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

Nuclear Genome Organization in Fungi: From Gene folding to Rabl Chromosomes

We discuss the current knowledge on the fungal genome organization, from the association of chromosomes within the nucleus to topological structures at individual genes and the genetic factors required for the hierarchical organization. Chromosome conformation capture followed by high-throughput sequencing (Hi-C) has elucidated how fungal genomes are globally organized in Rabl configuration where centromere or telomere bundles are associated with opposite faces of the nuclear envelope. Here, we explore the presence, in fungal taxa, of the typical proteins associated with genome organization in eukaryotes

The genome organization of <i>Neurospora crassa</i> at high resolution uncovers principles of fungal chromosome topology

AbstractThe eukaryotic genome must be precisely organized for its proper function, as genome topology impacts transcriptional regulation, cell division, replication, and repair, among other essential processes. Disruptions to human genome topology can lead to diseases, including cancer. The advent of chromosome conformation capture with high-throughput sequencing (Hi-C) to assess genome organization has revolutionized the study of nuclear genome topology; Hi-C has elucidated numerous genomic structures, including chromosomal territories, active/silent chromatin compartments, Topologically Associated Domains, and chromatin loops. While low-resolution heatmaps can provide important insights into chromosomal level contacts, high-resolution Hi-C datasets are required to reveal folding principles of individual genes. Of particular interest are high-resolution chromosome conformation datasets of organisms modeling the human genome. Here, we report the genome topology of the fungal model organism Neurospora crassaDpnMseDpnMs

Neurospora Importin α Is Required for Normal Heterochromatic Formation and DNA Methylation

<div><p>Heterochromatin and associated gene silencing processes play roles in development, genome defense, and chromosome function. In many species, constitutive heterochromatin is decorated with histone H3 tri-methylated at lysine 9 (H3K9me3) and cytosine methylation. In <i>Neurospora crassa</i>, a five-protein complex, DCDC, catalyzes H3K9 methylation, which then directs DNA methylation. Here, we identify and characterize a gene important for DCDC function, <i>dim-3</i> (<i>d</i>efective <i>i</i>n <i>m</i>ethylation-3), which encodes the nuclear import chaperone NUP-6 (Importin α). The critical mutation in <i>dim-3</i> results in a substitution in an ARM repeat of NUP-6 and causes a substantial loss of H3K9me3 and DNA methylation. Surprisingly, nuclear transport of all known proteins involved in histone and DNA methylation, as well as a canonical transport substrate, appear normal in <i>dim-3</i> strains. Interactions between DCDC members also appear normal, but the <i>nup-6dim-3</i> allele causes the DCDC members DIM-5 and DIM-7 to mislocalize from heterochromatin and NUP-6^<i>dim-3</i> itself is mislocalized from the nuclear envelope, at least in conidia. GCN-5, a member of the SAGA histone acetyltransferase complex, also shows altered localization in <i>dim-3</i>, raising the possibility that NUP-6 is necessary to localize multiple chromatin complexes following nucleocytoplasmic transport.</p></div