological conditions, the protein assembled into oligomers containing subunits in two packing arrangements. In one arrangement, the central region near Arg100 was exposed and susceptible to tryptic digestion at low enzyme concentrations (enzyme:substrate ratios = 1:5000 to 1:100). Also, in this arrangement, the proteins were susceptible to crosslinking by the bifunctional agent DTSSP. Proteins in the other arrangement were resistant to proteolysis at low enzyme concentrations. The central region of these resistant molecules was inaccessible to monospecific antibodies that recognized antigenic sites between residues 94 and 107 and these proteins were not crosslinked by DTSSP or EGS. Following incubation with trypsin, both the resistant molecules and the fragments derived from the susceptible proteins in the oligomer migrated as smaller complexes, suggesting that the regions digested by trypsin stabilized the oligomer unit. The results indicate that the central region of the HIV-1 CA protein plays a role in formation of higher-ordered structures. Moreover, the relative stability of the N- and C-terminal partial digestion fragments arising from cleavage at Arg100/Gly101 suggests that this exposed central region separates two structural domains of the protein.

Interactions of avian myeloblastosis virus nucleocapsid protein with nucleic acids.

The retroviral nucleocapsid protein (NC) associates, histone-like, with genomic RNA within the viral capsid. NC, an essential component of replication competent retroviruses, is also associated with events leading both to virus assembly and to reverse transcription. The nucleic acid binding properties of NC are key to understanding these properties, yet only a minimal biochemical description of NC-nucleic acid interactions is available. We have used the anisotropy of the intrinsic fluorescence of NC from avian myeloblastosis virus to quantify its binding to a variety of nucleic acids. Using salt back-titrations, the intrinsic equilibrium association constant per nucleic acid site, K(obs), was determined for NC binding to single- and double-stranded RNAs and DNAs. In 0.125 M NaCl, 40 mM HEPES at pH 7.0 and 27 degrees C, the log K(obs) ranged from 3.3 to 4.0 (average 3.7) for these nucleic acids. From the salt dependence of K(obs), it was estimated that, on balance, 1 ion was displaced upon formation of each complex; it is likely that cation displacement from nucleic acid is offset by anion binding by protein during complex formation. The logarithm of the mean intrinsic affinity in the absence of polyelectrolyte effects, log KT, was 3.1, corresponding to a delta G of -4.2 kcal/mol. K(obs), KT, and the number of displaced ions were independent of pH between pH 5.6 and 8.9, indicating that NC residues that titrate in this pH range are not contributing to binding. K(obs) and KT increase with temperature, in the range 15 to 47 degrees C. From van't Hoff analysis, entropy was found to be the driving force for formation of the NC-poly(rA) complex, even in the absence of the polyelectrolyte effect. The general nature of NC interactions with nucleic acids is shown by the similarity of the K(obs) values for RNAs and DNAs in both single-stranded and double-stranded structures. This ability of NC to interact with all types of nucleic acids may provide it with the necessary versatility to function like a histone in facilitating the packaging of viral RNA and yet function early in infection, where it has been ascribed a role in facilitating reverse transcription.

Retroviral nucleocapsid protein specifically recognizes the base and the ribose of mononucleotides and mononucleotide components.

The interaction of the retroviral nucleocapsid protein (NC) with nucleic acids forms the basis of its varied roles in the replication cycle, which include binding and condensing the viral RNA within the virion, stimulation of the early steps in reverse transcription, and dissociation from RNA in the replication complex. As part of an investigation of the NC binding site and of the forces that drive its interaction with nucleic acids, the relative affinities of NC from avian myeloblastosis virus were determined for a series of mononucleotides and mononucleotide components using a competitive displacement assay utilizing the extrinsic fluorescent probe bis-ANS [Secnik, J., Wang, Q., Chang, C.-M., & Jentoft, J.E. (1990) Biochemistry 29, 7991-7997]. The estimated binding affinities were unexpectedly similar for nucleotides, nucleosides, and bases (Ka greater than 10(6) M-1). AMP, UMP, GMP, and CMP bound to NC with essentially equal affinity, indicating that NC does not discriminate between bases. This is consistent with its role in coating, condensing, and packaging the RNA within virions. Nucleosides, bases, riboses, and ribose phosphate bind to NC with 1000-fold higher affinity than inorganic phosphate, indicating that the NC binding site includes elements that recognize nucleotide base and ribose components in addition to phosphate ions. However, the binding affinities of components are not additive, i.e., the Kapp values for adenine and deoxyribose are very similar to that for deoxyadenosine, indicating that the interaction between the NC subsite and the base and the sugar components is complex. The stoichiometry of the complex between bis-ANS and NC was established to be NC.(bis-ANS)3.(ABSTRACT TRUNCATED AT 250 WORDS)