A novel 15-subunit complex with the capacity to remodel the structure of chromatin, termed RSC, has been isolated from S. cerevisiae on the basis of homology to the SWI/SNF complex. At least three RSC subunits are related to SWI/SNF polypeptides: Sth1p, Rsc6p, and Rsc8p are significantly similar to Swi2/Snf2p, Swp73p, and Swi3p, respectively, and were identified by mass spectrometric and sequence analysis of peptide fragments. Like SWI/SNF, RSC exhibits a DNA-dependent ATPase activity stimulated by both free and nucleosomal DNA and a capacity to perturb nucleosome structure. RSC is, however, at least 10-fold more abundant than SWI/SNF complex and is essential for mitotic growth. Contrary to a report for SWII/SNF complex, no association of RSC (nor of SWI/SNF complex) with RNA polymerase II holoenzyme was detected.

The failure of cells to repair damaged DNA can result in genomic instability and cancer. To efficiently repair chromosomal DNA lesions, the repair machinery must gain access to the damaged DNA in the context of chromatin. Here we report that both the RSC and Swi/Snf ATP-dependent chromatin-remodeling complexes play key roles in double-strand break (DSB) repair, specifically by homologous recombination (HR). RSC and Swi/ Snf are each recruited to an in vivo DSB site but with distinct kinetics. We show that Swi/Snf is required earlier, at or preceding the strand invasion step of HR, while RSC is required following synapsis for completion of the recombinational repair event.Supplemental material is available at http://www.genesdev.org.

(7)(8)(9)(10), the GALl gene (which encodes galactokinase) (7), the ADH2 gene (which encodes alcohol dehydrogenase) (7,11,12), and Ty elements (13,14). These SWI and SNF proteins do not appear to contain any DNA-binding motifs, nor have these proteins exhibited any DNA-binding activity in vitro (7,9,15). SWI2/SNF2, however, contains a domain homologous to certain DNA-dependent ATPases, and a recombinant form of this domain exhibits DNAdependent ATPase activity (16)(17)(18). Genetic data suggest that these SWI and SNF proteins form a complex that is recruited to promoter regions through interactions with gene-specific DNA-binding proteins (7,9,19 Hepes-KOH, pH 7.6/20% glycerol/l mM dithiothreitol/l mM. EDTA/2 ,ug of chymostatin per ml/2 puM pepstatin A/0.6 pM leupeptin/2 mM benzamidine/1 mM phenylmethylsulfonyl fluoride/0.01% Nonidet P-40). The purification was monitored by imiunoblot analysis with antisera to SWI1/ADR6, SWI2/SNF2, and SWI3 proteins.Preparation of Recombinant SNF6 Protein. The SNF6 gene was transferred from the plasmid YCp5O-SNF6 to the Escherichia coli expression vector pETliA (Novagen) and tagged with six histidine residues at its carboxyl terminus by means of a polymerase chain reaction with the oligonucleotide primers BCSNF6N (5'-CCCCAGATCTTACATATGGGT-GTCATCAAGAAGAAA-3') and BCSNF6C (5'-CCCG-GATCCTCTAGACTAGTGATGGTGATGGTGATGTC-CAAAAAATACAGCATCAAGATCTCC-3'). The resulting product was digested with Nde I and BamHI and ligated to complementary sites in pETilA to form pET11A-SNF6HT. BL21 (DE3) cells (Novagen) containing the lysozyme-expressing plasmid pLysS (Novagen) were transformed with pET11A-SNF6HT. The transformant was grown at 300C in LB medium (2 liters) containing 100 pRg of ampicillin per ml and 25 ptg of chloramphenicol per ml to an optical density at 600 nm of 0.7. The culture was induced with 0.5 mM isopropyl P-D-thiogalactoside and grown an additional 2 hr.The cells were harvested by centrifugation at 5000 x g for 10 min, washed with 100 ml of 20 mM Hepes (pH 7.5), and suspended in 100 ml of buffer B (20 mM Hepes, pH 7.5/10% glycerol/5 mM 2-mercaptoethanol/l mM EDTA/2 pg of chymostatin per ml/2 pM pepstatin A/0.6 pM leupeptin/2 mM benzamidine/1 mM phenylmethylsulfonyl fluoride), containing 400 mM NaCl. The suspension was lysed by sonication at 40C and centrifuged at 10,000 x g for 10 min.The pellet, containing the majority of SNF6HT protein, was suspended in 10 ml of buffer-B containing 0.5% Triton X-100 and 1 M lithum chloride, incubated at 40C for 10 min, sonicated briefly, and centrifuged at 10,000 x g for 10 m' at 4°C. The pellet was washed three additional times in the same manner and suspended in 1 ml of buffer B containing 50 mM NaCl and 0.5% SDS. The resulting SNF6HT protein was judged to be roughly 95% pure by SDS/PAGE.Antisera. Approximately 4 mg of purified SNF6HT protein was subjected to electrophoresis in a 10% SDS/polyacrylamide gel. The gel was stained with Coomassie brilliant blue R-250, and a slice containing SNF6HT protein was removed. Rabbits were initially immuni...

The yeast SNF2 (SWI2) protein functions with SNF5, SNF6, SWI1, and SWI3 in the transcriptional activation of many differently regulated genes. These proteins appear to facilitate activation by gene-specific regulatory proteins. SNF2 is highly conserved among eukaryotes and defines a family of proteins with similarity to helicases and nucleic acid-dependent NTPases. Here, we present genetic and biochemical evidence that SNF2 has DNA-stimulated ATPase activity. Mutations in the nucleoside triphosphate (NTP)-binding motif and other conserved motifs impair SNF2 function. Swapping experiments with another member of this family indicate that the helicase-related domains are functionally interchangeable. Finally, bacterially expressed SNF2 protein has ATPase activity that is stimulated by double-stranded DNA, and mutation of the NTP-binding site abolishes this activity. Deletion analysis shows that the helicase-like region of SNF2 is necessary, but not sufficient, for transcriptional activation. In the yeast Saccharomyces cerevisiae, the SNF2/ SWI2, SNFS, SNF6, SWI1, and SWI3 proteins function in a related fashion in transcription of many diversely regulated genes (see Peterson and Herskowitz 1992). A role in transcriptional activation was indicated by evidence that LexA-SNF2, LexA-SNF5, and LexA-SNF6 hybrid proteins activate expression of a target gene when bound to DNA (Laurent et al. 1990(Laurent et al. , 1991