The focal accumulation of DNA repair factors, including the MRE11/Rad50/NBS1 (MRN) complex and the phosphohistone variant gamma-H2A.X, is a key cytological feature of the DNA damage response (DDR). Although these foci have been extensively studied by light microscopy, there is comparatively little known regarding their ultrastructure. Using correlative light microscopy and electron spectroscopic imaging (LM/ESI) we have characterised the ultrastructure of chromatin and DNA repair foci within the nuclei of normal human fibroblasts in response to DNA double-strand breaks (DSBs). The induction of DNA DSBs by etoposide leads to a global decrease in chromatin density, which is accompanied by the formation of invaginations of the nuclear envelope as revealed by live-cell microscopy. Using LM/ESI and the immunogold localisation of gamma-H2A.X and MRE11 within repair foci, we also observed decondensed 10 nm chromatin fibres within repair foci and the accumulation of large non-chromosomal protein complexes over three hours recovery from etoposide. At 18 h after etoposide treatment, we observed a close juxtapositioning of PML nuclear bodies and late repair foci of gamma-H2A.X, which exhibited a highly organised chromatin arrangement distinct from earlier repair foci. Finally, the dual immunogold labelling of MRE11 with either gamma-H2A.X or NBS1 revealed that gamma-H2A.X and the MRN complex are sub-compartmentalised within repair foci at the sub-micron scale. Together these data provide the first ultrastructural comparison of gamma-H2A.X and MRN DNA repair foci, which are structurally dynamic over time and strikingly similar in organisation.

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Transcription by RNA polymerase II is impeded by the nucleosomal organization of DNA; these negative effects are modulated at several stages of nucleosomal DNA transcription by FACT, a heterodimeric transcription factor. At promoters, FACT facilitates the binding of TATA-binding factor, while during transcription elongation FACT mediates the necessary destabilization of nucleosomes and subsequent restoration of nucleosome structure in the wake of the transcription elongation complex. Altered FACT activity can impair the fidelity of transcription initiation and affect transcription patterns. Using reporter genes we have identified new mutant versions of the Spt16 subunit of yeast FACT with dominant negative effects on the fidelity of transcription initiation. Two of these spt16 mutant alleles also affect cell integrity. Cells relying on these spt16 mutant alleles display sorbitol-remediated temperature sensitivity, altered sensitivity to detergent, and abnormal morphologies, and are further inhibited by the ssd1-d mutation. The overexpression of components of protein kinase C (Pkc1) signaling diminishes this spt16 ssd1-d temperature sensitivity, whereas gene deletions eliminating components of Pkc1 signaling further impair these spt16 mutant cells. Thus, the FACT subunit Spt16 and Pkc1 signaling have an overlapping essential function, with an unexpected role for FACT in the maintenance of cell integrity.

Twelve tetra-and di-tetra compound microsatellite loci are described for the rainbow smelt ( Osmerus mordax ). These loci were screened among a minimum of 79 individuals and the average number of alleles per locus was 16 with an average heterozygosity of 0.86. These microsatellites are being used to examine population structure and life-history evolution among natural populations of rainbow smelt.