Binding of XPA and RPA to damaged DNA investigated by fluorescence anisotropy

The proteins XPA and RPA are assumed to be involved in primary damage recognition of global genome nucleotide excision repair. XPA as well as RPA have been each reported to specifically bind DNA lesions, and ternary complex formation with damaged DNA has also been shown. We employed fluorescence anisotropy measurements to study the DNA-binding properties of XPA and RPA under true equilibrium conditions using damaged DNA probes carrying a terminal fluorescein modification as a reporter. XPA binds with low affinity and in a strongly salt-dependent manner to DNA containing a 1,3-d(GTG) intrastrand adduct of the anticancer drug cisplatin or a 6-nt mismatch (K(D) = 400 nM) with 3-fold preference for damaged vs undamaged DNA. At near physiological salt conditions binding is very weak (K(D) > 2 microM). RPA binds to damaged DNA probes with dissociation constants in the range of 20 nM and a nearly 15-fold preference over undamaged DNA. The presence of a cisplatin modification weakens the affinity of RPA for single-stranded DNA by more than 1 order of magnitude indicating that binding to the lesion itself is not a driving force in damage recognition. Our fluorescence anisotropy assays also show that the presence of XPA does not enhance the affinity of RPA for damaged DNA although both proteins interact. In contrast, cooperative binding of XPA and RPA is observed in EMSA. Our results point to a damage-sensing function of the XPA-RPA complex with RPA mediating the important DNA contacts.     

Neuroprotection in ischemia: blocking calcium-permeable acid-sensing ion channels

Ca2+ toxicity remains the central focus of ischemic brain injury. The mechanism by which toxic Ca2+ loading of cells occurs in the ischemic brain has become less clear as multiple human trials of glutamate antagonists have failed to show effective neuroprotection in stroke. Acidosis is a common feature of ischemia and is assumed to play a critical role in brain injury; however, the mechanism(s) remain ill defined. Here, we show that acidosis activates Ca2+ -permeable acid-sensing ion channels (ASICs), inducing glutamate receptor-independent, Ca2+ -dependent, neuronal injury inhibited by ASIC blockers. Cells lacking endogenous ASICs are resistant to acid injury, while transfection of Ca2+ -permeable ASIC1a establishes sensitivity. In focal ischemia, intracerebroventricular injection of ASIC1a blockers or knockout of the ASIC1a gene protects the brain from ischemic injury and does so more potently than glutamate antagonism. Thus, acidosis injures the brain via membrane receptor-based mechanisms with resultant toxicity of [Ca2+]i, disclosing new potential therapeutic targets for stroke.     

Functional TRPV4 channels are expressed in human airway smooth muscle cells

Hypotonic stimulation induces airway constriction in normal and asthmatic airways. However, the osmolarity sensor in the airway has not been characterized. TRPV4 (also known as VR-OAC, VRL-2, TRP12, OTRPC4), an osmotic-sensitive cation channel in the transient receptor potential (TRP) channel family, was recently cloned. In the present study, we show that TRPV4 mRNA was expressed in cultured human airway smooth muscle cells as analyzed by RT-PCR. Hypotonic stimulation induced Ca(2+) influx in human airway smooth muscle cells in an osmolarity-dependent manner, consistent with the reported biological activity of TRPV4 in transfected cells. In cultured muscle cells, 4alpha-phorbol 12,13-didecanoate (4-alphaPDD), a TRPV4 ligand, increased intracellular Ca(2+) level only when Ca(2+) was present in the extracellular solution. The 4-alphaPDD-induced Ca(2+) response was inhibited by ruthenium red (1 microM), a known TRPV4 inhibitor, but not by capsazepine (1 microM), a TRPV1 antagonist, indicating that 4-alphaPDD-induced Ca(2+) response is mediated by TRPV4. Verapamil (10 microM), an L-type voltage-gated Ca(2+) channel inhibitor, had no effect on the 4-alphaPDD-induced Ca(2+) response, excluding the involvement of L-type Ca(2+) channels. Furthermore, hypotonic stimulation elicited smooth muscle contraction through a mechanism dependent on membrane Ca(2+) channels in both isolated human and guinea pig airways. Hypotonicity-induced airway contraction was not inhibited by the L-type Ca(2+) channel inhibitor nifedipine (1 microM) or by the TRPV1 inhibitor capsazepine (1 microM). We conclude that functional TRPV4 is expressed in human airway smooth muscle cells and may act as an osmolarity sensor in the airway.     

Hill-Robertson interference in Drosophila melanogaster: reply to Marais,Mouchiroud and Duret

The usage of preferred codons in Drosophila melanogaster is reduced in regions of lower recombination. This is consistent with population genetics theory, whereby the effectiveness of selection on multiple targets is limited by stochastic effects caused by linkage. However, because the selectively preferred codons in D. melanogaster end in C or G, it has been argued that base-composition-biasing effects of recombination can account for the observed relationship between preferred codon usage and recombination rate (Marais et al., 2003). Here, we show that the correlation between base composition (of protein-coding and intron regions) and recombination rate holds only for lower values of the latter. This is consistent with a Hill-Robertson interference model and does not support a model whereby the entire effect of recombination on codon usage can be attributed to its potential role in generating compositional bias.     

Inferring the history of speciation from multilocus DNA sequence data: the case of Drosophila pseudoobscura and close relatives

The divergence of Drosophila pseudoobscura from its close relatives, D. persimilis and D. pseudoobscura bogotana, was examined using the pattern of DNA sequence variation in a common set of 50 inbred lines at 11 loci from diverse locations in the genome. Drosophila pseudoobscura and D. persimilis show a marked excess of low-frequency variation across loci, consistent with a model of recent population expansion in both species. The different loci vary considerably, both in polymorphism levels and in the levels of polymorphisms that are shared by different species pairs. A major question we address is whether these patterns of shared variation are best explained by gene flow or by persistence since common ancestry. A new test of gene flow, based on patterns of linkage disequilibrium, is developed. The results from these, and other tests, support a model in which D. pseudoobscura and D. persimilis have exchanged genes at some loci. However, the pattern of variation suggests that most gene flow, although occurring after speciation began, was not recent. There is less evidence of gene flow between D. pseudoobscura and D. p. bogotana. The results are compared with recent work on the genomic locations of genes that contribute to reproductive isolation between D. pseudoobscura and D. persimilis. We show that there is a good correspondence between the genomic regions associated with reproductive isolation and the regions that show little or no evidence of gene flow.     

Interactions between natural selection, recombination and gene density in the genes of Drosophila

In Drosophila, as in many organisms, natural selection leads to high levels of codon bias in genes that are highly expressed. Thus codon bias is an indicator of the intensity of one kind of selection that is experienced by genes and can be used to assess the impact of other genomic factors on natural selection. Among 13,000 genes in the Drosophila genome, codon bias has a slight positive, and strongly significant, association with recombination--as expected if recombination allows natural selection to act more efficiently when multiple linked sites segregate functional variation. The same reasoning leads to the expectation that the efficiency of selection, and thus average codon bias, should decline with gene density. However, this prediction is not confirmed. Levels of codon bias and gene expression are highest for those genes in an intermediate range of gene density, a pattern that may be the result of a tradeoff between the advantages for gene expression of close gene spacing and disadvantages arising from regulatory conflicts among tightly packed genes. These factors appear to overlay the more subtle effect of linkage among selected sites that gives rise to the association between recombination rate and codon bias.     

The XPC-HR23B complex displays high affinity and specificity for damaged DNA in a true-equilibrium fluorescence assay

The XPC-HR23B complex is a prime candidate for the initial damage recognition step during global genome nucleotide excision repair. A specific interaction between the XPC-HR23B complex and various types of damaged DNA substrates has been demonstrated in recent work by electrophoretic mobility shift assays or immunoprecipitation. Although these studies allowed the estimation of relative binding affinities for the different types of lesions, the presence of large amounts of competitor DNA or the need for glutaraldehyde fixation prevented the quantification of equilibrium constants. We have performed a quantitative study on the binding of XPC to damaged DNA using fluorescence anisotropy measurements. The XPC-HR23B complex binds with high affinity (K(D) approximately 1-3 nM) to fluorescent 36 bp DNA fragments containing a single cisplatin 1,3-intrastrand adduct or a six-nucleotide mispaired region. From stoichiometric titration experiments, it is concluded that approximately 70% of the XPC-HR23B preparation is active in DNA binding. Binding experiments employing fluorescent probes with a single defined photoproduct reveal a 30-fold preference of XPC for 6,4-photoproducts as compared to a cyclobutane dimer. Competition experiments with undamaged and damaged plasmid DNA indicate that the XPC-HR23B complex discriminates between damaged and undamaged sites with high specificity. The specificity factor is between 100 and 3000, depending on the number of nonspecific sites considered in the calculations. Upon addition of XPA to the XPC binding reaction mixtures, it was not possible to detect cooperative ternary complex formation on the platinated 36 bp probe.