Langer-Giedion syndrome associated with submucous cleft palate

We report a 4-year-old girl with characteristic features of the Langer-Giedion syndrome (trichorhinophalangeal syndrome type II) who also had submucous cleft palate. When she underwent a palatoplasty, a diagnosis of Langer-Giedion syndrome was made because of the characteristic facial features, multiple exostoses, and partial deletion of the long arm of chromosome 8. This is the first case of trichorhinophalangeal syndrome associated with cleft palate. We review the clinical alterations of trichorhinophalangeal syndromes and differential diagnosis of Langer-Giedion syndrome from trichorhinophalangeal syndrome type I and hereditary multiple exostoses. We also describe the importance of trichorhinophalangeal syndrome in plastic surgery.     

Extending the understanding of mutagenicity: structural insights into primer-extension past a benzo[a]pyrene diol epoxide-DNA adduct

DNA polymerase enzymes employ a number of innate fidelity mechanisms to ensure the faithful replication of the genome. However, when confronted with DNA damage, their fidelity mechanisms can be evaded, resulting in a mutation that may contribute to the carcinogenic process. The environmental carcinogen benzo[a]pyrene is metabolically activated to reactive intermediates, including the tumorigenic (+)-anti-benzo[a]pyrene diol epoxide, which can attack DNA at the exocyclic amino group of guanine to form the major (+)-trans-anti-[BP]-N(2)-dG adduct. Bulky adducts such as (+)-trans-anti-[BP]-N(2)-dG primarily block DNA replication, but are occasionally bypassed and cause mutations if paired with an incorrect base. In vitro standing-start primer-extension assays show that the preferential insertion of A opposite (+)-trans-anti-[BP]-N(2)-dG is independent of the sequence context, but the primer is extended preferentially when dT is positioned opposite the damaged base in a 5'-CG*T-3' sequence context. Regardless of the base positioned opposite (+)-trans-anti-[BP]-N(2)-dG, extension of the primer past the lesion site poses the greatest block to polymerase progression. In order to gain insight into primer-extension of each base opposite (+)-trans-anti-[BP]-N(2)-dG, we carried out molecular modeling and 1.25 ns unrestrained molecular dynamics simulations of the adduct in the +1 position of the template within the replicative pol I family T7 DNA polymerase. Each of the four bases was modeled at the 3' terminus of the primer, incorporated opposite the adduct, and the next-to-be replicated base was in the active site with its Watson-Crick partner as the incoming nucleotide. As in our studies of nucleotide incorporation, (+)-trans-anti-[BP]-N(2)-dG was modeled in the syn conformation in the +1 position, with the BP moiety on the open major groove side of the primer-template duplex region, leaving critical protein-DNA interactions intact. The present work revealed that the efficiency of primer-extension past this bulky adduct opposite each of the four bases in the 5'-CG*T-3' sequence can be rationalized by the stability of interactions between the polymerase protein, primer-template DNA and incoming nucleotide. However, the relative stabilization of each nucleotide opposite (+)-trans-anti-[BP]-N(2)-dG in the +1 position (T > G > A > or = C) differed from that when the adduct and partner were the nascent base-pair (A > T > or = G > C). In addition, extension past (+)-trans-anti-[BP]-N(2)-dG may pose a greater block to a high fidelity DNA polymerase than does nucleotide incorporation opposite the adduct because the presence of the modified base-pair in the +1 position is more disruptive to the polymerase-DNA interactions than it is within the active site itself. The dN:(+)-trans-anti-[BP]-N(2)-dG base-pair is strained to shield the bulky aromatic BP moiety from contact with the solvent in the +1 position, causing disruption of protein-DNA interactions that would likely result in decreased extension of the base-pair. These studies reveal in molecular detail the kinds of specific structural interactions that determine the function of a processive DNA polymerase when challenged by a bulky DNA adduct.     

Structural and thermodynamic features of spiroiminodihydantoin damaged DNA duplexes

Oxidation of guanine or 8-oxo-7,8-dihydroguanine can produce spiroiminodihydantoin (Sp) R and S stereoisomers. Both in vitro and in vivo experiments have shown that the Sp stereoisomers are highly mutagenic, causing G --> C and G --> T transversion mutations. Therefore, they are of interest as potential endogenous cancer causing lesions. However, their structural properties in DNA duplexes remain to be elucidated. We have employed computational methods to study the Sp lesions in 11-mer DNA duplexes with A, C, G, and T partners. Molecular dynamics simulations have been carried out to obtain ensembles of structures, and the trajectories were employed to analyze the structures and compute free energies. The structural and thermodynamic analyses reveal that the Sp stereoisomers energetically favor positioning in the B-DNA major groove, with minor groove conformers also low energy in some cases, depending on the partner base. The R and S stereoisomers adopt opposite orientations with respect to the 5' to 3' direction of the modified strand. Both syn and anti glycosidic bond conformations are energetically feasible, with partner base and stereochemistry determining the preference. The lesions adversely impact base stacking and Watson-Crick hydrogen bonding interactions in the duplex, and cause groove widening. The chemical nature of the partner base determines specific hydrogen bonding and stacking properties of the damaged duplexes. The structural characteristics may relate to observed mutagenic properties of the Sp stereoisomers, including possible stereoisomer-dependent differences.     

Altering DNA polymerase incorporation fidelity by distorting the dNTP binding pocket with a bulky carcinogen-damaged template

Fidelity of DNA polymerases is predominantly governed by an induced fit mechanism in which the incoming dNTP in the ternary complex fits tightly into a binding pocket whose geometry is determined by the nature of the templating base. However, modification of the template with a bulky carcinogen may alter the dNTP binding pocket and thereby the polymerase incorporation fidelity. High fidelity DNA polymerases, such as bacteriophage T7 DNA polymerase, are predominantly blocked by bulky chemical lesions on the template strand during DNA replication. However, some mutagenic bypass can occur, which may lead to carcinogenesis. Experimental studies have shown that a DNA covalent adduct derived from (+)-anti-BPDE [(+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene], a carcinogenic metabolite of benzo[a]pyrene (BP), primarily blocks Sequenase 2.0, an exo(-) T7 DNA polymerase; however, a mismatched dATP can be preferentially inserted opposite the damaged adenine templating base within the active site of the polymerase [Chary, P., and Lloyd, R. S. (1995) Nucleic Acids Res. 23, 1398-1405]. The goal of this work is to elucidate structural features that contribute to DNA polymerase incorporation fidelity in the presence of this bulky covalent adduct and to interpret the experimental findings on a molecular level. We have carried out molecular modeling and molecular dynamics simulations with AMBER 6.0, investigating a T7 DNA polymerase primer-template closed ternary complex containing this 10S (+)-trans-anti-[BP]-N(6)-dA adduct in the templating position within the polymerase active site. All four incoming dNTPs were studied. The simulations show that the BP ring system fits well into an open pocket on the major groove side of the modified template adenine with anti glycosidic bond conformation, without disturbing critical polymerase-DNA interactions. However, steric hindrance between the BP ring system and the primer-template DNA causes displacement of the modified template adenine, so that the dNTP base binding pocket is enlarged. This alteration can explain the experimentally observed preference for incorporation of dATP opposite this lesion. These studies also rationalize the observed lower probabilities of incorporation of the other three nucleotides. Our results suggest that the differences in incorporation of dGTP, dCTP, and dTTP are due to the effects of imperfect geometric complementarity. Thus, the simulations suggest that altered DNA polymerase incorporation fidelity can result from adduct-induced changes in the dNTP base binding pocket geometry. Furthermore, plausible structural explanations for the observed effects of [BP]-N(6)-dA adduct stereochemistry on the observed stalling patterns are proposed.     

Substrate discrimination by formamidopyrimidine-DNA glycosylase: distinguishing interactions within the active site

Reactive oxygen species are byproducts of normal aerobic respiration and ionizing radiation, and they readily react with DNA to form a number of base lesions, including the mutagenic 8-oxo-7,8-dihydroguanine (8-oxoG), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), 4,6-diamino-5-formamidopyrimidine (FapyA), and 8-oxo-7,8-dihydroadenine (8-oxoA). Such oxidative lesions are removed by the base excision repair pathway, which is initiated by DNA glycosylases such as the formamidopyrimidine-DNA glycosylase (Fpg) in Escherichia coli. The 8-oxoG, FapyG, and FapyA lesions are bound and excised by Fpg, while structurally similar 8-oxoA is excised by Fpg very poorly. We carried out molecular modeling and molecular dynamics simulations to interpret substrate discrimination within the active site of E. coli Fpg. Lys-217 and Met-73 were identified as residues playing important roles in the recognition of the oxidized imidazole ring in the substrate bases, and the Watson-Crick edge of the damaged base plays a role in optimally positioning the base within the active site. The recognition and excision of FapyA likely result from the opened imidazole ring, while 8-oxoA's lack of flexibility and closed imidazole ring may contribute to Fpg's inability to excise this base. Different interactions between each base and the enzyme specificity pocket account for differential treatment of the various lesions by this enzyme, and thus elucidate the structure-function relationship involved in an initial step of base excision repair.     

Preferred WMSA catalytic mechanism of the nucleotidyl transfer reaction in human DNA polymerase �� elucidates error-free bypass of a bulky DNA lesion

Human DNA Pol κ is a polymerase enzyme, specialized for near error-free bypass of certain bulky chemical lesions to DNA that are derived from environmental carcinogens present in tobacco smoke, automobile exhaust and cooked food. By employing ab initio QM/MM–MD (Quantum Mechanics/Molecular Mechanics–Molecular Dynamics) simulations with umbrella sampling, we have determined the entire free energy profile of the nucleotidyl transfer reaction catalyzed by Pol κ and provided detailed mechanistic insights. Our results show that a variant of the Water Mediated and Substrate Assisted (WMSA) mechanism that we previously deduced for Dpo4 and T7 DNA polymerases is preferred for Pol κ as well, suggesting its broad applicability. The hydrogen on the 3′-OH primer terminus is transferred through crystal and solvent waters to the γ-phosphate of the dNTP, followed by the associative nucleotidyl transfer reaction; this is facilitated by a proton transfer from the γ-phosphate to the α,β-bridging oxygen as pyrophosphate leaves, to neutralize the evolving negative charge. MD simulations show that the near error-free incorporation of dCTP opposite the major benzo[a]pyrene—derived dG lesion is compatible with the WMSA mechanism, allowing for an essentially undisturbed pentacovalent phosphorane transition state, and explaining the bypass of this lesion with little mutation by Pol κ.     

Structural, energetic and dynamic properties of guanine(C8)-thymine(N3) cross-links in DNA provide insights on susceptibility to nucleotide excision repair

The one-electron oxidation of guanine in DNA by carbonate radical anions, a decomposition product of peroxynitrosocarbonate which is associated with the inflammatory response, can lead to the formation of intrastrand cross-links between guanine and thymine bases [Crean et al. (Oxidation of single-stranded oligonucleotides by carbonate radical anions: generating intrastrand cross-links between guanine and thymine bases separated by cytosines. Nucleic Acids Res. 2008; 36: 742-755.)]. These involve covalent bonds between the C8 positions of guanine (G*) and N3 of thymine (T*) in 5'-d(…G*pT*…) and 5'-d(…G*pCpT*…) sequence contexts. We have performed nucleotide excision repair (NER) experiments in human HeLa cell extracts which show that the G*CT* intrastrand cross-link is excised with approximately four times greater efficiency than the G*T* cross-link embedded in 135-mer DNA duplexes. In addition, thermal melting studies reveal that both lesions significantly destabilize duplex DNA, and that the destabilization induced by the G*CT* cross-link is considerably greater. Consistent with this difference in NER, our computations show that both lesions dynamically distort and destabilize duplex DNA. They disturb Watson-Crick base-pairing and base-stacking interactions, and cause untwisting and minor groove opening. These structural perturbations are much more pronounced in the G*CT* than in the G*T* cross-link. Our combined experimental and computational studies provide structural and thermodynamic understanding of the features of the damaged duplexes that produce the most robust NER response.     

Flavonoids Modulate the Proliferation of Neospora caninum in Glial Cell Primary Cultures

Neospora caninum (Apicomplexa; Sarcocystidae) is a protozoan that causes abortion in cattle, horses, sheep, and dogs as well as neurological and dermatological diseases in dogs. In the central nervous system of dogs infected with N. caninum, cysts were detected that exhibited gliosis and meningitis. Flavonoids are polyphenolic compounds that exhibit antibacterial, antiparasitic, antifungal, and antiviral properties. In this study, we investigated the effects of flavonoids in a well-established in vitro model of N. caninum infection in glial cell cultures. Glial cells were treated individually with 10 different flavonoids, and a subset of cultures was also infected with the NC-1 strain of N. caninum. All of the flavonoids tested induced an increase in the metabolism of glial cells and many of them increased nitrite levels in cultures infected with NC-1 compared to controls and uninfected cultures. Among the flavonoids tested, 3'',4''-dihydroxyflavone, 3'',4'',5,7-tetrahydroxyflavone (luteolin), and 3,3'',4'',5,6-pentahydroxyflavone (quercetin), also inhibited parasitophorous vacuole formation. Taken together, our findings show that flavonoids modulate glial cell responses, increase NO secretion, and interfere with N. caninum infection and proliferation.