(NAS Colloquium) Links Between Recombination and Replication: Vital Roles of Recombination

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Colloquium on Links Between Recombination and Replication: Vital Roles of Recombination

Colloquium

The architecture of the human Rad54-DNA complex provides evidence for protein translocation along DNA

Dejan Ristic*^†, Claire Wyman*^†, Coen Paulusma*, and Roland Kanaar*^‡^§

*Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands; and ^‡Department of Radiation Oncology, University Hospital Rotterdam/Daniel, PO Box 5201, 3008 AE Rotterdam, The Netherlands

Proper maintenance and duplication of the genome require accurate recombination between homologous DNA molecules. In eukaryotic cells, the Rad51 protein mediates pairing between homologous DNA molecules. This reaction is assisted by the Rad54 protein. To gain insight into how Rad54 functions, we studied the interaction of the human Rad54 (hRad54) protein with double-stranded DNA. We have recently shown that binding of hRad54 to DNA induces a change in DNA topology. To determine whether this change was caused by a protein-constrained change in twist, a protein-constrained change in writhe, or the introduction of unconstrained plectonemic supercoils, we investigated the hRad54-DNA complex by scanning force microscopy. The architecture of the observed complexes suggests that movement of the hRad54 protein complex along the DNA helix generates unconstrained plectonemic supercoils. We discuss how hRad54-induced superhelical stress in the target DNA may function to facilitate homologous DNA pairing by the hRad51 protein directly. In addition, the induction of supercoiling by hRad54 could stimulate recombination indirectly by displacing histones and/or other proteins packaging the DNA into chromatin. This function of DNA translocating motors might be of general importance in chromatin metabolism.

Recombination between homologous DNA molecules is important for maintenance and faithful duplication of the genome (1–4). Homologous recombination is a major pathway for the accurate repair of DNA double-strand breaks (DSBs) that arise from exposure to exogenous DNA-damaging agents such as ionizing radiation, which is commonly used in antitumor therapies. Furthermore, homologous recombination processes programmed DSB intermediates during meiosis. Finally, homologous recombination plays a major role in reestablishing DNA replication forks that are stalled or have collapsed because of the presence of spontaneous or induced DNA damage in one of the template strands of the DNA double helix (5–12).

Extensive genetic and biochemical experiments have revealed that DSB repair mediated by homologous recombination in yeast, chicken, and mammalian cells occurs through the close cooperation of the RAD52 group of proteins, including Rad51, Rad52, and Rad54 (13, 14). A key member of this group is the Rad51 protein. Rad51 protomers assemble a nucleoprotein filament on the single-stranded DNA tails that form at the break site. This filament pairs with homologous double-stranded DNA, resulting in a joint molecule. Joint molecules are pivotal intermediates in recombination because they allow the broken DNA to use the intact homologous double-stranded DNA as a repair template (15). The Rad52 and Rad54 proteins serve as accessory factors in Rad51-mediated joint molecule formation. The details of the molecular mechanisms through which Rad52 and Rad54 stimulate joint molecule formation are not well understood. Rad52 has been shown to increase the rate of annealing of complementary single-stranded DNA molecules, to bind to DNA ends, to stimulate homologous paring by Rad51, and to overcome the inhibitory effect of the single-stranded DNA-binding protein RPA on Rad51 nucleoprotein filament formation (16–22). Rad54 can interact with Rad51 (23–26) and has ATPase activity (27, 28). Importantly, the ATPase activity of Rad54 specifically requires the presence of double-stranded DNA. It is not active in the presence of single-stranded DNA (27, 28). This cofactor specificity is opposite to that of Rad51 (29). Because the initial substrate of Rad51 during homologous recombination is single-stranded DNA (13), it is likely that the substrate for Rad54 is the double-stranded homologous repair template.

To understand how the Rad54 protein assists Rad51 during joint molecule formation we have investigated the interaction of the human Rad54 (hRad54) protein with double-stranded DNA. We have recently demonstrated that binding of hRad54 to double-stranded DNA induces a change in the topology of the DNA (26), as is also observed for yeast Rad54 homologues (30–33). In the topological experiments, singly nicked plasmid DNA is incubated with hRad54 protein. Subsequently, the nick is closed by the addition of DNA ligase. The ligation will fix any change in linking number (∆Lk) in the DNA that is induced by protein binding. Lk describes the number of times that the two strands of the DNA double helix wind around each other. Lk is a topological parameter of double-stranded DNA that is made up of two geometrical parameters, twist (Tw) and writhe (Wr) (34). Tw and Wr give information about the shape of the DNA. The local winding of the two strands of the double helix is described by Tw, whereas Wr describes the number of times that the axis of the double helix winds around itself. The relationship between these three parameters is expressed by the equation Lk=Tw+Wr (35). Therefore, to mechanistically interpret the ∆Lk induced by hRad54 binding it is necessary to determine whether protein binding changes Tw or Wr.

One well-characterized class of proteins that has the ability to change the Lk of DNA is topoisomerases (36–38). Topoisomerases induce a ∆Lk by a strand passage mechanism because these enzymes can break and rejoin DNA strands. In contrast, no strand breakage and rejoining activity has been detected for hRad54 (26). Therefore, the ∆Lk measured by the assay described above could be caused by the hRad54 protein constraining either Tw or Wr. Protein-constrained ∆Tw and ∆Wr result

This paper results from the National Academy of Sciences colloquium, “Links Between Recombination and Replication: Vital Roles of Recombination,” held November 10–12, 2000, in Irvine, CA.

Abbreviations: ∆Lk, change in linking number; SFM, scanning force microscopy; Tw, twist; Wr, writhe; ATPγS, adenosine 5′-[γ-thio]triphosphate.

^†	D.R. and C.W. contributed equally to this work.
^§	To whom reprint requests should be addressed. E-mail: kanaar@gen.fgg.eur.nl.

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