Human Rad51 filaments on double- and single-stranded DNA: Correlating regular and irregular forms with recombination function

Department of Cell Biology and Genetics, Erasmus Medical Center PO Box 1738, 3000 DR Rotterdam, The Netherlands.
Nucleic Acids Research (Impact Factor: 9.11). 02/2005; 33(10):3292-302. DOI: 10.1093/nar/gki640
Source: PubMed

ABSTRACT Recombinase proteins assembled into helical filaments on DNA are believed to be the catalytic core of homologous recombination. The assembly, disassembly and dynamic rearrangements of this structure must drive the DNA strand exchange reactions of homologous recombination. The sensitivity of eukaryotic recombinase activity to reaction conditions in vitro suggests that the status of bound nucleotide cofactors is important for function and possibly for filament structure. We analyzed nucleoprotein filaments formed by the human recombinase Rad51 in a variety of conditions on double-stranded and single-stranded DNA by scanning force microscopy. Regular filaments with extended double-stranded DNA correlated with active in vitro recombination, possibly due to stabilizing the DNA products of these assays. Though filaments formed readily on single-stranded DNA, they were very rarely regular structures. The irregular structure of filaments on single-stranded DNA suggests that Rad51 monomers are dynamic in filaments and that regular filaments are transient. Indeed, single molecule force spectroscopy of Rad51 filament assembly and disassembly in magnetic tweezers revealed protein association and disassociation from many points along the DNA, with kinetics different from those of RecA. The dynamic rearrangements of proteins and DNA within Rad51 nucleoprotein filaments could be key events driving strand exchange in homologous recombination.

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Available from: John van Noort, Jul 08, 2015
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    • "Rad51 exhibits ssDNA and dsDNA-stimulated ATPase activity, which drives the nucleation and extension of the Rad51 nucleoprotein filament. There is clear evidence that only Rad51-ssDNA nucleoprotein filament is able to catalyze DNA joint formation, supporting the assessment that Rad51 is recruited to ssDNA generated by nucleolytic processing of DNA termini (Ristic et al., 2005). Formation of Rad51 nucleoprotein filament also depends on a large number of factors controlling the effectiveness of HRR. "
    DNA Repair - On the Pathways to Fixing DNA Damage and Errors, 09/2011; , ISBN: 978-953-307-649-2
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    • "It is interesting to observe that yeast Rad51 is completely dependent on ScRad54 for D-loop formation [114], whereas human RAD51 can form D-loops in the absence of hRAD54 [115] [116]. This suggests that Rad54 function may relate to the DNA binding properties of its cognate Rad51 protein [118] [119] [120]. At present, it is difficult to distinguish between the latter two models. "
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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 assembly onto single-stranded DNA is greatly affected by dissociation such that the growth profiles differ [26]. This implies that the RAD51 filament on single-stranded DNA is a very dynamic structure and indeed it can hardly be visualized (without fixation) as a defined structure by SFM or EM [24]. The dynamic nature of recombinase filaments in ATP hydrolysis permitting environments is likely to play an important role in later steps of strand exchange reactions. "
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    ABSTRACT: Homologous recombination, the exchange of DNA strands between homologous DNA molecules, is involved in repair of many structural diverse DNA lesions. This versatility stems from multiple ways in which homologous DNA strands can be rearranged. At the core of homologous recombination are recombinase proteins such as RecA and RAD51 that mediate homology recognition and DNA strand exchange through formation of a dynamic nucleoprotein filament. Four stages in the life cycle of nucleoprotein filaments are filament nucleation, filament growth, homologous DNA pairing and strand exchange, and filament dissociation. Progression through this cycle requires a sequence of recombinase-DNA and recombinase protein-protein interactions coupled to ATP binding and hydrolysis. The function of recombinases is controlled by accessory proteins that allow coordination of strand exchange with other steps of homologous recombination and that tailor to the needs of specific aberrant DNA structures undergoing recombination. Accessory proteins are also able to reverse filament formation thereby guarding against inappropriate DNA rearrangements. The dynamic instability of the recombinase-DNA interactions allows both positive and negative action of accessory proteins thereby ensuring that genome maintenance by homologous recombination is not only flexible and versatile, but also accurate.
    DNA repair 10/2010; 9(12):1264-72. DOI:10.1016/j.dnarep.2010.09.014 · 3.36 Impact Factor