poliota-dependent lesion bypass in vitro

Based upon phylogenetic relationships, the broad Y-family of DNA polymerases can be divided into various subfamilies consisting of UmuC (polV)-like; DinB (polIV/polκ)-like; Rev1-like, Rad30A (polη)-like and Rad30B (polι)-like polymerases. The polIV/polκ-like polymerases are most ubiquitous, having been identified in bacteria, archaea and eukaryotes. In contrast, the polV-like polymerases appear restricted to bacteria (both Gram positive and Gram negative). Rev1 and polη-like polymerases are found exclusively in eukaryotes, and to date, polι-like polymerases have only been identified in higher eukaryotes. In general, the in vitro properties of polymerases characterized within each sub-family are quite similar. An exception to this rule occurs with the polι-like polymerases, where the enzymatic properties of Drosophila melanogaster polι are more similar to that of Saccharomyces cerevisiae and human polη than to the related human polι. For example, like polη, Drosophila polι can bypass a cis-syn thymine–thymine dimer both accurately and efficiently, while human polι bypasses the same lesion inefficiently and with low-fidelity. Even in cases where human polι can efficiently insert a base opposite a lesion (such as a synthetic abasic site, the 3′T of a 6-4-thymine–thymine pyrimidine–pyrimidone photoproduct or opposite benzo[a]pyrene diol epoxide deoxyadenosine adducts), further extension is often limited. Thus, although polι most likely arose from a genetic duplication of polη millions of years ago as eukaryotes evolved, it would appear that polι from humans (and possibly all mammals) has been further subjected to evolutionary pressures that have “tailored” its enzymatic properties away from lesion bypass and towards other function(s) specific for higher eukaryotes. The identification of such functions and the role that mammalian polι plays in lesion bypass in vivo, should hopefully be forthcoming with the construction of human cell lines deleted for polι and the identification of mice deficient in polι.     

Hepatic ABCG5 and ABCG8 overexpression increases hepatobiliary sterol transport but does not alter aortic atherosclerosis in transgenic mice

The individual roles of hepatic versus intestinal ABCG5 and ABCG8 in sterol transport have not yet been investigated. To determine the specific contribution of liver ABCG5/G8 to sterol transport and atherosclerosis, we generated transgenic mice that overexpress human ABCG5 and ABCG8 in the liver but not intestine (liver G5/G8-Tg) in three different genetic backgrounds: C57Bl/6, apoE-KO, and low density lipoprotein receptor (LDLr)-KO. Hepatic overexpression of ABCG5/G8 enhanced hepatobiliary secretion of cholesterol and plant sterols by 1.5-2-fold, increased the amount of intestinal cholesterol available for absorption and fecal excretion by up to 27%, and decreased the accumulation of plant sterols in plasma by approximately 25%. However, it did not alter fractional intestinal cholesterol absorption, fecal neutral sterol excretion, hepatic cholesterol concentrations, or hepatic cholesterol synthesis. Consequently, overexpression of ABCG5/G8 in only the liver had no effect on the plasma lipid profile, including cholesterol, HDL-C, and non-HDL-C, or on the development of proximal aortic atherosclerosis in C57Bl/6, apoE-KO, or LDLr-KO mice. Thus, liver ABCG5/G8 facilitate the secretion of liver sterols into bile and serve as an alternative mechanism, independent of intestinal ABCG5/G8, to protect against the accumulation of dietary plant sterols in plasma. However, in the absence of changes in fractional intestinal cholesterol absorption, increased secretion of sterols into bile induced by hepatic overexpression of ABCG5/G8 was not sufficient to alter hepatic cholesterol balance, enhance cholesterol removal from the body or to alter atherogenic risk in liver G5/G8-Tg mice. These findings demonstrate that overexpression of ABCG5/G8 in the liver profoundly alters hepatic but not intestinal sterol transport, identifying distinct roles for liver and intestinal ABCG5/G8 in modulating sterol metabolism.     

Sequence context-dependent replication of DNA templates containing UV-induced lesions by human DNA polymerase iota

Humans possess four Y-family polymerases: pols eta, iota, kappa and the Rev1 protein. The pivotal role that pol eta plays in protecting us from UV-induced skin cancers is unquestioned given that mutations in the POLH gene (encoding pol eta), lead to the sunlight-sensitive and cancer-prone xeroderma pigmentosum variant phenotype. The roles that pols iota, kappa and Rev1 play in the tolerance of UV-induced DNA damage is, however, much less clear. For example, in vitro studies in which the ability of pol iota to bypass UV-induced cyclobutane pyrimidine dimers (CPDs) or 6-4 pyrimidine-pyrimidone (6-4PP) lesions has been assayed, are somewhat varied with results ranging from limited misinsertion opposite CPDs to complete lesion bypass. We have tested the hypothesis that such discrepancies might have arisen from different assay conditions and local sequence contexts surrounding each UV-photoproduct and find that pol iota can facilitate significant levels of unassisted highly error-prone bypass of a T-T CPD, particularly when the lesion is located in a 3'-A[T-T]A-5' template sequence context and the reaction buffer contains no KCl. When encountering a T-T 6-4PP dimer under the same assay conditions, pol iota efficiently and accurately inserts the correct base, A, opposite the 3'T of the 6-4PP by factors of approximately 10(2) over the incorporation of incorrect nucleotides, while incorporation opposite the 5'T is highly mutagenic. Pol kappa has been proposed to function in the bypass of UV-induced lesions by helping extend primers terminated opposite CPDs. However, we find no evidence that the combined actions of pol iota and pol kappa result in a significant increase in bypass of T-T CPDs when compared to pol iota alone. Our data suggest that under certain conditions and sequence contexts, pol iota can bypass T-T CPDs unassisted and can efficiently incorporate one or more bases opposite a T-T 6-4PP. Such biochemical activities may, therefore, be of biological significance especially in XP-V cells lacking the primary T-T CPD bypassing enzyme, pol eta.     

Cellular pregnenolone esterification by acyl-CoA:cholesterol acyltransferase

Pregnenolone (PREG) can be converted to PREG esters (PE) by the plasma enzyme lecithin: cholesterol acyltransferase (LCAT), and by other enzyme(s) with unknown identity. Acyl-CoA:cholesterol acyltransferase 1 and 2 (ACAT1 and ACAT2) convert various sterols to steryl esters; their activities are activated by cholesterol. PREG is a sterol-like molecule, with 3-β-hydroxy moiety at steroid ring A, but with much shorter side chain at steroid ring D. Here we show that without cholesterol, PREG is a poor ACAT substrate; with cholesterol, the V(max) for PREG esterification increases by 100-fold. The binding affinity of ACAT1 for PREG is 30-50-fold stronger than that for cholesterol; however, PREG is only a substrate but not an activator, while cholesterol is both a substrate and an activator. These results indicate that the sterol substrate site in ACAT1 does not involve significant sterol-phospholipid interaction, while the sterol activator site does. Studies utilizing small molecule ACAT inhibitors show that ACAT plays a key role in PREG esterification in various cell types examined. Mice lacking ACAT1 or ACAT2 do not have decreased PREG ester contents in adrenals, nor do they have altered levels of the three major secreted adrenal steroids in serum. Mice lacking LCAT have decreased levels of PREG esters in the adrenals. These results suggest LCAT along with ACAT1/ACAT2 contribute to control pregnenolone ester content in different cell types and tissues.     

Influence of brassinosteroids on initiation of the root gravitropic response in Pisum sativum seedlings

In roots of Pisum sativum seedlings, the average lag-time required for initiation of the gravitropic response was reduced proportionally to the concentration of 24-epibrassinolide (EBL) added to the root solution (range of 10⁻¹³ to 10⁻⁸ M concentrations). A treatment with clotrimazole, a compound inhibiting steroid synthesis, prevents initiation of the gravitropic response. This effect was partly reverted by addition of EBL. From analysis of variability in the populations, it is suggested that BR conditions the root curvature through a gravitropic-induced change in sensitivity to the PGRs regulating cell elongation.     

Comprehensive mutagenesis of HIV-1 protease: a computational geometry approach

A computational geometry technique based on Delaunay tessellation of protein structure, represented by C(alpha) atoms, is used to study effects of single residue mutations on sequence-structure compatibility in HIV-1 protease. Profiles of residue scores derived from the four-body statistical potential are constructed for all 1881 mutants of the HIV-1 protease monomer and compared with the profile of the wild-type protein. The profiles for an isolated monomer of HIV-1 protease and the identical monomer in a dimeric state with an inhibitor are analyzed to elucidate changes to structural stability. Protease residues shown to undergo the greatest impact are those forming the dimer interface and flap region, as well as those known to be involved in inhibitor binding.     

Efficient initiation of S-phase in yeast requires Cdc40p, a protein involved in pre-mRNA splicing

The S. cerevisiae CDC40 gene was originally identified as a cell-division-specific gene that is essential only at elevated temperatures. Cells carrying mutations in this gene arrest with a large bud and a single nucleus with duplicated DNA content. Cdc40p is also required for spindle establishment or maintenance. Sequence analysis reveals that CDC40 is identical to PRP17, a gene involved in pre-mRNA splicing. In this paper, we show that Cdc40p is required at all temperatures for efficient entry into S-phase and that cell cycle arrest associated with cdc40 mutations is independent of all the known checkpoint mechanisms. Using immunofluorescence, we show that Cdc40p is localized to the nuclear membrane, weakly associated with the nuclear pore. Our results point to a link between cell cycle progression, pre-mRNA splicing, and mRNA export. Received: 9 April 1998 / Accepted: 10 August 1998     

Fidelity of Dpo4: effect of metal ions, nucleotide selection and pyrophosphorolysis

We report the crystal structures of a translesion DNA polymerase, Dpo4, complexed with a matched or mismatched incoming nucleotide and with a pyrophosphate product after misincorporation. These structures suggest two mechanisms by which Dpo4 may reject a wrong incoming nucleotide with its preformed and open active site. First, a mismatched replicating base pair leads to poor base stacking and alignment of the metal ions and as a consequence, inhibits incorporation. By replacing Mg2+ with Mn2+, which has a relaxed coordination requirement and tolerates misalignment, the catalytic efficiency of misincorporation increases dramatically. Mn2+ also enhances translesion synthesis by Dpo4. Subtle conformational changes that lead to the proper metal ion coordination may, therefore, be a key step in catalysis. Second, the slow release of pyrophosphate may increase the fidelity of Dpo4 by stalling mispaired primer extension and promoting pyrophosphorolysis that reverses the polymerization reaction. Indeed, Dpo4 has robust pyrophosphorolysis activity and degrades the primer strand in the presence of pyrophosphate. The correct incoming nucleotide allows DNA synthesis to overcome pyrophosphorolysis, but an incorrect incoming nucleotide does not.     

Application of singular spectrum-based change-point analysis to EMG-onset detection

While many approaches have been proposed to identify the signal onset in EMG recordings, there is no standardized method for performing this task. Here, we propose to use a change-point detection procedure based on singular spectrum analysis to determine the onset of EMG signals. This method is suitable for automated real-time implementation, can be applied directly to the raw signal, and does not require any prior knowledge of the EMG signal’s properties. The algorithm proposed by Moskvina and Zhigljavsky (2003) was applied to EMG segments recorded from wrist and trunk muscles. Wrist EMG data was collected from 9 Parkinson’s disease patients with and without tremor, while trunk EMG data was collected from 13 healthy able-bodied individuals. Along with the change-point detection analysis, two threshold-based onset detection methods were applied, as well as visual estimates of the EMG onset by trained practitioners. In the case of wrist EMG data without tremor, the change-point analysis showed comparable or superior frequency and quality of detection results, as compared to other automatic detection methods. In the case of wrist EMG data with tremor and trunk EMG data, performance suffered because other changes occurring in these signals caused larger changes in the detection statistic than the changes caused by the initial muscle activation, suggesting that additional criteria are needed to identify the onset from the detection statistic other than its magnitude alone. Once this issue is resolved, change-point detection should provide an effective EMG-onset detection method suitable for automated real-time implementation.