Rad51 Protein Stimulates the Branch Migration Activity of Rad54 Protein * □ S

Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102-1192, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 08/2008; 283(36):24698-706. DOI: 10.1074/jbc.M800839200
Source: PubMed


The Rad51 and Rad54 proteins play important roles during homologous recombination in eukaryotes. Rad51 forms a nucleoprotein
filament on single-stranded DNA and performs the initial steps of double strand break repair. Rad54 belongs to the Swi2/Snf2
family of ATP-dependent DNA translocases. We previously showed that Rad54 promotes branch migration of Holliday junctions.
Here we find that human Rad51 (hRad51) significantly stimulates the branch migration activity of hRad54. The stimulation appears
to be evolutionarily conserved, as yeast Rad51 also stimulates the branch migration activity of yeast Rad54. We further investigated
the mechanism of this stimulation. Our results demonstrate that the stimulation of hRad54-promoted branch migration by hRad51
is driven by specific protein-protein interactions, and the active form of the hRad51 filament is more stimulatory than the
inactive one. The current results support the hypothesis that the hRad51 conformation state has a strong effect on interaction
with hRad54 and ultimately on the function of hRad54 in homologous recombination.

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    • "Binding to its preferred substrate also stimulated the ATPase activity of hRAD54 about three to five-fold over dsDNA [124] [125]. Inclusion of hRAD51 protein also stimulated hRAD54 branch migration activity [126], likely through the stimulation of the Rad54 ATPase activity [75] [116]. hRAD54 was also able to drive branch migration through regions of heterology when using junction substrates reconstituted from oligonucleotides or in reconstituted recombination reactions with hRAD51 [124] [127]. "
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    ABSTRACT: Homologous recombination is a central pathway to maintain genomic stability and is involved in the repair of DNA damage and replication fork support, as well as accurate chromosome segregation during meiosis. Rad54 is a dsDNA-dependent ATPase of the Snf2/Swi2 family of SF2 helicases, although Rad54 lacks classical helicase activity and cannot carry out the strand displacement reactions typical for DNA helicases. Rad54 is a potent and processive motor protein that translocates on dsDNA, potentially executing several functions in recombinational DNA repair. Rad54 acts in concert with Rad51, the central protein of recombination that performs the key reactions of homology search and DNA strand invasion. Here, we will review the role of the Rad54 protein in homologous recombination with an emphasis on mechanistic studies with the yeast and human enzymes. We will discuss how these results relate to in vivo functions of Rad54 during homologous recombination in somatic cells and during meiosis. This article is part of a Special Issue entitled: Snf2/Swi2 ATPase structure and function.
    Biochimica et Biophysica Acta 06/2011; 1809(9):509-23. DOI:10.1016/j.bbagrm.2011.06.006 · 4.66 Impact Factor
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    • "RAD51, a major protein of HR, interacts physically and functionally with both RAD54 and BLM; moreover RAD51 is known to stimulate various activities of RAD54 including branch migration (25,37,55). Here, we asked whether RAD51 can stimulate fork regression promoted by RAD54 and BLM. "
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    ABSTRACT: DNA lesions cause stalling of DNA replication forks, which can be lethal for the cell. Homologous recombination (HR) plays an important role in DNA lesion bypass. It is thought that Rad51, a key protein of HR, contributes to the DNA lesion bypass through its DNA strand invasion activity. Here, using model stalled replication forks we found that RAD51 and RAD54 by acting together can promote DNA lesion bypass in vitro through the 'template-strand switch' mechanism. This mechanism involves replication fork regression into a Holliday junction ('chicken foot structure'), DNA synthesis using the nascent lagging DNA strand as a template and fork restoration. Our results demonstrate that RAD54 can catalyze both regression and restoration of model replication forks through its branch migration activity, but shows strong bias toward fork restoration. We find that RAD51 modulates this reaction; by inhibiting fork restoration and stimulating fork regression it promotes accumulation of the chicken foot structure, which we show is essential for DNA lesion bypass by DNA polymerase in vitro. These results indicate that RAD51 in cooperation with RAD54 may have a new role in DNA lesion bypass that is distinct from DNA strand invasion.
    Nucleic Acids Research 11/2010; 39(6):2153-64. DOI:10.1093/nar/gkq1139 · 9.11 Impact Factor
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    • "Rad54 belongs to the Snf2/Swi2 protein family. It possesses a robust DNA-dependent ATPase activity, uses ATP hydrolysis to supercoil DNA and cooperates with the Rad51 recombinase in DNA joint formation (Smirnova et al., 2004; Rossi and Mazin, 2008). This gene has been mapped at chromosome 1p32 by fluorescent in situ hybridization (FISH) (Kanaar et al., 1996). "
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    ABSTRACT: The hRAD54 gene is a key member of the RAD52 epistasis group involved in repair of double-strand breaks (DSB) by homologous recombination (HR). Thus, alterations of the normal function of these genes could generate genetic instability, shifting the normal process of the cell cycle, leading the cells to develop into cancer. In this work we analyzed exon 18 of the hRAD54 gene, which has been previously reported by our group to carry a silent polymorphism, 2290 C/T (Ala730Ala), associated to meningiomas. We performed a PCR-SSCP method to detect the polymorphism in 239 samples including leukemia and normal control population. The results revealed that the 2290 C/T polymorphism has frequencies of 0.1 for the leukemia and 0.1 for the control group. These frequencies show no statistical differences. Additionally, we dissected the leukemia group in chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL) to evaluate the polymorphism. The frequencies found in these subgroups were 0.14 for CML and 0.05 for ALL. We found statistically significant differences between CML patients and the control group (p < 0.05) but we did not find significant differences between ALL and the control group (p > 0.05). These results suggest a possible link between the 2290 C/T polymorphism of the hRAD54 gene and CML.
    Genetics and Molecular Biology 10/2010; 33(4):646-9. DOI:10.1590/S1415-47572010005000095 · 1.20 Impact Factor
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