Cryptic protein interactions regulate DNA replication initiation

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/147744/1/mmi14142_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/147744/2/mmi14142-sup-0001-SupInfo.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/147744/3/mmi14142.pd

DdcA antagonizes a bacterial DNA damage checkpoint

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/147820/1/mmi14151.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/147820/2/mmi14151_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/147820/3/mmi14151-sup-0001-Supinfo.pd

Addressing the Requirements of High‐Sensitivity Single‐Molecule Imaging of Low‐Copy‐Number Proteins in Bacteria

Single‐molecule fluorescence super‐resolution imaging and tracking provide nanometer‐scale information about subcellular protein positions and dynamics. These single‐molecule imaging experiments can be very powerful, but they are best suited to high‐copy number proteins where many measurements can be made sequentially in each cell. We describe artifacts associated with the challenge of imaging a protein expressed in only a few copies per cell. We image live Bacillus subtilis in a fluorescence microscope, and demonstrate that under standard single‐molecule imaging conditions, unlabeled B. subtilis cells display punctate red fluorescent spots indistinguishable from the few PAmCherry fluorescent protein single molecules under investigation. All Bacillus species investigated were strongly affected by this artifact, whereas we did not find a significant number of these background sources in two other species we investigated, Enterococcus faecalis and Escherichia coli. With single‐molecule resolution, we characterize the number, spatial distribution, and intensities of these impurity spots.Bright spots: A single‐molecule‐like fluorescent background signal is reported in Bacillus subtilis cells, and the density and fluorescence intensity of these spots are quantified in several Bacillus species and other Gram‐negative and Gram‐positive organisms.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/144710/1/cphc201600035_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/144710/2/cphc201600035.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/144710/3/cphc201600035-sup-0001-misc_information.pd

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution of existing proteins, and the assembly of new protein complexes can be stimulated by a variety of DNA lesions and mismatched DNA base pairs. Fluorescence microscopy has been used as a powerful experimental tool for visualizing and quantifying these and other responses to DNA lesions and to monitor DNA replication status within the complex subcellular architecture of a living cell. Translational fusions between fluorescent reporter proteins and components of the DNA replication and repair machinery have been used to determine the cues that target DNA repair proteins to their cognate lesions in vivo and to understand how these proteins are organized within bacterial cells. In addition, transcriptional and translational fusions linked to DNA damage inducible promoters have revealed which cells within a population have activated genotoxic stress responses. In this review, we provide a detailed protocol for using fluorescence microscopy to image the assembly of DNA repair and DNA replication complexes in single bacterial cells. In particular, this work focuses on imaging mismatch repair proteins, homologous recombination, DNA replication and an SOS-inducible protein in Bacillus subtilis. All of the procedures described here are easily amenable for imaging protein complexes in a variety of bacterial species

Mismatch repair causes the dynamic release of an essential DNA polymerase from the replication fork

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/86887/1/MMI_7841_sm_SuppInfor.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/86887/2/j.1365-2958.2011.07841.x.pd

Structure of the Endonuclease Domain of MutL: Unlicensed to Cut

DNA mismatch repair corrects errors that have escaped polymerase proofreading, increasing replication fidelity 100- to 1000-fold in organisms ranging from bacteria to humans. The MutL protein plays a central role in mismatch repair by coordinating multiple protein-protein interactions that signal strand removal upon mismatch recognition by MutS. Here we report the crystal structure of the endonuclease domain of Bacillus subtilis MutL. The structure is organized in dimerization and regulatory subdomains connected by a helical lever spanning the conserved endonuclease motif. Additional conserved motifs cluster around the lever and define a Zn2+-binding site that is critical for MutL function in vivo. The structure unveils a powerful inhibitory mechanism to prevent undesired nicking of newly replicated DNA and allows us to propose a model describing how the interaction with MutS and the processivity clamp could license the endonuclease activity of MutL. The structure also provides a molecular framework to propose and test additional roles of MutL in mismatch repair.American Cancer Society (Research Professor)Natural Sciences and Engineering Research Council of Canada (NSERC scholarship)National Institutes of Health (U.S.) (CA21615)National Institutes of Health (U.S.) (GM45190)Natural Sciences and Engineering Research Council of Canada (NSERC, 288295)Deutsche Forschungsgemeinschaft (FR-1495/4-1)University of Michigan (Start-up funds

Tidal Dissipation in the Early Eocene and Implications for Ocean Mixing

The tidally driven vertical diffusivity in the abyssal ocean during the early Eocene (55 Ma) is investigated using an established tidal model. A weak tide is predicted in the Eocene ocean, except in the Pacific. Consequently, the integrated global tidal dissipation rate is a mere 1.44TW, of which 40% dissipate in the Pacific. However, due to a stronger abyssal vertical stratification the predicted Eocene vertical diffusivities are consistently larger than at present. The results support the hypothesis that altered tidal dissipation may play a role in explaining the maintenance of past climate regimes, especially the anomalously warm temperatures in the southwest Pacific in the Eocene, and the low dissipation rates may be important for lunar evolution history

DNA Damage and Reactive Nitrogen Species are Barriers to Vibrio cholerae Colonization of the Infant Mouse Intestine

Ingested Vibrio cholerae pass through the stomach and colonize the small intestines of its host. Here, we show that V. cholerae requires at least two types of DNA repair systems to efficiently compete for colonization of the infant mouse intestine. These results show that V. cholerae experiences increased DNA damage in the murine gastrointestinal tract. Agreeing with this, we show that passage through the murine gut increases the mutation frequency of V. cholerae compared to liquid culture passage. Our genetic analysis identifies known and novel defense enzymes required for detoxifying reactive nitrogen species (but not reactive oxygen species) that are also required for V. cholerae to efficiently colonize the infant mouse intestine, pointing to reactive nitrogen species as the potential cause of DNA damage. We demonstrate that potential reactive nitrogen species deleterious for V. cholerae are not generated by host inducible nitric oxide synthase (iNOS) activity and instead may be derived from acidified nitrite in the stomach. Agreeing with this hypothesis, we show that strains deficient in DNA repair or reactive nitrogen species defense that are defective in intestinal colonization have decreased growth or increased mutation frequency in acidified nitrite containing media. Moreover, we demonstrate that neutralizing stomach acid rescues the colonization defect of the DNA repair and reactive nitrogen species defense defective mutants suggesting a common defense pathway for these mutants

Non-Linear Neuronal Responses as an Emergent Property of Afferent Networks: A Case Study of the Locust Lobula Giant Movement Detector

In principle it appears advantageous for single neurons to perform non-linear operations. Indeed it has been reported that some neurons show signatures of such operations in their electrophysiological response. A particular case in point is the Lobula Giant Movement Detector (LGMD) neuron of the locust, which is reported to locally perform a functional multiplication. Given the wide ramifications of this suggestion with respect to our understanding of neuronal computations, it is essential that this interpretation of the LGMD as a local multiplication unit is thoroughly tested. Here we evaluate an alternative model that tests the hypothesis that the non-linear responses of the LGMD neuron emerge from the interactions of many neurons in the opto-motor processing structure of the locust. We show, by exposing our model to standard LGMD stimulation protocols, that the properties of the LGMD that were seen as a hallmark of local non-linear operations can be explained as emerging from the dynamics of the pre-synaptic network. Moreover, we demonstrate that these properties strongly depend on the details of the synaptic projections from the medulla to the LGMD. From these observations we deduce a number of testable predictions. To assess the real-time properties of our model we applied it to a high-speed robot. These robot results show that our model of the locust opto-motor system is able to reliably stabilize the movement trajectory of the robot and can robustly support collision avoidance. In addition, these behavioural experiments suggest that the emergent non-linear responses of the LGMD neuron enhance the system's collision detection acuity. We show how all reported properties of this neuron are consistently reproduced by this alternative model, and how they emerge from the overall opto-motor processing structure of the locust. Hence, our results propose an alternative view on neuronal computation that emphasizes the network properties as opposed to the local transformations that can be performed by single neurons