A delicate balance: TGF-beta and the tumor microenvironment

The activated form of TGF-beta is a known regulator of epithelial cell autonomous tumor initiation, progression, and metastasis. Recent studies have also indicated that TGF-beta mediates interactions between cancer cells and their local tumor microenvironment. Specifically, the loss of TGF-beta signaling in stromal components including fibroblasts and T-cells can result in an "activated" microenvironment that supports and even initiates transformation of adjacent epithelial cells. TGF-beta signaling in cancer can be regulated through mechanisms involving ligand activation and expression of essential components within the pathway including the receptors and downstream effectors. TGF-beta signaling in the tumor microenvironment significantly impacts carcinoma initiation, progression, and metastasis via epithelial cell autonomous and interdependent stromal-epithelial interactions in vivo.     

Reactive oxygen species and temperature stresses: A delicate balance between signaling and destruction

Temperature stress can have a devastating effect on plant metabolism, disrupting cellular homeostasis, and uncoupling major physiological processes. A direct result of stress-induced cellular changes is the enhanced accumulation of toxic compounds in cells that include reactive oxygen species (ROS). Although a considerable amount of work has shown a direct link between ROS scavenging and plant tolerance to temperature stress, recent studies have shown that ROS could also play a key role in mediating important signal transduction events. Thus, ROS, such as superoxide (O₂^–), are produced by NADPH oxidases during abiotic stress to activate stress-response pathways and induce defense mechanisms. The rates and cellular sites of ROS production during temperature stress could play a central role in stress perception and protection. ROS levels, as well as ROS signals, are thought to be controlled by the ROS gene network of plants. It is likely that in plants this network is interlinked with the different networks that control temperature stress acclimation and tolerance. In this review paper, we attempt to summarize some of the recent studies linking ROS and temperature stress in plants and propose a model for the involvement of ROS in temperature stress sensing and defense.     

The phylogeny and evolution of deoxyribonuclease II: an enzyme essential for lysosomal DNA degradation

Deoxyribonuclease II (DNase II) is an endonuclease with optimal activity at low pH, localized within the lysosomes of higher eukaryotes. The origin of this enzyme remains in dispute, and its phylogenetic distribution leaves many questions about its subsequent evolutionary history open. Earlier studies have documented its presence in various metazoans, as well as in Dictyostelium, Trichomonas and, anomalously, a single genus of bacteria (Burkholderia). This study makes use of searches of the genomes of various organisms against known DNase II query sequences, in order to determine the likely point of origin of this enzyme among cellular life forms. Its complete absence from any other bacteria makes prokaryotic origin unlikely. Convincing evidence exists for DNase II homologs in Alveolates such as Paramecium, Heterokonts such as diatoms and water molds, and even tentative matches in green algae. Apparent absences include red algae, plants, fungi, and a number of parasitic organisms. Based on this phylogenetic distribution and hypotheses of eukaryotic relationships, the most probable explanation is that DNase II has been subject to multiple losses. The point of origin is debatable, though its presence in Trichomonas and perhaps in other evolutionarily basal "Excavate" protists such as Reclinomonas, strongly support the hypothesis that DNase II arose as a plesiomorphic trait in eukaryotes. It probably evolved together with phagocytosis, specifically to facilitate DNA degradation and bacteriotrophy. The various absences in many eukaryotic lineages are accounted for by loss of phagotrophic function in intracellular parasites, in obligate autotrophs, and in saprophytes.     

DNA manipulators: caught in the act

DNA is a dynamic molecule that undergoes constant changes in the cell through interactions with numerous proteins. Several classes of enzyme are specialized in promoting DNA rearrangements, including site-specific recombinases, DNA helicases, transposases and DNA topoisomerases. Recent structures of protein-DNA reaction intermediates trapped in various states of DNA remodeling, complemented by biochemical and biophysical functional studies, have enhanced our understanding of their respective mechanistic pathways.     

Caught in the act: visualization of an intermediate in the DNA base-flipping pathway induced by HhaI methyltransferase

Rotation of a DNA or RNA nucleotide out of the double helix and into a protein pocket (‘base flipping’) is a mechanistic feature common to some DNA/RNA-binding proteins. Here, we report the structure of HhaI methyltransferase in complex with DNA containing a south-constrained abasic carbocyclic sugar at the target site in the presence of the methyl donor byproduct AdoHcy. Unexpectedly, the locked south pseudosugar appears to be trapped in the middle of the flipping pathway via the DNA major groove, held in place primarily through Van der Waals contacts with a set of invariant amino acids. Molecular dynamics simulations indicate that the structural stabilization observed with the south-constrained pseudosugar will not occur with a north-constrained pseudosugar, which explains its lowered binding affinity. Moreover, comparison of structural transitions of the sugar and phosphodiester backbone observed during computational studies of base flipping in the M.HhaI–DNA–AdoHcy ternary complex indicate that the south-constrained pseudosugar induces a conformation on the phosphodiester backbone that corresponds to that of a discrete intermediate of the base-flipping pathway. As previous crystal structures of M.HhaI ternary complex with DNA displayed the flipped sugar moiety in the antipodal north conformation, we suggest that conversion of the sugar pucker from south to north beyond the middle of the pathway is an essential part of the mechanism through which flipping must proceed to reach its final destination. We also discuss the possibility of the south-constrained pseudosugar mimicking a transition state in the phosphodiester and sugar moieties that occurs during DNA base flipping in the presence of M.HhaI.     

UBA 1: an essential yeast gene encoding ubiquitin-activating enzyme.

All known functions of ubiquitin are mediated through its covalent attachment to other proteins. The post-translational formation of ubiquitin--protein conjugates is preceded by an ATP-requiring step in which the carboxyl terminus of ubiquitin is adenylated and subsequently joined, through a thiolester bond, to a cysteine residue in the ubiquitin-activating enzyme, also known as E1. We report the isolation and functional analysis of the gene (UBA1) for the ubiquitin-activating enzyme of the yeast Saccharomyces cerevisiae. UBA1 encodes a 114 kd protein whose amino acid sequence contains motifs characteristic of nucleotide-binding sites. Expression of catalytically active UBA1 protein in E. coli, which lacks the ubiquitin system, confirmed that the yeast UBA1 gene encodes a ubiquitin-activating enzyme. Deletion of the UBA1 gene is lethal, demonstrating that the formation of ubiquitin--protein conjugates is essential for cell viability.     

DNA enzyme generated by a novel single-stranded DNA expression vector inhibits expression of the essential bacterial cell division gene ftsZ

Rapid emergence of antibiotic-resistant bacterial pathogens has created urgent demand for the discovery and development of new antibacterial agents directed toward novel targets. Antisense oligodeoxynucleotides (AS-ODN) and their modified forms have been utilized to block gene expression in bacterial cells, showing potential for developing highly specific and efficacious antibacterial agents. In this study, a tetracycline-regulated expression vector was developed for generating single-stranded DNA (ssDNA) of a desired target sequence in bacterial cells. This inducible ssDNA expression vector was tested for producing a DNA enzyme designed to specifically cleave ftsZ mRNA. Our results indicate that the expressed DNA enzyme molecules not only repress ftsZ gene expression and but also inhibit bacterial cell proliferation. Although we believe that the cleavage of ftsZ mRNA by the expressed DNA enzyme molecules is responsible for the inhibitory effects on ftsZ gene expression and bacterial cell proliferation, the antisense mechanism could also be responsible for the biological effects. The ability of this ssDNA expression system to selectively modulate gene expression may provide a powerful strategy in determining the contribution of a given gene product to bacterial growth or pathogenesis and opens a new venue for developing antibacterial agents.     

DNA Damage Response as an Anti-Cancer Barrier: Damage Threshold and the Concept of 'Conditional Haploinsufficiency'

DNA damage response (DDR) emerges as a biological tumorigenesis barrier in early stages of cancer development, and a selective pressure that favors outgrowth of malignant clones with defects in the genome maintenance machinery, such as mutations of p53 and other DDR components. Recent studies indicate that the DDR barrier is not alarmed universally among early noninvasive lesions, but rather responds to high-risk tumorigenic threats that occur in high-grade, pre-malignant lesions that are generally more likely to develop into bona fide malignancies. In addition, while the DDR barrier appears to operate in major types of cancer, such as carcinomas of the lung, breast and colon, DDR activation is rare at any stage of progression among testicular germ-cell tumors. Together with observations that several, but not all oncogenic insults are capable of activating the DDR machinery, these new results point to existence of a critical threshold of such oncogene-induced DNA damage. It seems that only cells and lesions that experience DNA replication stress and DNA damage above such threshold activate the cellular senescence or cell death pathways within the DDR machinery. The higher load of DNA damage may also contribute to cancer predisposition in families with inherited heterozygous defects in the DDR barrier, such as in ATM, BRCA1, BRCA2, p53 and other genes. We propose that carriers of such DDR defects may be more prone to malignancy due to ‘conditional haploinsufficiency’: such partial defects may be asymptomatic in normal tissues, yet they may become manifest under conditions of supra-threshold endogenous DNA damage in oncogene-driven pre-malignant lesions.     

A plant gene for photolyase: an enzyme catalyzing the repair of UV-light-induced DNA damage

Photolyases are thought to be critical components of the defense of plants against damage to DNA by solar ultraviolet light, but nothing is known about their molecular or enzymatic nature. The molecular cloning of a photolyase from mustard ( Sinapis alba ) described here is intended to increase the knowledge about this important repair mechanism in plant species at a molecular level. The gene encodes a polypeptide of 501 amino acids with a predicted molecular mass of 57 kDa. There is a strong sequence similarity to bacterial and yeast photolyases, with a close relationship to enzymes with a deazaflavin chromophor. The plant photolyase is shown to be functional in Escherichia coli which also indicates conservation of photolyases during evolution. It is demonstrated that photolyase expression in plants is light induced, thus providing good evidence for the adaptation of plants to their environment in order to diminish the harmful effects of sunlight.     

Replication-Dependent DNA Damage Response Triggered by Roscovitine Induces an Uncoupling of DNA Replication Proteins

The cyclin-dependent kinase (CDK) inhibitor roscovitine is under evaluation in clinical trials for its antiproliferative properties. Roscovitine arrests cell cycle progression in G1 and in G2 phase by inhibiting CDK2 and CDK1, and possibly CDK7 and CDK9. However, the effects of CDK2 inhibition in S-phase cells have been not fully investigated. Here, we show that a short-term treatment with roscovitine is sufficient to inhibit DNA synthesis, and to activate a DNA damage checkpoint response, as indicated by phosphorylation of p53-Ser15, replication protein A, and histone H2AX. Analysis of DNA replication proteins loaded onto DNA during S phase showed that the amount of proliferating cell nuclear antigen (PCNA), a cofactor of DNA replication enzymes, was significantly reduced by roscovitine. In contrast, chromatin-bound levels of DNA polymerase δ, DNA ligase I and CDK2, were stabilized. Checkpoint inhibition with caffeine could rescue PCNA disassembly only partially, pointing to additional effects due to CDK2 inhibition and the presence of replication stress. These results suggest that in S-phase cells, roscovitine induces checkpoint-dependent and -independent effects, leading to stabilization of replication forks and an uncoupling between PCNA and PCNA-interacting proteins.     

Domain requirements for DNA unwinding by mycobacterial UvrD2, an essential DNA helicase

Mycobacterial UvrD2 is a DNA-dependent ATPase with 3' to 5' helicase activity. UvrD2 is an atypical helicase, insofar as its N-terminal ATPase domain resembles the superfamily I helicases UvrD/PcrA, yet it has a C-terminal HRDC domain, which is a feature of RecQ-type superfamily II helicases. The ATPase and HRDC domains are connected by a CxxC-(14)-CxxC tetracysteine module that defines a new clade of UvrD2-like bacterial helicases found only in Actinomycetales. By characterizing truncated versions of Mycobacterium smegmatis UvrD2, we show that whereas the HRDC domain is not required for ATPase or helicase activities in vitro, deletion of the tetracysteine module abolishes duplex unwinding while preserving ATP hydrolysis. Replacing each of the CxxC motifs with a double-alanine variant AxxA had no effect on duplex unwinding, signifying that the domain module, not the cysteines, is crucial for function. The helicase activity of a truncated UvrD2 lacking the tetracysteine and HRDC domains was restored by the DNA-binding protein Ku, a component of the mycobacterial NHEJ system and a cofactor for DNA unwinding by the paralogous mycobacterial helicase UvrD1. Our findings indicate that coupling of ATP hydrolysis to duplex unwinding can be achieved by protein domains acting in cis or trans. Attempts to disrupt the M. smegmatis uvrD2 gene were unsuccessful unless a second copy of uvrD2 was present elsewhere in the chromosome, indicating that UvrD2 is essential for growth of M. smegmatis.     

DNA topoisomerases: single gyrase caught in the act

A recent study has analysed the action of bacterial DNA gyrase on a single substrate DNA molecule, discriminating the initial DNA wrapping and subsequent supercoiling steps in the reaction cycle.     

Construction of a vaccinia virus deficient in the essential DNA repair enzyme uracil DNA glycosylase by a complementing cell line.

The vaccinia virus D4R open reading frame, encoding the essential DNA repair enzyme uracil DNA glycosylase, was expressed in two permanent cell lines, the rabbit kidney cell line RK13 and the human fibroblast cell line 293. The temperature-sensitive vaccinia virus mutant ts4149, which maps within D4R, was able to grow under restrictive conditions in both of these transformed cell lines. Cell clones complemented D4R function to various degrees, demonstrating complementation of an essential vaccinia virus gene by a cell line constitutively expressing the essential function. Thus, the complementing host cells allowed the rescue of a virus defective in the D4R gene, demonstrating that this system may be used for the propagation of defective cytoplasmic DNA viruses. The defective virus grew to high yields only in the engineered cell lines. The data support the hypothesis that early gene products, such as uracil DNA glycosylase, supplied in trans can fully complement essential viral functions.     

Activation of the DNA Damage Response by Telomere Attrition: A Passage to Cellular Senescence

Critical telomere shortening induces senescence in many normal human cell types grown in culture. Recentdata have revealed that dysfunctional telomeres can resemble certain forms of DNA damage, and point to a role for DNA damage signaling in the establishment and maintenance of telomere-initiated senescence. Here, we review these new observations and highlight potential avenues of future research. We consider the identities of the key DNA damage response factors involved in senescence and discuss a model for the molecular events occurring in pre-senescent cells that ultimately lead to a permanent cell cycle arrest phenotype.     

DNA Damage Profiling in Motor Neurons: A Single-Cell Analysis by Comet Assay

We developed a method to measure DNA damage in single motor neurons (MN). A cell fraction enriched in viable -motor neurons was isolated from adult rat spinal cord. This cell preparation was used to measure the vulnerability of the MN genome to different reactive oxygen species (ROS). MN were exposed in vitro to hydrogen peroxide, nitric oxide and peroxynitrite. Specific types of DNA lesions (e.g., abasic sites, single-strand breaks, and double-strand breaks) were measured using single-cell gel electrophoresis (comet assay). The MN genome was very susceptible to attack by ROS. Different ROS induced different DNA damage profiles in MN. MN were also isolated from adult rats with sciatic nerve avulsions to show that DNA damage emerges early during their degeneration in vivo. This study demonstrates that the comet assay is a feasible method for profiling DNA lesions in the genome of single MN. Viable mature MN can be isolated and used for in vitro models of MN genotoxicity and can be isolated from in vivo models of MN degeneration for profiling DNA damage on a single-cell basis.