MYCBP2 Is a Guanosine Exchange Factor for Ran Protein and Determines Its Localization in Neurons of Dorsal Root Ganglia

The small GTPase Ran coordinates retrograde axonal transport in neurons, spindle assembly during mitosis, and the nucleo-cytoplasmic transport of mRNA. Its localization is tightly regulated by the GTPase-activating protein RanGAP1 and the nuclear guanosine exchange factor (GEF) RCC1. We show that loss of the neuronal E3 ubiquitin ligase MYCBP2 caused the up-regulation of Ran and RanGAP1 in dorsal root ganglia (DRG) under basal conditions and during inflammatory hyperalgesia. SUMOylated RanGAP1 physically interacted with MYCBP2 and inhibited its E3 ubiquitin ligase activity. Stimulation of neurons induced a RanGAP1-dependent translocation of MYCBP2 to the nucleus. In the nucleus of DRG neurons MYCBP2 co-localized with Ran and facilitated through its RCC1-like domain the GDP/GTP exchange of Ran. In accordance with the necessity of a GEF to promote GTP-binding and nuclear export of Ran, the nuclear localization of Ran was strongly increased in MYCBP2-deficient DRGs. The finding that other GEFs for Ran besides RCC1 exist gives new insights in the complexity of the regulation of the Ran signaling pathway.     

Metabolic Engineering of Corynebacterium glutamicum for Methanol Metabolism

Methanol is already an important carbon feedstock in the chemical industry, but it has found only limited application in biotechnological production processes. This can be mostly attributed to the inability of most microbial platform organisms to utilize methanol as a carbon and energy source. With the aim to turn methanol into a suitable feedstock for microbial production processes, we engineered the industrially important but nonmethylotrophic bacterium Corynebacterium glutamicum toward the utilization of methanol as an auxiliary carbon source in a sugar-based medium. Initial oxidation of methanol to formaldehyde was achieved by heterologous expression of a methanol dehydrogenase from Bacillus methanolicus, whereas assimilation of formaldehyde was realized by implementing the two key enzymes of the ribulose monophosphate pathway of Bacillus subtilis: 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase. The recombinant C. glutamicum strain showed an average methanol consumption rate of 1.7 ± 0.3 mM/h (mean ± standard deviation) in a glucose-methanol medium, and the culture grew to a higher cell density than in medium without methanol. In addition, [¹³C]methanol-labeling experiments revealed labeling fractions of 3 to 10% in the m + 1 mass isotopomers of various intracellular metabolites. In the background of a C. glutamicum Δald ΔadhE mutant being strongly impaired in its ability to oxidize formaldehyde to CO₂, the m + 1 labeling of these intermediates was increased (8 to 25%), pointing toward higher formaldehyde assimilation capabilities of this strain. The engineered C. glutamicum strains represent a promising starting point for the development of sugar-based biotechnological production processes using methanol as an auxiliary substrate.     

Botrytis pseudocinerea Is a Significant Pathogen of Several Crop Plants but Susceptible to Displacement by Fungicide-Resistant B. cinerea Strains

Botrytis cinerea is one of the most important pathogens worldwide, causing gray mold on a large variety of crops. Botrytis pseudocinerea has been found previously to occur together with B. cinerea in low abundance in vineyards and strawberry fields. Here, we report B. pseudocinerea to be common and sometimes dominant over B. cinerea on several fruit and vegetable crops in Germany. On apples with calyx end rot and on oilseed rape, it was the major gray mold species. Abundance of B. pseudocinerea was often negatively correlated with fungicide treatments. On cultivated strawberries, it was frequently found in spring but was largely displaced by B. cinerea following fungicide applications. Whereas B. cinerea strains with multiple-fungicide resistance were common in these fields, B. pseudocinerea almost never developed resistance to any fungicide even though resistance mutations occurred at similar frequencies in both species under laboratory conditions. The absence of resistance to quinone outside inhibitors in B. pseudocinerea was correlated with an intron in cytB preventing the major G143A resistance mutation. Our work indicates that B. pseudocinerea has a wide host range similar to that of B. cinerea and that it can become an important gray mold pathogen on cultivated plants.     

Exploring Vertical Transmission of Bifidobacteria from Mother to Child

Passage through the birth canal and consequent exposure to the mother''s microbiota is considered to represent the initiating event for microbial colonization of the gastrointestinal tract of the newborn. However, a precise evaluation of such suspected vertical microbiota transmission has yet to be performed. Here, we evaluated the microbiomes of four sample sets, each consisting of a mother''s fecal and milk samples and the corresponding infant''s fecal sample, by means of amplicon-based profiling supported by shotgun metagenomics data for two key samples. Notably, targeted genome reconstruction from microbiome data revealed vertical transmission of a Bifidobacterium breve strain and a Bifidobacterium longum subsp. longum strain from mother to infant, a notion confirmed by strain isolation and genome sequencing. Furthermore, PCR analyses targeting unique genes from these two strains highlighted their persistence in the infant gut at 6 months. Thus, this study demonstrates the existence of specific bifidobacterial strains that are common to mother and child and thus indicative of vertical transmission and that are maintained in the infant for at least relatively short time spans.     

The Translesion Polymerase ζ Has Roles Dependent on and Independent of the Nuclease MUS81 and the Helicase RECQ4A in DNA Damage Repair in Arabidopsis

DNA polymerase zeta catalytic subunit REV3 is known to play an important role in the repair of DNA damage induced by cross-linking and methylating agents. Here, we demonstrate that in Arabidopsis (Arabidopsis thaliana), the basic polymerase activity of REV3 is essential for resistance protection against these different types of damaging agents. Interestingly, its processivity is mainly required for resistance to interstrand and intrastrand cross-linking agents, but not alkylating agents. To better define the role of REV3 in relation to other key factors involved in DNA repair, we perform epistasis analysis and show that REV3-mediated resistance to DNA-damaging agents is independent of the replication damage checkpoint kinase ataxia telangiectasia-mutated and rad3-related homolog. REV3 cooperates with the endonuclease MMS and UV-sensitive protein81 in response to interstrand cross links and alkylated bases, whereas it acts independently of the ATP-dependent DNA helicase RECQ4A. Taken together, our data show that four DNA intrastrand cross-link subpathways exist in Arabidopsis, defined by ATP-dependent DNA Helicase RECQ4A, MMS and UV-sensitive protein81, REV3, and the ATPase Radiation Sensitive Protein 5A.The DNA of all living organisms is constantly exposed to damaging factors, and therefore a number of DNA damage repair and bypass mechanisms have evolved. DNA lesions that interfere with the replication machinery constitute a particular challenge for cells (Schröpfer et al., 2014a); if not repaired in a timely manner, such damage can result in the stalling or collapse of replication forks, which in turn can lead to cell death. Furthermore, one-sided double-strand breaks (DSBs) can occur when the replication fork encounters a single-strand break. Lesions within one DNA strand, such as alkylations or DNA intrastrand cross links, can be bypassed by postreplicative repair (PRR), a process that is best understood in yeast (Saccharomyces cerevisiae). This mechanism does not lead to repair of the lesion but prevents fatal long-lasting stalling of the replication fork. PRR can be divided into two branches: the error-prone pathway and the error-free pathway (for review, see Goodman and Woodgate, 2013; Haynes et al., 2015; Jansen et al., 2015). It is known from yeast that both branches of PRR are controlled by the Radiation sensitivity protein6 (Rad6) and Mms-Ubc13 E3 ubiquitin-conjugating enzyme complexes, which ubiquitinate the replicative processivity factor Proliferating Cellular Nuclear Antigen1. The monoubiquitination of PCNA at Lys-164 by Rad6-Rad18 initiates the error-prone pathway, whereas polyubiquitination additionally requires Mms2, Ubc13, and Rad5, and triggers the error-free PRR branch (Hoege et al., 2002; Moldovan et al., 2007; Lee and Myung, 2008). There are two possible competing models postulated for the error-free bypass of lesions at the replication fork, both of which depend on template-switch mechanisms; if the lesion concerns only one of the two sister strands, the undamaged strand can be used as the template for bypassing the lesion. One of the two models features the so-called overshoot synthesis, whereby the newly synthesized strand on the undamaged parental strand is elongated further than the strand blocked by the lesion. Regression of the replication fork then leads to the formation of a special type of four-way junction called a chicken-foot structure. This regression mechanism is thought to be accomplished by helicases, such as the RecQ helicase Bloom Syndrome Protein (BLM) in humans (Croteau et al., 2014). AtRECQ4A is the respective BLM homolog in Arabidopsis (Arabidopsis thaliana), and this enzyme has the ability to regress replication forks in vitro (Hartung et al., 2007, 2008; Schröpfer et al., 2014b). The second error-free subpathway entails invasion of the newly synthesized strand on the blocked sister chromatid into the complementary newly replicated strand on the other sister chromatid. Such a step forms a displacement loop-like structure in which synthesis over the damaged region can occur. In yeast, both error-free pathways are dependent on the multifunctional protein Rad5, which is known to recruit PRR factors and also exhibits helicase activity itself (Blastyák et al., 2007). We previously identified AtRAD5A as a functional Arabidopsis homolog of Rad5 (Chen et al., 2008). Interestingly, AtRAD5A is required for efficient repair by homologous recombination via the synthesis-dependent strand-annealing mechanism, a pathway that in some steps is related to the invasion model of PRR (Mannuss et al., 2010).The error-prone pathway is based on the function of translesion synthesis (TLS) polymerases, which promote replication through DNA lesions (Prakash et al., 2005). In a mechanism termed polymerase switch, the replicative polymerase is exchanged by such a TLS polymerase at a damaged site. After incorporation of a nucleotide opposite the damaged base by the TLS polymerase, a second polymerase switch exchanges the TLS polymerase for the replicative polymerase so that replication can proceed (Prakash and Prakash, 2002; Lehmann et al., 2007). TLS polymerases possess no 5′-3′-exonuclease activity, and therefore act in a potentially mutagenic manner. Nevertheless, depending on the damage incurred and the TLS polymerase used, damage bypass can be error free (Haracska et al., 2000; McCulloch et al., 2004).Polymerases can be divided into at least six families based on their amino acid sequences and crystal structures: A, B, C, D, X, and Y. All of them share the common structure analogous to a right hand grasping DNA with palm, finger, and thumb domains (Steitz, 1999). The amino acid sequences of the finger and thumb domains of different polymerase families are highly variable, whereas the palm domains share high similarity. The palm domain forms the largest part of the polymerase active site and contains highly conserved Asp residues that have been postulated to be involved in the catalytic activity of the enzyme (Joyce and Steitz, 1995; Steitz, 1999).Most TLS polymerases belong to the Y family of polymerases, a class of specially structured enzymes that catalyze replication over damaged templates (Ohmori et al., 2001; Sale et al., 2012). Although some polymerases of the A, B, or X family can also exhibit TLS activity, this is often not their primary function (Prakash et al., 2005). DNA Polymerase Zeta (POLζ) is a B family polymerase and consists of a DNA Polymerase Zeta subunit REV3-REV7 heterodimer, in which REV3 is the catalytic subunit with its accessory subunit and processivity factor REV7 (Nelson et al., 1996). Recent studies in yeast and human cells have shown that POLζ contains two additional subunits, Pol31 and Pol32 in yeast, orthologs to human POLD2 and POLD3, which are known to be accessory subunits of the replicative polymerase POLδ (Johnson et al., 2012; Lee et al., 2014). REV3 contains three regions that are highly conserved between organisms: an N-terminal region, a REV7 binding domain, and a B family-type polymerase domain. The polymerase domain carries the six common conserved regions, I to VI (IV-II-VI-III-I-V), of which I is the most and VI the least conserved region. The A (II), B (III), and C (I) motifs, located within regions I, II, and III (Wong et al., 1988), form the active site of the enzyme, and each harbors an essential Asp residue that coordinates two catalytic metal ions. Deficiency of REV3 in mice is embryo lethal (Bemark et al., 2000; Esposito et al., 2000), and vertebrate cells depleted in REV3 show hypersensitivity to various DNA-damaging agents, including UV and ionizing irradiation, cisplatin, MMS, and mitomycin C (MMC; Sonoda et al., 2003; Sharma and Canman, 2012). In Arabidopsis, rev3 mutants exhibit no obvious phenotype under standard growth conditions, but are hypersensitive to UV-B and gamma irradiation, MMC, MMS, and cisplatin (Sakamoto et al., 2003).Our previous work demonstrated the existence of several different pathways in Arabidopsis involved in repairing the DNA damage induced by cross-linking and methylating agents. These independent pathways are defined by the ATPase RAD5A, the helicase RECQ4A, and MMS and UV-sensitive protein81 (MUS81; Mannuss et al., 2010). The structure-specific endonuclease MUS81 together with its noncatalytic subunit (Mms4 in yeast, Eme1 in Schizosaccharomyces pombe, and MMS4 or Crossover Junction Endonuclease (EME1) in humans and plants) functions in the rescue of stalled replication forks. The enzyme is able to cleave the stalled fork at the lesion site, which leads to a one-sided DSB that is repaired by homologous recombination (HR) to restore the stalled fork (Hanada et al., 2006). We previously showed that mus81 transfer DNA (T-DNA) insertion lines in Arabidopsis are highly sensitive to treatment with MMS, cisplatin, hydroxyurea, ionizing irradiation, and MMC. We also found that AtMUS81 can form a complex with its heterologous binding partners AtEME1A or AtEME1B that is able to process intricate DNA structures, such as nicked Holliday junctions, which might also form at stalled replication forks (Hartung et al., 2006; Geuting et al., 2009).In the current study, we address whether fully functional polymerase activity is required for the repair of DNA damage induced by alkylating and cross-link-inducing agents. Moreover, we sought to clarify whether REV3 cooperates with other key factors identified in Arabidopsis in the repair of these different types of damage. Indeed, it has already been shown that AtREV3 and AtRAD5A do not cooperate in the repair of such DNA damage, confirming independent pathways of error-prone and error-free PRR in plants (Wang et al., 2011). However, as plants, animals, and yeast differ in their DNA cross-link repair machinery (e.g. Mannuss et al., 2010; Knoll et al., 2012; Dangel et al., 2014; Herrmann et al., 2015), it is of particular importance to define the role of REV3 in relation to MUS81 and RECQ4A.     

Dynamic and Thermodynamic Response of the Ras Protein Cdc42Hs upon Association with the Effector Domain of PAK3

Human cell division cycle protein 42 (Cdc42Hs) is a small, Rho-type guanosine triphosphatase involved in multiple cellular processes through its interactions with downstream effectors. The binding domain of one such effector, the actin cytoskeleton-regulating p21-activated kinase 3, is known as PBD46. Nitrogen-15 backbone and carbon-13 methyl NMR relaxation was measured to investigate the dynamical changes in activated GMPPCP·Cdc42Hs upon PBD46 binding. Changes in internal motion of the Cdc42Hs, as revealed by methyl axis order parameters, were observed not only near the Cdc42Hs–PBD46 interface but also in remote sites on the Cdc42Hs molecule. The binding-induced changes in side-chain dynamics propagate along the long axis of Cdc42Hs away from the site of PBD46 binding with sharp distance dependence. Overall, the binding of the PBD46 effector domain on the dynamics of methyl-bearing side chains of Cdc42Hs results in a modest rigidification, which is estimated to correspond to an unfavorable change in conformational entropy of approximately − 10 kcal mol^− 1 at 298 K. A cluster of methyl probes closest to the nucleotide-binding pocket of Cdc42Hs becomes more rigid upon binding of PBD46 and is proposed to slow the catalytic hydrolysis of the γ phosphate moiety. An additional cluster of methyl probes surrounding the guanine ring becomes more flexible on binding of PBD46, presumably facilitating nucleotide exchange mediated by a guanosine exchange factor. In addition, the Rho insert helix, which is located at a site remote from the PBD46 binding interface, shows a significant dynamic response to PBD46 binding.