Rad54: the Swiss Army knife of homologous recombination?

Abstract

Homologous recombination (HR) is a ubiquitous cellular pathway that mediates transfer of genetic information between homologous or near homologous (homeologous) DNA sequences. During meiosis it ensures proper chromosome segregation in the first division. Moreover, HR is critical for the tolerance and repair of DNA damage, as well as in the recovery of stalled and broken replication forks. Together these functions preserve genomic stability and assure high fidelity transmission of the genetic material in the mitotic and meiotic cell divisions. This review will focus on the Rad54 protein, a member of the Snf2-family of SF2 helicases, which translocates on dsDNA but does not display strand displacement activity typical for a helicase. A wealth of genetic, cytological, biochemical and structural data suggests that Rad54 is a core factor of HR, possibly acting at multiple stages during HR in concert with the central homologous pairing protein Rad51.

Figure 1

Pathways of DSBR by HR. HR can be conceptually divided into three stages: Pre-synapsis (1,2), synapsis (3,4) and post-synapsis (5–11). The proteins identified to function at the individual stages are listed, alternative human nomenclature is listed in brackets. / indicates alternative nomenclature in different organisms (Xrs2/Nbs1: S.cerevisiae Xrs2, Schizosaccharomyces pombe and human Nbs1; Mms4/Eme1: S.cerevisiae and human Mms4, S.pombe and human Eme1). Three different pathways emanate from the postulated D-loop intermediate (4), the product of DNA strand invasion by the Rad51-ssDNA filament. DSBR (steps 5–8) engages both ends of the DSB to form a double Holliday junction intermediate (dHJ), which can be resolved into crossover and non-crossover products. SDSA (step 10) retracts the invading strand after DNA synthesis on the target duplex to anneal the newly synthesized strand with the tail of the second end, leading to localized conversion without crossover. BIR (step 11) was proposed to assemble a replication fork at the D-loop to copy the entire chromosome arm distal to the DSB site leading to long gene conversion events.

Figure 2

Rad54 protein structure and phylogenetic comparison. (A) Schematic alignment of Rad54 proteins from Homo sapiens (HUMAN), Mus musculus (MOUSE), Gallus gallus (CHICK), Zebrafish Brachydanio rerio/Danio rerio (BRARE), D.melanogaster (DROME) and S.cerevisiae (YEAST). The seven conserved motor motifs are highlighted in yellow. * The database sequence appears incomplete and some residues from the N-terminus of chicken Rad54 are missing. (B) Structural model of human Rad54 based on the X-ray crystal structure of zebrafish Rad54 (PDB code: 1Z3I) (47). Rad54 from human and zebrafish share 78.4% identity. The colors in the structural model reflect the colors in the primary structural scheme of the human Rad54 core. The N-terminal domain (NTD) and C-terminal domain (CTD) are shown in yellow and cyan, respectively. Snf2-specific helical domains are depicted in red (HD1) and green (HD2). The RecA-like α/β domains are shown in blue (lobe 1, containing motifs I, IA, II, III) and magenta (lobe 2, containing motifs IV, V, VI). The linear structural model (top) is aligned and at the same scale as the representations in A. The dotted lines at the N- and C-termini indicate residues not present in the crystal structure, The X-ray crystal structure of zebrafish Rad54 was used in structural alignment in ICMLite (). The molecular modeling and model evaluation was performed using the methods described in (109). The image of the structural model was generated using PyMol ().

Figure 3

Mechanistic models for Rad54 function in HR. The mechanistic models were derived from analysis of reconstituted in vitro recombination reactions and biochemical analysis of the Rad54 protein. For more details see text. Shown is one processed DSB end with a 3′-ending ssDNA tail that invades a nucleosomal duplex target DNA. Pre-synapsis: Rad54 was found to mediate formation or to stabilize Rad51 filaments on ssDNA. The pre-synaptic function does not require Rad54 ATPase activity and requires Rad51 binding to ATP but not hydrolysis. Synapsis: Rad54 augments the ability of Rad51-ssDNA filaments to form joint molecules, possibly involving translocating the Rad51-ssDNA filament along duplex DNA or inducing strand separation through induction of topological change. Rad54 also exhibits chromatin remodeling activity that may clear nucleosomes or other proteins from the pairing site. The synaptic function requires the Rad54 ATPase activity but not the Rad51 ATPase activity. Post-synapsis: Rad54 was identified to catalyze heteroduplex extension (branch migration) and can dissociate the Rad51–dsDNA product complex, possibly to allow DNA polymerase access to the invading 3′-OH end to prime DNA synthesis. Post-synapsis requires the ATPase activities of both the Rad54 and Rad51 proteins. The exact oligomeric structure of Rad54 in its interaction with the Rad51-ssDNA and Rad51–dsDNA filaments is not known, so it is unclear how many molecules of Rad54 are represented by the symbol drawn in the figure. Rad54 is likely to act as an oligomer on DNA (58), and an oligomeric Rad54 particle has been directly visualized at the terminus of Rad51–dsDNA filaments by electron microscopy (81). This figure is derived from Figure 2 of reference (12).