The hereditary disease Cockayne syndrome (CS) is characterized by a complex clinical phenotype. CS cells are abnormally sensitive to ultraviolet radiation and are defective in the repair of transcriptionally active genes. The cloned CSB gene encodes a member of a protein family that includes the yeast Snf2 protein, a component of the transcriptional regulator Swi/Snf. We report the cloning of the CSA cDNA, which can encode a WD repeat protein. Mutations in the cDNA have been identified in CS-A cell lines. CSA protein interacts with CSB protein and with p44 protein, a subunit of the human RNA polymerase II transcription factor IIH. These observations suggest that the products of the CSA and CSB genes are involved in transcription.

We have constructed a strain of Saccharomyces cerevisiae with a deletion of the YKL510 open reading frame, which was initially identified in chromosome XI as a homolog of the RAD2 nucleotide excision repair gene (A. Jacquier, P. Legrain, and B. Dujon, Yeast 8:121-132, 1992). The mutant strain exhibits increased sensitivity to UV light and to the alkylating agent methylmethane sulfonate but not to ionizing radiation. We have renamed the YKL510 open reading frame the RAD27 gene, in keeping with the accepted nomenclature for radiationsensitive yeast mutants. Epistasis analysis indicates that the gene is in the RAD6 group of genes, which are involved in DNA damage tolerance. The mutant strain also exhibits increased plasmid loss, increased spontaneous mutagenesis, and a temperature-sensitive lethality whose phenotype suggests a defect in DNA replication. Levels of the RAD27 gene transcript are cell cycle regulated in a manner similar to those for several other genes whose products are known to be involved in DNA replication. We discuss the possible role of Rad27 protein in DNA repair and replication.

The human basal transcription factor TFIIH plays a central role in two distinct processes. TFIIH is an obligatory component of the RNA polymerase II (RNAP II) transcription initiation complex. Additionally, it is believed to be the core structure around which some if not all the components of the nucleotide excision repair (NER) machinery assemble to constitute a nucleotide excision repairosome. At least two of the subunits of TFIIH (XPB and XPD proteins) are implicated in the disease xeroderma pigmentosum (XP). We have exploited the availability of the cloned XPB, XPD, p62, p44, and p34 genes (all of which encode polypeptide subunits of TFIIH) to examine interactions between in vitro-translated polypeptides by co-immunoprecipitation. Additionally we have examined interactions between TFIIH components, the human NER protein XPG, and the CSB protein which is implicated in Cockayne syndrome (CS). Our analyses demonstrate that the XPB, XPD, p44, and p62 proteins interact with each other. XPG protein interacts with multiple subunits of TFIIH and with CSB protein.

Photofootprinting in vivo ofGALl reveals an activation-dependent pattern between the UASG and the TATA box, in a sequence not required for transcriptional activation by GAL4. The pattern results from a nucleosome whose position depends on sequences within the UASG. In the wild-type gene, activation by GAL4 and derivatives disrupts this nucleosome. This activity is independent of interactions with DNA-bound core transcription factors and is proportional to the strength of the activator. Presence of the nucleosome correlates with low basal transcription levels under various conditions, suggesting a role in limiting basal expression. We propose a role for the GAL4 activation domain in displacing a nucleosome and suggest that this is part of the mechanism by which GAL4 activates transcription in vivo.

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