Accumulation of premutagenic DNA lesions in mice defective in removal of oxidative base damage

DNA damage generated by oxidant byproducts of cellular metabolism has been proposed as a key factor in cancer and aging. Oxygen free radicals cause predominantly base damage in DNA, and the most frequent mutagenic base lesion is 7,8-dihydro-8-oxoguanine (8-oxoG). This altered base can pair with A as well as C residues, leading to a greatly increased frequency of spontaneous G·C→T·A transversion mutations in repair-deficient bacterial and yeast cells. Eukaryotic cells use a specific DNA glycosylase, the product of the OGG1 gene, to excise 8-oxoG from DNA. To assess the role of the mammalian enzyme in repair of DNA damage and prevention of carcinogenesis, we have generated homozygous ogg1−/− null mice. These animals are viable but accumulate abnormal levels of 8-oxoG in their genomes. Despite this increase in potentially miscoding DNA lesions, OGG1-deficient mice exhibit only a moderately, but significantly, elevated spontaneous mutation rate in nonproliferative tissues, do not develop malignancies, and show no marked pathological changes. Extracts of ogg1 null mouse tissues cannot excise the damaged base, but there is significant slow removal in vivo from proliferating cells. These findings suggest that in the absence of the DNA glycosylase, and in apparent contrast to bacterial and yeast cells, an alternative repair pathway functions to minimize the effects of an increased load of 8-oxoG in the genome and maintain a low endogenous mutation frequency.

Targeted deletion of alkylpurine-DNA-N-glycosylase in mice eliminates repair of 1,N6-ethenoadenine and hypoxanthine but not of 3,N4-ethenocytosine or 8-oxoguanine

It has previously been reported that 1,N6-ethenoadenine (ɛA), deaminated adenine (hypoxanthine, Hx), and 7,8-dihydro-8-oxoguanine (8-oxoG), but not 3,N4-ethenocytosine (ɛC), are released from DNA in vitro by the DNA repair enzyme alkylpurine-DNA-N-glycosylase (APNG). To assess the potential contribution of APNG to the repair of each of these mutagenic lesions in vivo, we have used cell-free extracts of tissues from APNG-null mutant mice and wild-type controls. The ability of these extracts to cleave defined oligomers containing a single modified base was determined. The results showed that both testes and liver cells of these knockout mice completely lacked activity toward oligonucleotides containing ɛA and Hx, but retained wild-type levels of activity for ɛC and 8-oxoG. These findings indicate that (i) the previously identified ɛA-DNA glycosylase and Hx-DNA glycosylase activities are functions of APNG; (ii) the two structurally closely related mutagenic adducts ɛA and ɛC are repaired by separate gene products; and (iii) APNG does not contribute detectably to the repair of 8-oxoG.

Semirational design of active tumor suppressor p53 DNA binding domain with enhanced stability

We have designed a p53 DNA binding domain that has virtually the same binding affinity for the gadd45 promoter as does wild-type protein but is considerably more stable. The design strategy was based on molecular evolution of the protein domain. Naturally occurring amino acid substitutions were identified by comparing the sequences of p53 homologues from 23 species, introducing them into wild-type human p53, and measuring the changes in stability. The most stable substitutions were combined in a multiple mutant. The advantage of this strategy is that, by substituting with naturally occurring residues, the function is likely to be unimpaired. All point mutants bind the consensus DNA sequence. The changes in stability ranged from +1.27 (less stable Q165K) to −1.49 (more stable N239Y) kcal mol−1, respectively. The changes in free energy of unfolding on mutation are additive. Of interest, the two most stable mutants (N239Y and N268D) have been known to act as suppressors and restored the activity of two of the most common tumorigenic mutants. Of the 20 single mutants, 10 are cancer-associated, though their frequency of occurrence is extremely low: A129D, Q165K, Q167E, and D148E are less stable and M133L, V203A and N239Y are more stable whereas the rest are neutral. The quadruple mutant (M133LV203AN239YN268D), which is stabilized by 2.65 kcal mol−1 and Tm raised by 5.6°C is of potential interest for trials in vivo.

Protein phosphatase 2A is required for the initiation of chromosomal DNA replication

Protein phosphatase 2A (PP2A) is an abundant, multifunctional serine/threonine-specific phosphatase that stimulates simian virus 40 DNA replication. The question as to whether chromosomal DNA replication also depends on PP2A was addressed by using a cell-free replication system derived from Xenopus laevis eggs. Immunodepletion of PP2A from Xenopus egg extract resulted in strong inhibition of DNA replication. PP2A was required for the initiation of replication but not for the elongation of previously engaged replication forks. Therefore, the initiation of chromosomal DNA replication depends not only on phosphorylation by protein kinases but also on dephosphorylation by PP2A.

A Cell-free System to Study Regulation of Focal Adhesions and of the Connected Actin Cytoskeleton

Assembly and modulation of focal adhesions during dynamic adhesive processes are poorly understood. We describe here the use of ventral plasma membranes from adherent fibroblasts to explore mechanisms regulating integrin distribution and function in a system that preserves the integration of these receptors into the plasma membrane. We find that partial disruption of the cellular organization responsible for the maintenance of organized adhesive sites allows modulation of integrin distribution by divalent cations. High Ca2+ concentrations induce quasi-reversible diffusion of β1 integrins out of focal adhesions, whereas low Ca2+ concentrations induce irreversible recruitment of β1 receptors along extracellular matrix fibrils, as shown by immunofluorescence and electron microscopy. Both effects are independent from the presence of actin stress fibers in this system. Experiments with cells expressing truncated β1 receptors show that the cytoplasmic portion of β1 is required for low Ca2+-induced recruitment of the receptors to matrix fibrils. Analysis with function-modulating antibodies indicates that divalent cation-mediated receptor distribution within the membrane correlates with changes in the functional state of the receptors. Moreover, reconstitution experiments show that purified α-actinin colocalizes and redistributes with β1 receptors on ventral plasma membranes depleted of actin, implicating binding of α-actinin to the receptors. Finally, we found that recruitment of exogenous actin is specifically restricted to focal adhesions under conditions in which new actin polymerization is inhibited. Our data show that the described system can be exploited to investigate the mechanisms of integrin function in an experimental setup that permits receptor redistribution. The possibility to uncouple, under cell-free conditions, events involved in focal adhesion and actin cytoskeleton assembly should facilitate the comprehension of the underlying molecular mechanisms.

Microdomain-dependent Regulation of Lck and Fyn Protein-Tyrosine Kinases in T Lymphocyte Plasma Membranes

Src family protein-tyrosine kinases are implicated in signaling via glycosylphosphatidylinositol (GPI)-anchored receptors. Both kinds of molecules reside in opposite leaflets of the same sphingolipid-enriched microdomains in the lymphocyte plasma membrane without making direct contact. Under detergent-free conditions, we isolated a GPI-enriched plasma membrane fraction, also containing transmembrane proteins, selectively associated with sphingolipid microdomains. Nonionic detergents released the transmembrane proteins, yielding core sphingolipid microdomains, limited amounts of which could also be obtained by detergent-free subcellular fractionation. Protein-tyrosine kinase activity in membranes containing both GPI-anchored and transmembrane proteins was much lower than in core sphingolipid microdomains but was strongly reactivated by nonionic detergents. The inhibitory mechanism acting on Lck and Fyn kinases in these membranes was independent of the protein-tyrosine phosphatase CD45 and was characterized as a mixed, noncompetitive one. We propose that in lymphocyte plasma membranes, Lck and Fyn kinases exhibit optimal activity when juxtaposed to the GPI- and sphingolipid-enriched core microdomains but encounter inhibitory conditions in surrounding membrane areas that are rich in glycerophospholipids and contain additional transmembrane proteins.

Nuclear Ferritin Protects DNA From UV Damage in Corneal Epithelial Cells

Previously, we identified the heavy chain of ferritin as a developmentally regulated nuclear protein of embryonic chicken corneal epithelial cells. The nuclear ferritin is assembled into a supramolecular form indistinguishable from the cytoplasmic form of ferritin found in other cell types and thus most likely has iron-sequestering capabilities. Free iron, via the Fenton reaction, is known to exacerbate UV-induced and other oxidative damage to cellular components, including DNA. Since corneal epithelial cells are constantly exposed to UV light, we hypothesized that the nuclear ferritin might protect the DNA of these cells from free radical damage. To test this possibility, primary cultures of cells from corneal epithelium and stroma, and from skin epithelium and stroma, were UV irradiated, and DNA strand breaks were detected by an in situ 3′-end labeling method. Corneal epithelial cells without nuclear ferritin were also examined. We observed that the corneal epithelial cells with nuclear ferritin had significantly less DNA breakage than other cell types examined. Furthermore, increasing the iron concentration of the culture medium exacerbated the generation of UV-induced DNA strand breaks in corneal and skin fibroblasts, but not in the corneal epithelial cells. Most convincingly, corneal epithelial cells in which the expression of nuclear ferritin was inhibited became much more susceptible to UV-induced DNA damage. Therefore, it seems that corneal epithelial cells have evolved a novel, nuclear ferritin-based mechanism for protecting their DNA against UV damage.

Efficient Trafficking of TGN38 from the Endosome to the trans-Golgi Network Requires a Free Hydroxyl Group at Position 331 in the Cytosolic Domain

TGN38 is one of the few known resident integral membrane proteins of the trans-Golgi network (TGN). Since it cycles constitutively between the TGN and the plasma membrane, TGN38 is ideally suited as a model protein for the identification of post-Golgi trafficking motifs. Several studies, employing chimeric constructs to detect such motifs within the cytosolic domain of TGN38, have identified the sequence 333YQRL336 as an autonomous signal capable of localizing reporter proteins to the TGN. In addition, one group has found that an upstream serine residue, S331, may also play a role in TGN38 localization. However, the nature and degree of participation of S331 in the localization of TGN38 remain uncertain, and the effect has been studied in chimeric constructs only. Here we investigate the role of S331 in the context of full-length TGN38. Mutations that abolish the hydroxyl moiety at position 331 (A, D, and E) lead to missorting of endocytosed TGN38 to the lysosome. Conversely, mutation of S331 to T has little effect on the endocytic trafficking of TGN38. Together, these findings indicate that the S331 hydroxyl group has a direct or indirect effect on the ability of the cytosolic tail of TGN38 to interact with trafficking and/or sorting machinery at the level of the early endosome. In addition, mutation of S331 to either A or D results in increased levels of TGN38 at the cell surface. The results confirm that S331 plays a critical role in the intracellular trafficking of TGN38 and further reveal that TGN38 undergoes a signal-mediated trafficking step at the level of the endosome.

Testosterone Signaling through Internalizable Surface Receptors in Androgen Receptor-free Macrophages

Testosterone acts on cells through intracellular transcription-regulating androgen receptors (ARs). Here, we show that mouse IC-21 macrophages lack the classical AR yet exhibit specific nongenomic responses to testosterone. These manifest themselves as testosterone-induced rapid increase in intracellular free [Ca2+], which is due to release of Ca2+ from intracellular Ca2+ stores. This Ca2+ mobilization is also inducible by plasma membrane-impermeable testosterone-BSA. It is not affected by the AR blockers cyproterone and flutamide, whereas it is completely inhibited by the phospholipase C inhibitor U-73122 and pertussis toxin. Binding sites for testosterone are detectable on the surface of intact IC-21 cells, which become selectively internalized independent on caveolae and clathrin-coated vesicles upon agonist stimulation. Internalization is dependent on temperature, ATP, cytoskeletal elements, phospholipase C, and G-proteins. Collectively, our data provide evidence for the existence of G-protein-coupled, agonist-sequestrable receptors for testosterone in plasma membranes, which initiate a transcription-independent signaling pathway of testosterone.

DNA Replication in Quiescent Cell Nuclei: Regulation by the Nuclear Envelope and Chromatin Structure

Quiescent nuclei from differentiated somatic cells can reacquire pluripotence, the capacity to replicate, and reinitiate a program of differentiation after transplantation into amphibian eggs. The replication of quiescent nuclei is recapitulated in extracts derived from activated Xenopus eggs; therefore, we have exploited this cell-free system to explore the mechanisms that regulate initiation of replication in nuclei from terminally differentiated Xenopus erythrocytes. We find that these nuclei lack many, if not all, pre-replication complex (pre-RC) proteins. Pre-RC proteins from the extract form a stable association with the chromatin of permeable nuclei, which replicate in this system, but not with the chromatin of intact nuclei, which do not replicate, even though these proteins cross an intact nuclear envelope. During extract incubation, the linker histones H1 and H10 are removed from erythrocyte chromatin by nucleoplasmin. We show that H1 removal facilitates the replication of permeable nuclei by increasing the frequency of initiation most likely by promoting the assembly of pre-RCs on chromatin. These data indicate that initiation in erythrocyte nuclei requires the acquisition of pre-RC proteins from egg extract and that pre-RC assembly requires the loss of nuclear envelope integrity and is facilitated by the removal of linker histone H1 from chromatin.

Inverse radiation dose-rate effects on somatic and germ-line mutations and DNA damage rates

The mutagenic effect of low linear energy transfer ionizing radiation is reduced for a given dose as the dose rate (DR) is reduced to a low level, a phenomenon known as the direct DR effect. Our reanalysis of published data shows that for both somatic and germ-line mutations there is an opposite, inverse DR effect, with reduction from low to very low DR, the overall dependence of induced mutations being parabolically related to DR, with a minimum in the range of 0.1 to 1.0 cGy/min (rule 1). This general pattern can be attributed to an optimal induction of error-free DNA repair in a DR region of minimal mutability (MMDR region). The diminished activation of repair at very low DRs may reflect a low ratio of induced (“signal”) to spontaneous background DNA damage (“noise”). Because two common DNA lesions, 8-oxoguanine and thymine glycol, were already known to activate repair in irradiated mammalian cells, we estimated how their rates of production are altered upon radiation exposure in the MMDR region. For these and other abundant lesions (abasic sites and single-strand breaks), the DNA damage rate increment in the MMDR region is in the range of 10% to 100% (rule 2). These estimates suggest a genetically programmed optimatization of response to radiation in the MMDR region.

A novel modifier gene for plasma von Willebrand factor level maps to distal mouse chromosome 11

Type 1 von Willebrand disease (VWD), characterized by reduced levels of plasma von Willebrand factor (VWF), is the most common inherited bleeding disorder in humans. Penetrance of VWD is incomplete, and expression of the bleeding phenotype is highly variable. In addition, plasma VWF levels vary widely among normal individuals. To identify genes that influence VWF level, we analyzed a genetic cross between RIIIS/J and CASA/Rk, two strains of mice that exhibit a 20-fold difference in plasma VWF level. DNA samples from F2 progeny demonstrating either extremely high or extremely low plasma VWF levels were pooled and genotyped for 41 markers spanning the autosomal genome. A novel locus accounting for 63% of the total variance in VWF level was mapped to distal mouse chromosome 11, which is distinct from the murine Vwf locus on chromosome 6. We designated this locus Mvwf for “modifier of VWF.” Additional genotyping of as many as 2407 meioses established a high resolution genetic map with gene order Cola1-Itg3a-Ngfr-Mvwf/Gip-Hoxb9-Hoxb1-Cbx·rs2-Cox5a-Gfap. The Mvwf candidate interval between Ngfr and Hoxb9 is ≈0.5 centimorgan (cM). These results demonstrate that a single dominant gene accounts for the low VWF phenotype of RIIIS/J mice in crosses with several other strains. The pattern of inheritance suggests a gain-of-function mutation in a unique component of VWF biosynthesis or processing. Characterization of the human homologue for Mvwf may have relevance for a subset of type 1 VWD cases and may define an important genetic factor modifying penetrance and expression of mutations at the VWF locus.

Thermodynamic basis of the enhanced specificity of structured DNA probes

Molecular beacons are DNA probes that form a stem-and-loop structure and possess an internally quenched fluorophore. When they bind to complementary nucleic acids, they undergo a conformational transition that switches on their fluorescence. These probes recognize their targets with higher specificity than probes that cannot form a hairpin stem, and they easily discriminate targets that differ from one another by only a single nucleotide. Our results show that molecular beacons can exist in three different states: bound to a target, free in the form of a hairpin structure, and free in the form of a random coil. Thermodynamic analysis of the transitions between these states reveals that enhanced specificity is a general feature of conformationally constrained probes.

Submillimolar levels of calcium regulates DNA structure at the dinucleotide repeat (TG/AC)n

Submillimolar levels of calcium, similar to the physiological total (bound + free) intranuclear concentration (0.01–1 mM), induced a conformational change within d(TG/AC)n, one of the frequent dinucleotide repeats of the mammalian genome. This change is calcium-specific, because no other tested cation induced it and it was detected as a concentration-dependent transition from B- to a non-B-DNA conformation expanding from 3′ end toward the 5′ of the repeat. Genomic footprinting of various rat brain regions revealed the existence of similar non-B-DNA conformation within a d(TG/AC)28 repeat of the endogenous enkephalin gene only in enkephalin-expressing caudate nucleus and not in the nonexpressing thalamus. Binding assays demonstrated that DNA could bind calcium and can compete with calmodulin for calcium.

DNA annealing by Rad52 Protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA

Homologous recombination in Saccharomyces cerevisiae depends critically on RAD52 function. In vitro, Rad52 protein preferentially binds single-stranded DNA (ssDNA), mediates annealing of complementary ssDNA, and stimulates Rad51 protein-mediated DNA strand exchange. Replication protein A (RPA) is a ssDNA-binding protein that is also crucial to the recombination process. Herein we report that Rad52 protein effects the annealing of RPA–ssDNA complexes, complexes that are otherwise unable to anneal. The ability of Rad52 protein to promote annealing depends on both the type of ssDNA substrate and ssDNA binding protein. RPA allows, but slows, Rad52 protein-mediated annealing of oligonucleotides. In contrast, RPA is almost essential for annealing of longer plasmid-sized DNA but has little effect on the annealing of poly(dT) and poly(dA), which are relatively long DNA molecules free of secondary structure. These results suggest that one role of RPA in Rad52 protein-mediated annealing is the elimination of DNA secondary structure. However, neither Escherichia coli ssDNA binding protein nor human RPA can substitute in this reaction, indicating that RPA has a second role in this process, a role that requires specific RPA–Rad52 protein interactions. This idea is confirmed by the finding that RPA, which is complexed with nonhomologous ssDNA, inhibits annealing but the human RPA–ssDNA complex does not. Finally, we present a model for the early steps of the repair of double-strand DNA breaks in yeast.