SUMMARY Rhabdomyosarcoma is a soft-tissue sarcoma with molecular and cellular features of developing skeletal muscle. Rhabdomyosarcoma has two major histological subtypes, embryonal and alveolar, each with distinct clinical, molecular, and genetic features. Genomic analysis show that embryonal tumors have more structural and copy number variations than alveolar tumors. Mutations in the RAS/NF1 pathway are significantly associated with intermediate- and high-risk embryonal rhabdomyosarcomas (ERMS). In contrast, alveolar rhabdomyosarcoma (ARMS) have fewer genetic lesions overall and no known recurrently mutated cancer consensus genes. To identify therapeutics for ERMS, we developed and characterized orthotopic xenografts of tumors that were sequenced in our study. High throughput screening of primary cultures derived from those xenografts identified oxidative stress as a pathway of therapeutic relevance for ERMS.

The Saccharomyces cerevisiae kinase Bur1 is involved in coupling transcription elongation to chromatin modification, but not all important Bur1 targets in the elongation complex are known. Using a chemical genetics strategy wherein Bur1 kinase was engineered to be regulated by a specific inhibitor, we found that Bur1 phosphorylates the Spt5 C-terminal repeat domain (CTD) both in vivo and in isolated elongation complexes in vitro. Deletion of the Spt5 CTD or mutation of the Spt5 serines targeted by Bur1 reduces recruitment of the PAF complex, which functions to recruit factors involved in chromatin modification and mRNA maturation to elongating polymerase II (Pol II). Deletion of the Spt5 CTD showed the same defect in PAF recruitment as rapid inhibition of Bur1 kinase activity, and this Spt5 mutation led to a decrease in histone H3K4 trimethylation. Brief inhibition of Bur1 kinase activity in vivo also led to a significant decrease in phosphorylation of the Pol II CTD at Ser-2, showing that Bur1 also contributes to Pol II Ser-2 phosphorylation. Genetic results suggest that Bur1 is essential for growth because it targets multiple factors that play distinct roles in transcription.

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We have identified a terminator for transcription by RNA polymerase I in the genes coding for rRNA of the yeast Saccharomyces cerevisiae. The terminator is located 108 bp downstream of the 3' end of the mature 25S rRNA and shares several characteristics with previously studied polymerase I terminators in the vertebrates.For example, the yeast terminator is orientation dependent, is inhibited by its own sequence, and forms RNA 3' ends 17 2 bp upstream of an essential protein binding site. The recognition sequence for binding of the previously cloned REB1 protein (Q. Ju, B. E. Morrow, and J. R. Warner, Mol. Cell. Biol. 10:5226-5234, 1990) is an essential component of the terminator. In addition, the efficiency of termination depends upon sequence context extending at least 12 bp upstream of the REB1 site.Studies with both mouse and frog systems have shown that termination of transcription by RNA polymerase I requires the binding of a termination protein to a specific sequence in the DNA template. In mice, the protein has been called TTF1, and its specific binding site is an 18-bp-long element called the Sal box (10) (the element fortuitously contains an SalI restriction site). In frogs, the binding protein has been christened Rib2 (21) and it binds to the site GACTTGCNC (19). In both systems, the terminator sequence will only cause termination when situated in the normal orientation and ceases to function when reversed. Partially purified TllF1 stimulates termination when added to a crude extract, and termination is abolished when an excess of an oligonucleotide containing the Sal box sequence is added to the reaction. Likewise, partially purified Rib2 causes specific DNase I footprints over the terminator element, and both footprinting and termination are abolished by competition with oligonucleotides containing the terminator sequence. Further progress in understanding the mechanism of termination has been hampered by the inability so far to completely purify either Tl F1 or Rib2 or to clone the relevant cDNAs coding for these proteins.In the hope that we could circumvent the problems of the vertebrate systems, we have begun to study termination by RNA polymerase I in the yeast Saccharomyces cerevisiae. The yeasts offer at least two potential advantages over the vertebrates: first, it is easier to obtain the large amounts of extract needed for biochemical purification, and second, the possibility of using the well-developed genetics of yeasts to supplement or replace biochemical approaches exists. The recent development of yeast extracts capable of supporting accurate initiation by RNA polymerase I in vitro (20,27,28) also offers the opportunity to develop in vitro assays for both DNA and protein elements involved in termination.In this paper, we show that the yeast whole-cell extract developed by Schultz et al. (28) is capable of supporting RNA 3'-end formation of RNA polymerase I transcripts. One of these sites has features similar to the previously characterized vertebrate polymerase I terminators. Further...

Insertion and deletion of small heteroduplex loops are common mutations in DNA, but why some loops are prone to mutation and others are efficiently repaired is unknown. Here we report that the mismatch recognition complex, MSH2/MSH3, discriminates between a repair-competent and a repair-resistant loop by sensing the conformational dynamics of their junctions. MSH2/MSH3 binds, bends, and dissociates from repair-competent loops to signal downstream repair. Repair-resistant Cytosine-Adenine-Guanine (CAG) loops adopt a unique DNA junction that traps nucleotide-bound MSH2/MSH3, and inhibits its dissociation from the DNA. We envision that junction dynamics is an active participant and a conformational regulator of repair signaling, and governs whether a loop is removed by MSH2/MSH3 or escapes to become a precursor for mutation.