POL32, a subunit of the Saccharomyces cerevisiae DNA polymerase δ, defines a link between DNA replication and the mutagenic bypass repair pathway

Pol32 is a subunit of Saccharomyces cerevisiae DNA polymerase δ required in DNA replication and repair. To gain insight into the function of Pol32 and to determine in which repair pathway POL32 may be involved, we extended the analysis of the pol32Δ mutant with respect to UV and methylation sensitivity, UV-induced mutagenesis; and we performed an epistasis analysis of UV sensitivity by combining the pol32Δ with mutations in several genes for postreplication repair (RAD6 group), nucleotide excision repair (RAD3 group) and recombinational repair (RAD52 group). These studies showed that pol32Δ is deficient in UV-induced mutagenesis and place POL32 in the error-prone RAD6/REV3 pathway. We also found that the increase in the CAN1 spontaneous forward mutation of different rad mutators relies entirely or partially on a functional POL32 gene. Moreover, in a two-hybrid screen, we observed that Pol32 interacts with Srs2, a DNA helicase required for DNA replication and mutagenesis. Simultaneous deletion of POL32 and SRS2 dramatically decreases cellular viability at 15 °C and greatly increases cellular sensitivity to hydroxyurea at the permissive temperature. Based on these findings, we propose that POL32 defines a link between the DNA polymerase and helicase activities, and plays a role in the mutagenic bypass repair pathway. Received: 25 May 2000 / Accepted: 3 July 2000     

Molecular properties of the lys1 + gene and the regulation of α-aminoadipate reductase in Schizosaccharomyces pombe

The -aminoadipate pathway for the biosynthesis of lysine is unique to fungi. Molecular properties of the cloned lys1 ⁺ gene and the regulation of the encoded -aminoadipate reductase (AAR) were investigated in the fission yeast Schizosaccharomyces pombe. A 5.2-kb HindIII-EcoRI fragment of S. pombe DNA, containing a functional lys1 ⁺ gene and a promoter, was subcloned to make the 10.7-kb plasmid pLYS1H. A nested 1.778-kb HindIII-EcoRI DNA fragment that complemented the lys1-131 mutant phenotype was sequenced from the plasmid pLYS1D, and shown to contain an open reading frame (ORF) of 470 amino acids, preceded by putative POLII promoter elements (TATA and CCAAT box elements, and two potential yeast GCN4-binding motifs) within 368 bp upstream of the start codon. This ORF shared with the corresponding region of the isofunctional AAR of Saccharomyces cerevisiae 49% amino-acid identity (62% similarity) overall, within which were smaller regions of marked sequence conservation. One such region coincided (95% identity) with a putative AMP-binding domain motif identified in the AAR of S. cerevisiae. In wild-type S. pombe, AAR activity from cells grown in lysine-supplemented minimal or YEPD media was less than the activity of cells grown in minimal mediu. The AAR of S. pombe was more sensitive to feedback inhibition by lysine in vitro than the AAR of S. cerevisiae. These results show the effects of extensive evolutionary divergence on the structure and expression of a pivotal enzyme in the -aminoadipate pathway. Presumably, delineated regions of strong sequence conservation correspond to discrete domains essential to AAR function.     

The mode of action of 1-β-d-arabinofuranosylcytosine in inhibiting DNA repair; new evidence using a sensitive assay for repair DNA synthesis and ligation in permeable cells

Mammalian cells permeabilised by treatment with saponin are capable of UV excision repair. We have developed an assay system which permits measurement of the later stages of repair, i.e. repair synthesis and ligation. Incomplete repair sites are accumulated in UV-irradiated cells by incubating them with DNA synthesis inhibitors hydroxyurea and aphidicolin. On removal of the inhibitors at the time of permeabilisation, these incomplete sites, detected as DNA breaks, are rapidly ligated in a reaction that requires deoxyribonucleoside triphosphates and is blocked by aphidicolin. Thus ligation is possible only after a significant amount of DNA synthesis. We have used the assay to clarify the mode of inhibition of DNA repair by 1-β-d-arabinofuranosylcytosine (ara C), another DNA polymerase inhibitor. It is well known that incomplete repair sites accumulated in whole cells with ara C are ligated at a slow rate, if at all. The hypothesis that ara C blocks or reduces further polymerisation after its incorporation into repair patches is disproved by our demonstration that, in permeable cells, the accumulated DNA breaks are ligated very rapidly. The likely explanation of the action of ara C is that, once phosphorylated, it remains in the cell as ara CTP and continues to inhibit polymerisation through competition with dCTP; in permeable cells, it readily leaks out.     

Rev1 promotes replication through UV lesions in conjunction with DNA polymerases η, ι, and κ but not DNA polymerase ζ

Translesion synthesis (TLS) DNA polymerases (Pols) promote replication through DNA lesions; however, little is known about the protein factors that affect their function in human cells. In yeast, Rev1 plays a noncatalytic role as an indispensable component of Polζ, and Polζ together with Rev1 mediates a highly mutagenic mode of TLS. However, how Rev1 functions in TLS and mutagenesis in human cells has remained unclear. Here we determined the role of Rev1 in TLS opposite UV lesions in human and mouse fibroblasts and showed that Rev1 is indispensable for TLS mediated by Polη, Polι, and Polκ but is not required for TLS by Polζ. In contrast to its role in mutagenic TLS in yeast, Rev1 promotes predominantly error-free TLS opposite UV lesions in humans. The identification of Rev1 as an indispensable scaffolding component for Polη, Polι, and Polκ, which function in TLS in highly specialized ways opposite a diverse array of DNA lesions and act in a predominantly error-free manner, implicates a crucial role for Rev1 in the maintenance of genome stability in humans.     

Genetic interaction between α4 and β2 subunits of high affinity nicotinic receptor: analysis in schizophrenia

Cholinergic dysfunction is one of the hypotheses for the cognitive deficits of schizophrenia. Neurocognitive deficits, which are well-described clinical features of schizophrenia, may be remediated by nicotine; therefore investigations of nicotinic receptor subtypes is of considerable clinical interest. We typed polymorphisms in CHRNA4 and CHRNB2 genes controlling the expression of neuronal high-affinity nicotinic receptors in 117 Canadian families having at least one schizophrenic patient. Using a family-based association strategy, we performed allele, haplotype and interaction analysis of these two loci. In the families tested, the two cholinergic genes interact to affect schizophrenia in combination (P=0.010), while neither was sufficient alone to confer susceptibility. Our present study provided the first line of direct evidence suggesting that the CHRNA4 gene combined with CHRNB2 receptor gene may be linked to schizophrenia.Vincenzo De Luca and Sophocles Voineskos have contributed equally to this work     

Does mitochondrial DNA play a role in Parkinson's disease? A review of cybrid and other supportive evidence

SIGNIFICANCE: Mitochondria are currently believed to play an important role in the neurodysfunction and neurodegeneration that underlie Parkinson's disease (PD). RECENT ADVANCES: While it increasingly appears that mitochondrial dysfunction in PD can have different causes, it has been proposed that mitochondrial DNA (mtDNA) may account for or drive mitochondrial dysfunction in the majority of the cases. If correct, the responsible mtDNA signatures could represent acquired mutations, inherited mutations, or population-distributed polymorphisms. CRITICAL ISSUES AND FUTURE DIRECTIONS: This review discusses the case for mtDNA as a key mediator of PD, and especially focuses on data from studies of PD cytoplasmic hybrid (cybrid) cell lines.     

Integrin α1/β1 and α2/β1 as a receptor for IgA1 in human glomerular mesangial cells in IgA nephropathy

IgA nephropathy (IgAN) is characterized by mesangial deposition of IgA1 and galactose-deficient IgA1 is expected to play a pathogenic role. However, the identity of the receptor for IgA1 is still controversial. Hence, the aim of this study was to explore the receptor for galactose-deficient IgA1. Human monoclonal IgA1 was treated with exoglycosidase and FITC-conjugated control, asialo- and agalactosyl-IgA1 was used as a probe to detect the receptor in cultured human mesangial cells. Tumor necrosis factor-α or transforming growth factor-β1 treatment accelerated IgA1-binding on mesangial cells, and these effects were diminished by the addition of dexamethasone, whereas these changes were not dependent on galactose-deficiency of IgA1. According to comprehensive gene expression analysis, we focused on integrin β1. Pre-treatment by Mn(2+), which activates integrin by changing its structure, enhanced the binding of IgA1 in cultured mesangial cells. Furthermore, pre-incubation with collagens specifically enhanced binding of IgA1 in the cultured human mesangial cells without activation by Mn(2+). Collagen type IV distributed in the mesangial region of the glomeruli as well as Bowman's capsule and tubular basal membrane in IgAN patients, and the IgA1 with collagen type IV induced proliferative signals on mesangial cells by phosphorylating extracellular signal-regulated kinase more effectively than the IgA1 alone. Immunoprecipitation assay revealed the binding of IgA1 and integrin α1/β1 and α2/β1 heterodimer and down-regulation of integrin α1, α2 and β1 expression in human mesangial cells induced by each specific small interfering RNA diminished the ability to bind IgA1 probe. Integrin α1/β1 and α2/β1 would be a candidate receptor for IgA1.     

The role of integrin α2 in cell and matrix therapy that improves perfusion,viability and function of infarcted myocardium

Injectable delivery matrices hold promise in enhancing engraftment and the overall efficacy of cardiac cell therapies; however, the mechanisms responsible remain largely unknown. Here we studied the interaction of a collagen matrix with circulating angiogenic cells (CACs) in a mouse myocardial infarction model. CACs + matrix treatment enhanced CAC engraftment, and improved myocardial perfusion, viability and function compared to cells or matrix alone. Integrin-linked kinase (ILK) was up-regulated in matrix-cultured CACs. Integrin α2β1 blocking prevented ILK up-regulation, significantly reduced the adhesion, proliferation, and paracrine properties of matrix-cultured CACs, and negated the benefits of CACs + matrix therapy in vivo. Furthermore, integrin α5 was essential for the angiogenic potential of CACs on matrix. These findings indicate that the synergistic therapeutic effect of CACs + matrix therapy in MI requires the matrix to enhance CAC function via α2β1 and α5 integrin signaling mechanisms, rather than simply delivering the cells.     

Distribution pattern of matrix metalloproteinases 1, 2, 3, and 9, tissue inhibitors of matrix metalloproteinases 1 and 2, and α2-macroglobulin in cases of generalized AA- and AL amyloidosis

Matrix metalloproteinases (MMPs) degrade basement membranes and connective tissue and play an essential role in the homeostasis of the extracellular matrix which is disrupted by the deposition of amyloid. This immunohistochemical study investigated the distribution pattern of matrix metalloproteinases (MMP-1, -2, -3, and -9) and their inhibitors [alpha 2-macroglobulin (alpha 2-M), tissue inhibitors of MMPs (TIMP)-1, and TIMP-2] in human AA- and AL amyloid deposits. Specimens of liver, kidney, and spleen from 22 autopsy cases were investigated. Nine patients had suffered from generalized AA amyloidosis, eight from generalized AL amyloidosis, and five from rheumatoid arthritis or tuberculosis with no histological evidence of amyloid. In all amyloidotic and non-amyloidotic patients, each protease and protease inhibitor was detected in almost every organ investigated. In the amyloidotic cases, there was no indication that a specific protease or protease inhibitor was absent or expressed, but a difference was observed in their spatial distribution patterns. The most noticeable difference was found in immunostaining of amyloid. Only MMP-1, -2, and -3, and alpha 2-M were present in AA amyloid deposits, and only TIMP-1 and TIMP-2 were found in deposits of AL amyloid. This is the first study to show that MMP-1, -2, and -3 are present in AA amyloid deposits. They may be involved in tissue remodeling or in proteolysis of the precursor and fibril proteins.     

Expression patterns of nicotinic subunits α2, α7, α8, and β1 affect the kinetics and pharmacology of ACh-induced currents in adult bee olfactory neuropiles

Acetylcholine (ACh) is the main excitatory neurotransmitter of the insect brain, where nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission. In the honeybee Apis mellifera, nAChRs are expressed in diverse structures including the primary olfactory centers of the brain, the antennal lobes (ALs) and the mushroom bodies (MBs), where they participate in olfactory information processing. To understand the nature and properties of the nAChRs involved in these processes, we performed a pharmacological and molecular characterization of nAChRs on cultured Kenyon cells of the MBs, using whole cell patch-clamp recordings combined with single-cell RT-PCR. In all cells, applications of ACh as well as nicotinic agonists such as nicotine and imidacloprid induced inward currents with fast desensitization. These currents were fully blocked by saturating doses of the antagonists α-bungarotoxin (α-BGT), dihydroxy-β-erythroidine (DHE), and methyllycaconitine (MLA) (MLA ≥ α-BGT ≥ DHE). Molecular analysis of ACh-responding cells revealed that of the 11 nicotinic receptor subunits encoded within the honeybee genome, α2, α8, and β1 subunits were expressed in adult Kenyon cells. Comparison with the expression pattern of adult AL cells revealed the supplementary presence of subunit α7, which could be responsible for the kinetic and pharmacological differences observed when comparing ACh-induced currents from AL and Kenyon cells. Together, our data demonstrate the existence of functional nAChRs on adult MB Kenyon cells that differ from nAChRs on AL cells in both their molecular composition and pharmacological properties, suggesting that changing receptor subsets could mediate different processing functions depending on the brain structure within the olfactory pathway.     

Characterization of hERG1a and hERG1b potassium channels—a possible role for hERG1b in the I Kr current

I (Kr) is the fast component of the delayed rectifier potassium currents responsible for the repolarization of the cardiac muscle. The molecular correlate underlying the I (Kr) current has been identified as the hERG1 channel. Recently, two splice variants of the hERG1 alpha-subunit, hERG1a and hERG1b, have been shown to be co-expressed in human cardiomyocytes. In this paper, we present the electrophysiological characterization of hERG1a, hERG1b, and co-expressed hERG1a/b channels in a mammalian expression system using the whole-cell patch clamp technique. We also quantified the messenger RNA (mRNA) levels of hERG1a and hERG1b in human cardiac tissue, and based on the expressed ratios, we evaluated the resulting currents in Xenopus laevis oocytes. Compared to hERG1a channels, activation was faster for both hERG1b and hERG1a/b channels. The deactivation kinetics was greatly accelerated in the presence of hERG1b, whereas no difference in the time constant of inactivation was observed. The voltage-dependent recovery from inactivation was also similar. However, the time constant of recovery from inactivation was significantly faster for hERG1b channels compared to hERG1a and hERG1a/b. Quantification of hERG1a and hERG1b mRNA in the human heart showed that hERG1b mRNA constitutes, on average, 19% in the right atrium and 12% in the left ventricle of the total hERG1 mRNA. Expression of the observed ratios of hERG1a to hERG1b in X. laevis oocytes showed that these ratios are indeed sufficient to change the deactivation phenotype markedly. The present work suggests that hERG1b is likely to play a role in the formation of the native I (Kr) current.     

A possible role for bcl-2 in regulating T-cell memory - a ‘balancing act’ between cell death and survival

One of the central questions in T-cell immunity is how the generation of T-cell memory may proceed simultaneously with the maintenance of T-cell homeostasis. In this article, we present a hypothesis that the regulation of bcl-2 gene expression within the activated CD45RO⁺ T-cell population may be one of the key factors in the balance between death and survival of these activated mature T cells, both in vitro and in vivo.