We investigated the role of chromatin in the catalysis of homologous strand pairing by Rad54 and Rad51. Rad54 is related to the ATPase subunits of chromatin-remodeling factors, whereas Rad51 is related to bacterial RecA. In the absence of superhelical tension, we found that the efficiency of strand pairing with chromatin is >100-fold higher than that with naked DNA. In addition, we observed that Rad54 and Rad51 function cooperatively in the ATP-dependent remodeling of chromatin. These findings indicate that Rad54 and Rad51 have evolved to function with chromatin, the natural substrate, rather than with naked DNA. Received August 13, 2002; revised version accepted September 5, 2002. In the eukaryotic nucleus, chromatin is an integral component of processes that use DNA. The packaging of DNA into chromatin is essential for the compaction and organization of nuclear DNA, but it also influences the functions of factors that interact with DNA. For instance, chromatin represses the basal transcription process, and sequence-specific DNA-binding activators along with coactivators (which include chromatin-remodeling factors and histone-modifying proteins) function to counteract chromatin-mediated transcriptional repression. Because chromatin is the natural substrate for DNA-using processes in the nucleus, it will ultimately be necessary to understand how chromatin affects each of these phenomena. To this end, we have undertaken a biochemical analysis of homologous recombination in chromatin.Homologous recombination occurs in the repair of DNA double-strand breaks as well as during meiosis. Genetic studies in Saccharomyces cerevisiae led to the identification of the RAD52 epistasis group of genes (which includes RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, MRE11, and XRS2) as components of the recombinational repair pathway (Game 1983;Petrini et al. 1997;Kanaar et al. 1998;Paques and Haber 1999;Cromie et al. 2001;Masson and West 2001). These genes are conserved from yeast to humans. A central protein in this pathway is Rad51, which is related to the bacterial RecA protein. Both Rad51 and RecA are able to mediate strand invasion and annealing to yield a D loop, which is a key step in the recombination process.In this reaction, Rad51 (or RecA) forms a nucleoprotein filament on single-stranded DNA in the presence of ATP, and this filament is used for homologous pairing with a double-stranded DNA molecule. The efficiency of strand pairing by Rad51 (between single-stranded DNA and homologous duplex DNA) has been shown to be stimulated by the presence of additional factors such as RP-A (Sugiyama et al. 1997;Baumann and West 1999), the Rad55-Rad57 heterodimer (Sung 1997a), Rad52 (Sung 1997b;Benson et al. 1998;New et al. 1998; Shinohara and Ogawa 1998), and Rad54 (Petukhova et al. 1998(Petukhova et al. , 1999 Mazin et al. 2000a,b;Van Komen et al. 2000).To study homologous recombination in the context of chromatin, we focused on the ability of purified recombinant Rad51 and Rad54 to catalyze D-loop formation between single...

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DNA replication is initiated by binding of initiation factors to the origin of replication. Nucleosomes are known to inhibit the access of the replication machinery to origin sequences. Recently, nucleosome remodelling factors have been identified that increase the accessibility of nucleosomal DNA to transcription regulators. To test whether the initiation of DNA replication from an origin covered by nucleosomes would also benefit from the action of nucleosome remodelling factors, we reconstituted SV40 DNA into chromatin in Drosophila embryo extracts. In the presence of T-antigen and ATP, a chromatin-associated cofactor allowed efficient replication from a nucleosomal origin in vitro. In search of the energy-dependent cofactor responsible we found that purified 'chromatin accessibility complex' (CHRAC) was able to alter the nucleosomal structure at the origin allowing the binding of T-antigen and efficient initiation of replication. These experiments provide evidence for the involvement of a nucleosome remodelling machine in structural changes at the SV40 origin of DNA replication in vitro.

RNA interference (RNAi) is implicated in maintaining tandem DNA arrays as constitutive heterochromatin. We used chromatin immunoprecipitation with antibodies to RNA polymerase II (RNAPol-ChIP) to test for transcription of the following repeat arrays in human cells: subtelomeric D4Z4, pericentromeric satellite 2, and centromeric satellite alpha. D4Z4 has a promoter-like sequence upstream of an ORF in its 3.3-kb repeat unit. A short D4Z4 array at 4q35 is linked to facioscapulohumeral muscular dystrophy (FSHD). By RNAPol-ChIP and RT-PCR, little or no transcription of D4Z4 was detected in FSHD and normal myoblasts; lymphoblasts from an FSHD patient, a control, and a patient with D4Z4 hypomethylation due to mutation of DNMT3B (ICF syndrome); and normal or cancer tissues. However, RNAPol-ChIP assays indicated transcription of D4Z4 in a chromosome 4-containing human-mouse somatic cell hybrid. ChIP and RT-PCR showed satellite DNA transcription in some cancers and lymphoblastoid cell lines, although only at a low level. Given the evidence for the involvement of RNAi in satellite DNA heterochromatinization, it is surprising that, at most, a very small fraction of satellite DNA was associated with RNA Pol II. In addition, our results do not support the previously hypothesized disease-linked differential transcription of D4Z4 sequences in short, FSHD-linked arrays.

The Swi2/Snf2-related protein Rad54 is a chromatin remodeling enzyme that is important for homologous strand pairing catalyzed by the eukaryotic recombinase Rad51. The chromatin remodeling and DNA-stimulated ATPase activities of Rad54 are significantly enhanced by Rad51. To investigate the functions of Rad54, we generated and analyzed a series of mutant Rad54 proteins. Notably, the deletion of an N-terminal motif (amino acid residues 2-9), which is identical in Rad54 in Drosophila, mice, and humans, results in a complete loss of chromatin remodeling and strand pairing activities, and partial inhibition of the ATPase activity. In contrast, this conserved N-terminal motif has no apparent effect on the ability of DNA to stimulate the ATPase activity or of Rad51 to enhance the DNA-stimulated ATPase activity. Unexpectedly, as the N terminus of Rad54 is progressively truncated, the mutant proteins regain partial chromatin remodeling activity as well as essentially complete DNAstimulated ATPase activity, both of which are no longer responsive to Rad51. These findings suggest that the Nterminal region of Rad54 contains an autoinhibitory activity that is relieved by Rad51.Chromatin is the natural form of the DNA substrate in a variety of processes that include transcription, replication, repair, and recombination. The participation of chromatin in these processes involves dynamic changes in its structure. There are two key strategies by which the structure of chromatin is altered. First, the components of chromatin are covalently modified. For instance, the DNA is methylated, such as at CpG residues, and the histones are acetylated, methylated, phosphorylated, ubiquitylated, and ADP-ribosylated (for reviews, see Refs. 1-4). Second, the structure of chromatin is reconfigured by ATP-utilizing chromatin remodeling factors, which disrupt histone-DNA contacts in the nucleosome and catalyze nucleosome mobility (for reviews, see Refs. 5-11).Homologous strand pairing is a key step in the repair of DNA double-strand breaks by homologous recombination (for reviews, see . In this reaction, the eukaryotic recombinase Rad51 assembles onto single-stranded DNA (which is generated at the site of a double-strand break) to give a nucleoprotein filament that, upon invasion and annealing to homologous double-stranded DNA, yields a structure termed a D loop. Rad51 belongs to the Rad52 epistasis group of genes (including Rad50, Rad51, Rad52, Rad54, Rad55, Rad57, Rad59, MRE11, and XRS2). These genes, which are conserved from yeast to humans, have been shown to be important for double-strand break repair by homologous recombination.In this study, we focus upon the Rad54 protein. Rad54 is a member of the Rad52 group of genes. In addition, the Rad54 protein is in the Snf2-like family of DNA-dependent ATPases (20), which includes the ATPase subunits of a variety of ATPdependent chromatin remodeling factors such as SWI/SNF, 1 RSC, NURF, ACF, CHRAC, INO80.COM, SWR1, NuRD, RSF, and others. Rad54 has been found to stimulate homologous strand pairing ...