Improved detection of DNA replication fork-associated proteins.

Innovative methods to retrieve proteins associated with actively replicating DNA have provided a glimpse into the molecular dynamics of replication fork stalling. We report that a combination of density-based replisome enrichment by isolating proteins on nascent DNA (iPOND2) and label-free quantitative mass spectrometry (iPOND2-DRIPPER) substantially increases both replication factor yields and the dynamic range of protein quantification. Replication protein abundance in retrieved nascent DNA is elevated up to 300-fold over post-replicative controls, and recruitment of replication stress factors upon fork stalling is observed at similar levels. The increased sensitivity of iPOND2-DRIPPER permits direct measurement of ubiquitination events without intervening retrieval of diglycine tryptic fragments of ubiquitin. Using this approach, we find that stalled replisomes stimulate the recruitment of a diverse cohort of DNA repair factors, including those associated with poly-K63-ubiquitination. Finally, we uncover the temporally controlled association of stalled replisomes with nuclear pore complex components and nuclear cytoskeleton networks.

A customizable series of microbiology lab activities exploring a foodborne outbreak to enhance student recruitment to a biology program.

As educators at a small university, we are constantly trying to find new and innovative ways of getting high school students interested in a degree in Biology at our school. Thus, we designed an outreach program to draw interested high school students to our campus and participate in a day-long outbreak investigation. The investigation is composed of six distinct activities, each taking between 15 min and 1 h of active time. These activities can be used in conjunction or individually to engage students with basic epidemiology and microbiology. The modules included in this recruitment event are outbreak interviews, DNA fingerprinting analysis, Gram staining, examination of microbial diagnostic tests, use of high-performance liquid chromatography to analyze toxins, and examination of potential food preparation contamination. Our first event was a success, with all participants reporting that they enjoyed their time at the University and found the faculty and staff helpful. One of the students even said, "I wish all school was like this." The goal of this event was to increase potential student interest and enrollment in our program. We hope that in sharing our experience here we can provide other instructors with a menu from which to pick and choose inexpensive, easy, and engaging activities for high school and introductory college students.

The PP2A/4/6 subfamily of phosphoprotein phosphatases regulates DAF-16 and confers resistance to environmental stress in postreproductive adult C. elegans.

Insulin and insulin-like growth factors are longevity determinants that negatively regulate Forkhead box class O (FoxO) transcription factors. In C. elegans mutations that constitutively activate DAF-16, the ortholog of mammalian FoxO3a, extend lifespan by two-fold. While environmental insults induce DAF-16 activity in younger animals, it also becomes activated in an age-dependent manner in the absence of stress, modulating gene expression well into late adulthood. The mechanism by which DAF-16 activity is regulated during aging has not been defined. Since phosphorylation of DAF-16 generally leads to its inhibition, we asked whether phosphatases might be necessary for its increased transcriptional activity in adult C. elegans. We focused on the PP2A/4/6 subfamily of phosphoprotein phosphatases, members of which had been implicated to regulate DAF-16 under low insulin signaling conditions but had not been investigated during aging in wildtype animals. Using reverse genetics, we functionally characterized all C. elegans orthologs of human catalytic, regulatory, and scaffolding subunits of PP2A/4/6 holoenzymes in postreproductive adults. We found that PP2A complex constituents PAA-1 and PPTR-1 regulate DAF-16 transcriptional activity during aging and that they cooperate with the catalytic subunit LET-92 to protect adult animals from ultraviolet radiation. PP4 complex members PPH-4.1/4.2, and SMK-1 also appear to regulate DAF-16 in an age-dependent manner, and together with PPFR-2 they contribute to innate immunity. Interestingly, SUR-6 but no other subunit of the PP2A complex was necessary for the survival of pathogen-infected animals. Finally, we found that PP6 complex constituents PPH-6 and SAPS-1 contribute to host defense during aging, apparently without affecting DAF-16 transcriptional activity. Our studies indicate that a set of PP2A/4/6 complexes protect adult C. elegans from environmental stress, thus preserving healthspan. Therefore, along with their functions in cell division and development, the PP2A/4/6 phosphatases also appear to play critical roles later in life.

Adaptive Engineering of Phytochelatin-based Heavy Metal Tolerance.

Metabolic engineering approaches are increasingly employed for environmental applications. Because phytochelatins (PC) protect plants from heavy metal toxicity, strategies directed at manipulating the biosynthesis of these peptides hold promise for the remediation of soils and groundwaters contaminated with heavy metals. Directed evolution of Arabidopsis thaliana phytochelatin synthase (AtPCS1) yields mutants that confer levels of cadmium tolerance and accumulation greater than expression of the wild-type enzyme in Saccharomyces cerevisiae, Arabidopsis, or Brassica juncea. Surprisingly, the AtPCS1 mutants that enhance cadmium tolerance and accumulation are catalytically less efficient than wild-type enzyme. Metabolite analyses indicate that transformation with AtPCS1, but not with the mutant variants, decreases the levels of the PC precursors, glutathione and γ-glutamylcysteine, upon exposure to cadmium. Selection of AtPCS1 variants with diminished catalytic activity alleviates depletion of these metabolites, which maintains redox homeostasis while supporting PC synthesis during cadmium exposure. These results emphasize the importance of metabolic context for pathway engineering and broaden the range of tools available for environmental remediation.

RNF4 and PLK1 are required for replication fork collapse in ATR-deficient cells.

The ATR-CHK1 axis stabilizes stalled replication forks and prevents their collapse into DNA double-strand breaks (DSBs). Here, we show that fork collapse in Atr-deleted cells is mediated through the combined effects the sumo targeted E3-ubiquitin ligase RNF4 and activation of the AURKA-PLK1 pathway. As indicated previously, Atr-deleted cells exhibited a decreased ability to restart DNA replication following fork stalling in comparison with control cells. However, suppression of RNF4, AURKA, or PLK1 returned the reinitiation of replication in Atr-deleted cells to near wild-type levels. In RNF4-depleted cells, this rescue directly correlated with the persistence of sumoylation of chromatin-bound factors. Notably, RNF4 repression substantially suppressed the accumulation of DSBs in ATR-deficient cells, and this decrease in breaks was enhanced by concomitant inhibition of PLK1. DSBs resulting from ATR inhibition were also observed to be dependent on the endonuclease scaffold protein SLX4, suggesting that RNF4 and PLK1 either help activate the SLX4 complex or make DNA replication fork structures accessible for subsequent SLX4-dependent cleavage. Thus, replication fork collapse following ATR inhibition is a multistep process that disrupts replisome function and permits cleavage of the replication fork.

Thiol-based regulation of redox-active glutamate-cysteine ligase from Arabidopsis thaliana.

Glutathione biosynthesis is a key component in the network of plant stress responses that counteract oxidative damage and maintain intracellular redox environment. Using a combination of mass spectrometry and site-directed mutagenesis, we examined the response of Arabidopsis thaliana glutamate-cysteine ligase (GCL) to changes in redox environment. Mass spectrometry identified two disulfide bonds (Cys186-Cys406 and Cys349-Cys364) in GCL. Mutation of either Cys-349 or Cys-364 to a Ser reduced reaction rate by twofold, but substitution of a Ser for either Cys-186 or Cys-406 decreased activity by 20-fold and abrogated the response to changes in redox environment. Redox titrations show that the regulatory disulfide bond has a midpoint potential comparable with other known redox-responsive plant proteins. Mutation of Cys-102, Cys-251, Cys-349, or Cys-364 did not alter the response to redox environment, indicating that modulation of activity depends on the Cys186-Cys406 disulfide bond. In vivo analysis of GCL in Arabidopsis root extracts revealed that multiple oxidative stresses altered the distribution of oxidized (active) and reduced (inactive) enzyme and that this change correlated with increased GCL activity. The thiol-based regulation of GCL provides a posttranslational mechanism for modulating enzyme activity in response to in vivo redox environment and suggests a role for oxidative signaling in the maintenance of glutathione homeostasis in plants.