Homologous recombination can be conceptually divided into three stages: Pre-synapsis (1–2), synapsis (3–4), and post-synapsis (5–11). The Saccharomyces cerevisiae proteins identified to function at the individual stages are listed in black, the human proteins with alternate nomenclature are listed in blue in square brackets and human proteins not found in yeast are listed in blue without brackets. Three different pathways emanate from the D-loop intermediate (4), the product of DNA strand invasion by the Rad51-ssDNA filament. The dHJ (double Holliday junction; steps 5–8) pathway engages both ends of the DSB to ultimately form a double Holliday junction intermediate (dHJ), which can be resolved into crossover and non-crossover products. SDSA (synthesis-dependent strand annealing; step 10) retracts the invading strand after DNA synthesis on the target duplex to anneal the newly synthesized strand with the single strand resulting from resection of the second end, leading to localized conversion without crossover. BIR (break-induced replication; 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.

(A) Schematic alignment of Rad54 proteins and Rad54 paralogs from Homo Sapiens and S. cerevisiae. The motor domains are outlined in green (DEXDc) and red (HELICc) [172], the 7 conserved ATPase motifs are highlighted in yellow. (B) X-ray crystal structure of the zebra fish Danio rerio Rad54 protein, an N-terminal truncation representing amino acids 91–738, is shown with the DEXDc and HELICc domains colored as in (A) (PDB code:1Z3I) [79].

Schematic representation of possible stages, where nucleosomes on chromosomal DNA (1) may exert an effect on HR, potentially requiring chromatin remodeling, including resection (2), homology search (3), DNA strand invasion (4), branch migration (5), and recombination-associated DNA synthesis (6). The question mark in (2) indicates the possibility that nucleosomes may be associated with ssDNA after resection. The architecture of the Rad54 protein (monomer, multimer) in association with the Rad51-ssDNA filament or duplex DNA is unknown. For convenience, we represent here and in subsequent figures Rad54 associated with the Rad51-ssDNA filament as a monomer. The active translocating motor on duplex DNA is depicted here and in subsequent figures as a hexameric ring encircling the DNA in an interpretation of the single molecule and electron microscopic studies discussed in the text based on the analogy of processive, homo-hexameric DNA helicases.

A. Scheme of the three-stranded DNA strand exchange reaction between linear duplex DNA and circular single-stranded DNA. B. Scheme of the D-loop reaction between supercoiled, circular duplex DNA and a single-stranded DNA oligonucleotide, which is depicted with a heterologous duplex DNA tail. C. The dsDNA sliding model envisions that the Rad54 motor protein slides duplex DNA along the Rad51 presynaptic filament to enhance homology search. D. The dsDNA strand-opening model suggests that Rad54 translocation on the duplex target DNA results in strand opening that facilitates the conversion to stable joint molecules. In both models, Rad54 protein needs to transition from an ATPase inactive form when associated with the Rad51-ssDNA filament to an active ATPase on duplex DNA, a process which is not depicted in the figure. E. The Rad51-dsDNA complex dissociation model maintains that in the in vitro reaction some Rad51 will bind to the duplex DNA, which inhibits D-loop formation, unless Rad51 is dissociated from duplex DNA by Rad54 and entirely redistributed to bind to the ssDNA.

Schematic representation of Rad54 dissociating Rad51 from heteroduplex DNA after DNA strand invasion. The stippled blue arrow indicates new DNA synthesis by a DNA polymerase that gains access after Rad51 dissociation.

Schematic representation of Rad54 dissociating Rad51 dead-end complex on chromosomal duplex DNA.

Schematic representation of ATPase-independent stabilization of the Rad51-ssDNA filament by Rad54. The exact interaction of Rad54 with the Rad51-ssDNA filament, laterally or terminally, monomer or multimer is unknown.

Schematic representation of the potential roles of Rad54 in branch migration: A) enlarging D-loops, B) moving nicked (not shown) or closed Holliday junctions, C) migrating D-loops during recombination-associated DNA synthesis, D) dissociating D-loops, and E) regressing replication forks. For details see text.

A) Recruitment of the Mus81-Mms4 endonuclease by Rad54 to a D-loop and B) other recombination-mediated junctions by Rad54, including a DNA synthesis extended D-loop, a partial double Holliday junction after second end capture, or a nicked double Holliday junction. Note that all three junction intermediates depicted are excellent substrates for Mus81-Mms4 cleavage in vitro, whereas the mature double Holliday junction with four full-length ligated strands has not been reported to be cleaved by Mus81-Mms4 and a single Holliday junction is either not cleaved at all or very inefficiently [32, 152].

Inhibition of the Rad54-Rad51 interaction by Mek1-mediated phosphorylation of Rad54-T132 and Hed1 binding to Rad51.