One ring to rule them all? Another cellular responsibility for PCNA

To prevent duplication or loss of genomic regions during DNA replication, it is essential that the entire genome is copied precisely once every S phase. Cells achieve this by mutually exclusive regulation of origin firing and licensing. A crucial protein that is involved in origin licensing is chromatin licensing and DNA replication factor 1 (CDT1) and, therefore, activity of this protein must be strictly controlled. Four recent articles have demonstrated that proliferating cell nuclear antigen (PCNA), an essential sliding clamp used in replication and DNA repair, has a crucial role in this process by mediating the proteasomal degradation of CDT1.     

Batteries: what causes them to fail?

Rechargeable batteries do not die suddenly but gradually get weaker over time. The ability to hold charge diminishes through aging, usage (cycling), handling and environmental conditions. Batteries seem to have their own inherited life span. A fixed replacement plan, in which all batteries are replaced at a certain age, does not work well. Rather, each battery should periodically be analyzed and only those replaced whose capacity drops below an acceptable performance threshold. Implementing such a maintenance program reduces unexpected downtime caused by weak batteries and saves money. Unexpected downtime due to failing batteries can cause tense moments for the equipment operator. Some people may even seek legal compensation for personal damages suffered by a failing battery.     

One way to do experiments on gene conversion?

Summary Competent cells of B. subtilis were transfected with heteroduplex SPP1 DNA, made by annealing complementary strands of wild type and 21 plaque type mutant DNAs. The frequencies of cells yielding mutant and wild type, only wild type, and only mutant phages were determined by single burst analyses of transfected cells. The data obtained reveal that an effective mechanism is operating in B. subtilis, which converts heterozygous to homozygous molecules prior to their replication. This correction mechanism is asymmetric with regard to the strand which is preferentially corrected in a given heteroduplex pair. The direction of asymmetry thus defined depends on the marker introduced into a particular heteroduplex. The efficiency of correction varies with the markers used and is correlated to the position of markers in the genetic map. From this correlation, the direction of replication of the SPP1 genome is deduced. The frequency distribution of wild type and mutant phages in cells yielding both genotypes indicates that both strands of the input DNA contribute equally to the production of progeny, i.e. DNA replication is symmetric.     

One ring to bring them all—The role of Ku in mammalian non-homologous end joining

The repair of DNA double strand breaks is essential for cell survival and several conserved pathways have evolved to ensure their rapid and efficient repair. The non-homologous end joining pathway is initiated when Ku binds to the DNA break site. Ku is an abundant nuclear heterodimer of Ku70 and Ku80 with a toroidal structure that allows the protein to slide over the broken DNA end and bind with high affinity. Once locked into placed, Ku acts as a tool-belt to recruit multiple interacting proteins, forming one or more non-homologous end joining complexes that act in a regulated manner to ensure efficient repair of DNA ends. Here we review the structure and functions of Ku and the proteins with which it interacts during non-homologous end joining.     

Telomerase activation. One step on the road to cancer?

Ever since the discovery that telomeres are short in cancer cells and telomerase is activated in immortal cells, telomerase has been an oncogene wannabe. Oncogenes have been the glamour genes of molecular biology for 20 years, garnering flashy headlines and name recognition. More recently, tumor-suppressor genes have joined oncogenes on center stage. Recent evidence has shown that MYC upregulates the catalytic subunit of telomerase, TERT, and that TERT cooperates with HPV E7 in cell immortalization. This evidence now supports the placement of telomerase among the cancer gene elite.     

Flavonoid–membrane interactions: Involvement of flavonoid–metal complexes in raft signaling

Flavonoids are polyphenolic compounds produced by plants and delivered to the human body through food. Although the epidemiological analyses of large human populations did not reveal a simple correlation between flavonoid consumption and health, laboratory investigations and clinical trials clearly demonstrate the effectiveness of flavonoids in the prevention of cardiovascular, carcinogenic, neurodegenerative and immune diseases, as well as other diseases. At present, the abilities of flavonoids in the regulation of cell metabolism, gene expression, and protection against oxidative stress are well-known, although certain biophysical aspects of their functioning are not yet clear. Most flavonoids are poorly soluble in water and, similar to lipophilic compounds, have a tendency to accumulate in biological membranes, particularly in lipid rafts, where they can interact with different receptors and signal transducers and influence their functioning through modulation of the lipid-phase behavior. In this study, we discuss the enhancement in the lipophilicity and antioxidative activity of flavonoids after their complexation with transient metal cations. We hypothesize that flavonoid–metal complexes are involved in the formation of molecular assemblies due to the facilitation of membrane adhesion and fusion, protein–protein and protein–membrane binding, and other processes responsible for the regulation of cell metabolism and protection against environmental hazards.     

LRRK2 signaling pathways: the key to unlocking neurodegeneration?

Mutations in PARK8, encoding leucine-rich repeat kinase 2 (LRRK2), are a major cause of Parkinson's disease. We contrast data suggesting that changes in LRRK2 activity cause alterations in mitogen-activated protein kinase, translational control, tumor necrosis factor α/Fas ligand and Wnt signaling pathways with the cell biological functions of LRRK2 such as vesicle trafficking. Despite scarce in vivo data on cell signaling, involvement in diverse cell biological functions suggests a role for LRRK2 as an upstream regulator in events leading to neurodegeneration. To stimulate discussion and give direction for future research, we further suggest that despite the importance of the catalytic activity for cytotoxicity, the main cellular function of LRRK2 is linked to assembly of signaling complexes.     

The many functions of cohesin—different rings to rule them all?

EMBO J 31 9, 2076–2089 March132012EMBO J 31 9, 2090–2102 March132012It is well known that somatic and germ cells use different cohesin complexes to mediate sister chromatid cohesion, but why different isoforms of cohesin also co-exist within somatic vertebrate cells has remained a mystery. Two papers in this issue of The EMBO Journal have begun to address this question by analysing mouse cells lacking SA1, an isoform of a specific cohesin subunit.When one cell divides into two, many things have to go right for the two daughter cells to receive identical copies of their mother cell''s genome. It has long been recognized that sister chromatid cohesion, the physical connection established during DNA replication between newly synthesized sister DNA molecules, is one of these essential prerequisites for proper chromosome segregation. It is this cohesion that enables the bi-orientation of chromosomes on the mitotic or meiotic spindle, and thus makes their symmetrical segregation possible. Cohesion is mediated by cohesin, a multi-subunit protein complex, which is thought to connect sister DNA molecules by embracing them as a ring (Figure 1; reviewed in Peters et al, 2008). It is well established that cohesin complexes differ between somatic and germ cells, where they are needed for the proper separation of sister chromatids and of homologous chromosomes, respectively. What has been largely ignored, however, is that even within somatic vertebrate cells there are different forms of cohesin, containing mutually exclusive variable subunits: either SA1 or the closely related SA2 protein (also known as STAG1 and STAG2, respectively), and either Pds5A or the related Pds5B subunit (Peters et al, 2008). Why is that? Two papers from the Losada lab (Remeseiro et al, 2012a, 2012b) have begun to address this question by generating mouse cells lacking the SA1 gene, revealing unexpected insights into the functions of SA1 subunit-containing cohesin complexes (cohesin-SA1).Open in a separate windowFigure 1Schematic drawing illustrating how the SA1 and SA2 proteins interact in a mutually exclusive manner with three core subunits of cohesin (Smc1, Smc3, Rad21) that form a ring-like structure. It has been proposed that these complexes mediate cohesion by trapping the two sister DNA molecules inside the cohesin ring (above), and that cohesin rings might affect chromatin structure by forming or stabilizing intra-chromatid loops (below). Cohesin is thought to influence gene regulation at least in part by mediating chromatin looping.Although cohesin is best known for its role in sister chromatid cohesion, it is clearly also needed for homologous recombination-mediated DNA repair and for gene regulation. Much of what we know about these functions comes from studies in yeast and fruit flies, organisms with only a single SA1/SA2-related mitotic subunit (Scc3 in budding yeast), and only a single Pds5 subunit. It is therefore plausible that, like many other genes during vertebrate evolution, SA1/SA2 and Pds5A/Pds5B have arisen by gene duplication to constitute paralogs, with functional differences between them assumed to be subtle. Consistently, absence of either Pds5A or Pds5B causes only mild, if any, defects in sister chromatid cohesion, and mice lacking either protein can develop to term, although they die shortly after birth owing to multiple organ defects (Zhang et al, 2007, 2009). First indications that the situation may be different for the Scc3-related subunits came from Canudas and Smith (2009), who reported that RNAi depletion of SA1 and SA2 from HeLa cells caused defects in telomere and centromere cohesion, respectively. The generation of mice lacking either one or both alleles of the SA1 gene has now allowed a more systematic and thorough analysis of SA1 function (Remeseiro et al, 2012a, 2012b).One of the most striking results obtained in these studies is that most mice lacking SA1 die around day 12 of embryonic development, clearly showing that the function of SA1 cannot be fulfilled by SA2, despite the fact that SA2 is substantially more abundant in somatic cells than SA1 (Holzmann et al, 2010). What could this SA1-specific function be? Losada and colleagues report observations, which imply that SA1 does not have just one, but possibly several important functions in different processes. First, the authors confirm the previous observation that SA1 is required for cohesion specifically at telomeres, while likely collaborating with SA2 in chromosome arms or centromeric regions. Furthermore, telomeres have an unusual morphology in mitotic chromosomes lacking SA1 (Remeseiro et al, 2012a), reminiscent of a fragile-site phenotype previously reported in telomeres with DNA replication defects (Sfeir et al, 2009), and SA1 is indeed required for efficient telomere duplication. Depletion of sororin, a protein that is required for cohesin''s ability to mediate sister chromatid cohesion, also causes a fragile-site phenotype at telomeres. These findings imply that SA1''s role in telomere cohesion is important for efficient telomere replication, perhaps, as the authors speculate, because telomere cohesion may help to stabilize or re-start stalled replication forks, or because cohesion-dependent homologous recombination might be involved in repair of DNA double strand breaks created by collapsed replication forks. Interestingly, cells lacking SA1 frequently show chromosome bridges in anaphase, often fail to divide, and either die or become bi-nucleated. The exact origin of chromosome bridges is difficult to determine, but previous studies have found such bridges often associated with fragile sites on chromosomes; treatment with low doses of DNA replication inhibitors was shown to increase the frequency of such bridges (Chan et al, 2009), and similar observations were indeed made by Remeseiro et al (2012a) in mouse embryonic fibroblasts. It is therefore plausible that the telomere cohesion defect observed in SA1-lacking cells leads to incomplete telomere replication, which in turn results in the formation of anaphase chromosome bridges and subsequent cytokinesis defects. Losada and colleagues further speculate that these chromosome segregation defects could underlie the increased frequency of spontaneous development of various tumours in mice containing just one instead of two SA1 alleles (Remeseiro et al, 2012a). This is an attractive interpretation since tetraploidy and aneuploidy are thought to contribute to the rate with which tumour cells can evolve; however, Losada and colleagues report SA1 deficiency to cause defects also in other cohesin functions, which may therefore as well contribute to tumour formation.To further understand why SA1 cannot be fulfilled by SA2, Losada and colleagues also analysed the distribution of these proteins in the non-repetitive parts of the mouse genome by chromatin immunoprecipitation coupled to deep sequencing (ChIP-seq). The results of these experiments, published in the second of the two papers (Remeseiro et al, 2012b), raise the interesting possibility that cohesin-SA1 associates more frequently with gene promoters than cohesin-SA2. However, the fact that different antibodies have to be used for any ChIP-based comparison of the distribution of two proteins makes it difficult to know to what degree observed differences might be due to different antibody efficiency. Obviously, such limitations do not exist if the distribution of one and the same protein is analysed under different conditions, and in such an experimental setting, Remeseiro et al indeed make some striking observations. When SA1 is absent, SA2 does not detectably change in abundance, but its distribution in the genome does, in that more than half of all SA2-binding sites in SA1-deficient cells differ from those bound in wild-type cells. Most SA2-binding sites in SA1-deficient cells are in intergenic regions, and CTCF, a zinc finger protein often co-localizing with cohesin and implicated in its gene regulation function (Peters et al, 2008), appears to be absent at many of these sites. It presently remains a mystery why cohesin-SA2 changes its distribution so dramatically in the absence of SA1, but the observation that gene promoters are more frequently occupied by cohesin in the presence of SA1 than in its absence raises the possibility that cohesin-SA1 may have a specific role in gene regulation. This possibility is particularly interesting in light of a recent study that found hardly any change in gene expression upon re-expression of SA2 in SA2-deficient human glioblastoma cells (Solomon et al, 2011), despite the fact that cohesin is thought to regulate numerous genes. With this in mind, Remeseiro et al analysed gene expression in mouse cells and indeed found 549 genes to be mis-regulated in the absence of SA1, in striking contrast to the above-mentioned comparison of human SA2-deficient or proficient cells that found only 19 genes to change in expression levels (Solomon et al, 2011). Obviously direct comparisons will be essential to analyse further the specific roles of SA1 and SA2 in gene regulation, but the current evidence raises the interesting possibility that SA1 may have a particularly important role in gene regulation, whereas cohesin-SA2 is dedicated to creating arm and centromeric cohesive structures for chromosome segregation.That is not to say that cohesin-SA1 cannot mediate sister chromatid cohesion. It almost certainly can, as it is essential for cohesion at telomeres (Canudas and Smith, 2009; Remeseiro et al, 2012a). Likewise, it would be wrong to assume that we now fully understand why SA1 and SA2 co-exist in somatic vertebrate cells, and what their precise functions is. There are many things we do not understand. For example, if SA2 has little or no role in gene regulation, as the Solomon et al (2011) study implies, why does SA2 nevertheless interact directly with CTCF (Xiao et al, 2011), its gene regulation collaborator? How do cohesin-SA1 and cohesin-SA2 complexes further differ in their genomic distributions and their functions depending on whether they contain either Pds5A or Pds5B, constituting really not just two but four distinct cohesin complexes? The work by Losada and colleagues represents an important step towards understanding these questions, but there is still a long and presumably exciting way to go to understand how different cohesin complexes control the mammalian genome.     

What makes the mitochondria a killer? Can we condition them to be less destructive?

Cardioprotection, such as preconditioning and postconditioning, has been shown to result in a significant reduction in cell death. Many of the signaling pathways activated by cardioprotection have been elucidated, but there is still a lack of understanding of the mechanisms by which these signaling pathways reduce cell death. Mitochondria have been reported to be an important player in many types of apoptotic and necrotic cell death. If mitochondria play an important role in cell death, then it seems reasonable to consider that cardioprotective mechanisms might act, at least in part, by opposing mitochondrial cell death pathways. One of the major mechanisms of cell death in ischemia-reperfusion is suggested to be the opening of a large conductance pore in the inner mitochondrial membrane, known as the mitochondrial permeability transition pore. Inhibition of this mitochondrial pore appears to be one of the major mechanisms by which cardioprotection reduces cell death. Cardioprotection activates a number of signaling pathways that reduce the level of triggers (reactive oxygen species and calcium) or enhances inhibitors of the mitochondrial permeability transition pore at the start of reperfusion. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

Cytokine networks: do we understand them well enough to facilitate clinical benefits?

Cytokines are chiefly local transmitters. The cloning of these molecules has been driven by the belief that they will be of major therapeutic benefit, despite the problems inherent in their clinical administration systemically, a non-physiological route. This brief review discusses the clinical use of cytokines and the possibilities for clinical benefit of interfering with cytokine function. A major step forward in the past few years has been the realization that shed extracellular domains of cytokine receptors are the most common natural cytokine inhibitors, described for TNF, IFN gamma, IL-2, IL-4, IL-7, for example. These natural cytokine inhibitors may offer interesting therapeutic possibilities, as they are enhancing or "replacing" the natural anti-cytokine mechanisms.     

Whither T-suppressors: if they didn't exist would we have to invent them?

Arriving at an understanding of the role of suppressor T-cells (regulatory T-cells, CD4(+)CD25+) depends on whether their functional repertoire is somatically selected to be anti-Self or anti-Nonself. Immunologists are ambivalent; often publications espousing opposite views share an author. Here the arguments are detailed that the suppressor repertoire is not somatically selected to be anti-Self, but rather it is anti-Nonself. Therefore, suppression cannot regulate the Self-Nonself discrimination; its function is to regulate the magnitude and class of the anti-Nonself effector response.     

Cell wall growth during elongation and division: one ring to bind them?

The role of the cell division protein FtsZ in bacterial cell wall (CW) synthesis is believed to be restricted to localizing proteins involved in the synthesis of the septal wall. In this issue of Molecular Microbiology, the groups of Christine Jacobs-Wagner and Waldemar Vollmer provide compelling evidence that in Caulobacter crescentus, FtsZ plays an additional role in CW synthesis in non-dividing cells. During elongation (cell growth) FtsZ is responsible for the incorporation of CW material in a zone at the midcell by recruiting MurG, a protein involved in peptidoglycan (PG) precursor synthesis. This resembles earlier findings of FtsZ mediated PG synthesis activity in Escherichia coli. A role of FtsZ in PG synthesis during elongation forces a rethink of the current model of CW synthesis in rod-shaped bacteria.     

All signaling is local?

The mechanism of signal transmission following ligand stimulation of receptor tyrosine kinases in living cells is poorly understood. Recent studies have visualized the spatio-temporal pattern of EGF signaling, indicating that receptor density is an important factor in the mechanism of lateral propagation of local EGF signaling.