Replication Stress Shapes a Protective Chromatin Environment across Fragile Genomic Regions.

Recent integrative epigenome analyses highlight the importance of functionally distinct chromatin states for accurate cell function. How these states are established and maintained is a matter of intense investigation. Here, we present evidence for DNA damage as an unexpected means to shape a protective chromatin environment at regions of recurrent replication stress (RS). Upon aberrant fork stalling, DNA damage signaling and concomitant H2AX phosphorylation coordinate the FACT-dependent deposition of macroH2A1.2, a histone variant that promotes DNA repair by homologous recombination (HR). MacroH2A1.2, in turn, facilitates the accumulation of the tumor suppressor and HR effector BRCA1 at replication forks to protect from RS-induced DNA damage. Consequently, replicating primary cells steadily accrue macroH2A1.2 at fragile regions, whereas macroH2A1.2 loss in these cells triggers DNA damage signaling-dependent senescence, a hallmark of RS. Altogether, our findings demonstrate that recurrent DNA damage contributes to the chromatin landscape to ensure the epigenomic integrity of dividing cells.

Mutational analysis of the MS2 lysis protein L.

Small single-stranded nucleic acid phages effect lysis by expressing a single protein, the amurin, lacking muralytic enzymatic activity. Three amurins have been shown to act like 'protein antibiotics' by inhibiting cell-wall biosynthesis. However, the L lysis protein of the canonical ssRNA phage MS2, a 75 aa polypeptide, causes lysis by an unknown mechanism without affecting net peptidoglycan synthesis. To identify residues important for lytic function, randomly mutagenized alleles of L were generated, cloned into an inducible plasmid and the transformants were selected on agar containing the inducer. From a total of 396 clones, 67 were unique single base-pair changes that rendered L non-functional, of which 44 were missense mutants and 23 were nonsense mutants. Most of the non-functional missense alleles that accumulated in levels comparable to the wild-type allele are localized in the C-terminal half of L, clustered in and around an LS dipeptide sequence. The LS motif was used to align L genes from ssRNA phages lacking any sequence similarity to MS2 or to each other. This alignment revealed a conserved domain structure, in terms of charge, hydrophobic character and predicted helical content. None of the missense mutants affected membrane-association of L. Several of the L mutations in the central domains were highly conservative and recessive, suggesting a defect in a heterotypic protein-protein interaction, rather than in direct disruption of the bilayer structure, as had been previously proposed for L.

Analytical Ultracentrifugation (AUC): An Overview of the Application of Fluorescence and Absorbance AUC to the Study of Biological Macromolecules.

The biochemical and biophysical investigation of proteins, nucleic acids, and the assemblies that they form yields essential information to understand complex systems. Analytical ultracentrifugation (AUC) represents a broadly applicable and information-rich method for investigating macromolecular characteristics such as size, shape, stoichiometry, and binding properties, all in the true solution-state environment that is lacking in most orthogonal methods. Despite this, AUC remains underutilized relative to its capabilities and potential in the fields of biochemistry and molecular biology. Although there has been a rapid development of computing power and AUC analysis tools in this millennium, fewer advancements have occurred in development of new applications of the technique, leaving these powerful instruments underappreciated and underused in many research institutes. With AUC previously limited to absorbance and Rayleigh interference optics, the addition of fluorescence detection systems has greatly enhanced the applicability of AUC to macromolecular systems that are traditionally difficult to characterize. This overview provides a resource for novices, highlighting the potential of AUC and encouraging its use in their research, as well as for current users, who may benefit from our experience. We discuss the strengths of fluorescence-detected AUC and demonstrate the power of even simple AUC experiments to answer practical and fundamental questions about biophysical properties of macromolecular assemblies. We address the development and utility of AUC, explore experimental design considerations, present case studies investigating properties of biological macromolecules that are of common interest to researchers, and review popular analysis approaches. © 2020 The Authors.

Complete Genome Sequence of Citrobacter freundii Myophage Moon.

Citrobacter freundii and other Gram-negative opportunistic pathogens necessitate concern from the public health sector. Bacteriophages that kill such pathogens may be useful in the control and containment of these infections. Here, we describe the genome of a newly isolated T4-like myophage of C. freundii, Moon, and present its features.