A Conserved N-terminal Motif in Rad54 Is Important for Chromatin Remodeling and Homologous Strand Pairing

Abstract

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 ...

Synergistic action of the Saccharomyces cerevisiae homologous recombination factors Rad54 and Rad51 in chromatin remodeling

“…Importantly, we and others have presented evidence that the flexible N-terminus of Rad54 harbors the Rad51 interaction domain [28,29]. We have shown here that the histone H3 tail binding domain likewise resides within the N-terminus of Rad54.…”

“…The promotion of Rad51-mediated homologous DNA pairing and strand exchange by Rad54 requires the ATPase activity of the latter and physical interaction between these two HR factors [27][28][29]. Interestingly, Rad54 dissociates Rad51 from duplex DNA via its DNA translocase activity, a function believed to be important for the intracellular recycling of Rad51 and possibly for freeing the primer end in the D-loop structure to initiate the DNA synthesis reaction [30].…”

Yeast Recombination Factor Rdh54 Functionally Interacts with the Rad51 Recombinase and Catalyzes Rad51 Removal from DNA

“…This finding and the fact that Rdh54 neither binds nor functionally synergizes with the E. coli RecA protein (13) (this work) are consistent with the premise that specific complex formation between Rdh54 and Rad51 is a prerequisite for functional interactions between these two S. cerevisiae HR factors. Previous studies have shown that complex formation of Rad54 with Rad51 is required for functional synergy between these two recombination factors (14,33,38,39,(41)(42)(43)(44), and Rdh54 (13) (this work) resembles Rad54 in this regard.…”

“…Even though Rad51 removal in the in vitro setting is not absolutely contingent upon Rdh54 possessing the ability to bind Rad51, within cells, complex formation with Rad51 may well be necessary for efficient targeting of Rdh54 to chromosome locales where Rad51 is bound. As noted earlier, complex formation of Rad54 with Rad51 appears to be indispensable for functional synergy between them (14,33,38,39,(41)(42)(43)(44). It will be important to test whether variants of Rad54 that lack the ability to interact with Rad51 (38,39) are capable of clearing Rad51 from dsDNA.…”

Regulation of the Rhp26 ERCC6/CSB chromatin remodeler by a novel conserved leucine latch motif

“…These SNF2-like ATPases are broadly conserved throughout evolution and share a common core ATPase domain. Whereas the core motor ATPase domain provides the driving force for DNA translocation and chromatin-remodeling activity, emerging evidence suggests important roles of the flanking regions in mediating specific interactions with nucleosomes or other protein factors, substrate specificity, and the regulation of the function of the motor (2)(3)(4)(5)(6)(7).…”