ATP-dependent and independent functions of Rad54 in genome maintenance

Department of Cell Biology and Genetics, Cancer Genomics Center, 3000 CA Rotterdam, Netherlands.
The Journal of Cell Biology (Impact Factor: 9.83). 02/2011; 192(5):735-50. DOI: 10.1083/jcb.201011025
Source: PubMed


Rad54, a member of the SWI/SNF protein family of DNA-dependent ATPases, repairs DNA double-strand breaks (DSBs) through homologous recombination. Here we demonstrate that Rad54 is required for the timely accumulation of the homologous recombination proteins Rad51 and Brca2 at DSBs. Because replication protein A and Nbs1 accumulation is not affected by Rad54 depletion, Rad54 is downstream of DSB resection. Rad54-mediated Rad51 accumulation does not require Rad54's ATPase activity. Thus, our experiments demonstrate that SWI/SNF proteins may have functions independent of their ATPase activity. However, quantitative real-time analysis of Rad54 focus formation indicates that Rad54's ATPase activity is required for the disassociation of Rad54 from DNA and Rad54 turnover at DSBs. Although the non-DNA-bound fraction of Rad54 reversibly interacts with a focus, independent of its ATPase status, the DNA-bound fraction is immobilized in the absence of ATP hydrolysis by Rad54. Finally, we show that ATP hydrolysis by Rad54 is required for the redistribution of DSB repair sites within the nucleus.

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    • "Rad54 contains the classical seven motifs of the SF2 superfamily of helicases/translocases, which mark proteins that translocate on DNA in an ATP hydrolysis-driven fashion. Despite its SF2 membership, Rad54 likely has both ATPase-dependent and -independent roles in HR [8], [9]. The pre-synaptic stabilization of Rad51 filaments by Rad54 is ATPase-independent [10] and is likely mediated by Rad51-Rad54 physical interactions [11], [12], [13]. "
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    ABSTRACT: Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recombination (HR). Although its activity has been implicated in several steps of HR, its exact role(s) at each step are still not fully understood. We have identified a new interaction between Rad54 and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA). This interaction was only mildly weakened by the mutation of two key hydrophobic residues in the highly-conserved PCNA interaction motif (PIP-box) of Rad54 (Rad54-AA). Intriguingly, the rad54-AA mutant cells displayed sensitivity to DNA damage and showed HR defects similar to the null mutant, despite retaining its ability to interact with HR proteins and to be recruited to HR foci in vivo. We therefore surmised that the PCNA interaction might be impaired in vivo and was unable to promote repair synthesis during HR. Indeed, the Rad54-AA mutant was defective in primer extension at the MAT locus as well as in vitro, but additional biochemical analysis revealed that this mutant also had diminished ATPase activity and an inability to promote D-loop formation. Further mutational analysis of the putative PIP-box uncovered that other phenotypically relevant mutants in this domain also resulted in a loss of ATPase activity. Therefore, we have found that although Rad54 interacts with PCNA, the PIP-box motif likely plays only a minor role in stabilizing the PCNA interaction, and rather, this conserved domain is probably an extension of the ATPase domain III.
    PLoS ONE 12/2013; 8(12):e82630. DOI:10.1371/journal.pone.0082630 · 3.23 Impact Factor
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    • "Extensive biochemical analysis revealed that Rad54 exerts many functions during the recombination process [7], [15], [16]. While Rad54 can stabilize the Rad51-ssDNA filament in an ATPase-independent fashion [17]–[19], other functions of Rad54 require its ATPase activity, including DNA strand invasion, branch migration, chromatin remodeling, and Rad51 dissociation from heteroduplex DNA to allow access of DNA polymerases to the 3′-OH end of the invading strand [7], [15], [16], [20]. Genetic analysis established that the ATPase activity is essential for Rad54 function, and mutations in the Walker A box cause DNA damage sensitivity as extreme as the gene deletion [21], [22]. "
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    ABSTRACT: Rad54 is a dsDNA-dependent ATPase that translocates on duplex DNA. Its ATPase function is essential for homologous recombination, a pathway critical for meiotic chromosome segregation, repair of complex DNA damage, and recovery of stalled or broken replication forks. In recombination, Rad54 cooperates with Rad51 protein and is required to dissociate Rad51 from heteroduplex DNA to allow access by DNA polymerases for recombination-associated DNA synthesis. Sequence analysis revealed that Rad54 contains a perfect match to the consensus PIP box sequence, a widely spread PCNA interaction motif. Indeed, Rad54 interacts directly with PCNA, but this interaction is not mediated by the Rad54 PIP box-like sequence. This sequence is located as an extension of motif III of the Rad54 motor domain and is essential for full Rad54 ATPase activity. Mutations in this motif render Rad54 non-functional in vivo and severely compromise its activities in vitro. Further analysis demonstrated that such mutations affect dsDNA binding, consistent with the location of this sequence motif on the surface of the cleft formed by two RecA-like domains, which likely forms the dsDNA binding site of Rad54. Our study identified a novel sequence motif critical for Rad54 function and showed that even perfect matches to the PIP box consensus may not necessarily identify PCNA interaction sites.
    PLoS ONE 12/2013; 8(12):e82184. DOI:10.1371/journal.pone.0082184 · 3.23 Impact Factor
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    • "Meanwhile, Jakob et al. (2009a, 2009b) have used IR induced by heavy ion sources to show that repair foci have similar kinetics as undamaged loci, but do not appear to be constrained over several hours, suggesting that damaged DNA could travel large distances given enough time. Finally, damage induced by a particle irradiation is highly mobile and moved over large distances within minutes (Agarwal et al., 2011; Aten et al., 2004; Krawczyk et al., 2012). This latter situation is reminiscent of results obtained with uncapped telomeres in mouse embryonic cells (Dimitrova et al., 2008). "
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    ABSTRACT: Mechanistic analyses based on improved imaging techniques have begun to explore the biological implications of chromatin movement within the nucleus. Studies in both prokaryotes and eukaryotes have shed light on what regulates the mobility of DNA over long distances. Interestingly, in eukaryotes, genomic loci increase their movement in response to double-strand break induction. Break mobility, in turn, correlates with the efficiency of repair by homologous recombination. We review here the source and regulation of DNA mobility and discuss how it can both contribute to and jeopardize genome stability.
    Cell 03/2013; 152(6):1355-64. DOI:10.1016/j.cell.2013.02.010 · 32.24 Impact Factor
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