Homologous recombination is a ubiquitous DNA metabolic process involved in the repair and tolerance of DNA damage, the recovery of stalled or broken replication forks, and the faithful segregation of chromosomes during meiosis. A class of enzymes known as DNA-strand transferases carry out the signature reactions of recombination: homology search, DNA-strand invasion, and DNA-strand exchange. These proteins are found in all domains of life, and their active form is a right-handed protein–DNA filament that utilizes Watson–Crick base-pairing principles to exchange DNA strands.

Displacement loops (D-loops) are critical intermediates formed during homologous recombination. Rdh54 (a.k.a. Tid1), a Rad54 paralog in Saccharomyces cerevisiae, is well-known for its role with Dmc1 recombinase during meiotic recombination. Yet contrary to Dmc1, Rdh54/Tid1 is also present in somatic cells where its function is less understood. While Rdh54/Tid1 enhances the Rad51 DNA strand invasion activity in vitro, it is unclear how it interplays with Rad54. Here, we show that Rdh54/Tid1 inhibits D-loop formation by Rad51 and Rad54 in an ATPase-independent manner. Using a novel D-loop Mapping Assay, we further demonstrate that Rdh54/Tid1 uniquely restricts the length of Rad51-Rad54-mediated D-loops. The alterations in D-loop properties appear to be important for cell survival and mating-type switch in haploid yeast. We propose that Rdh54/Tid1 and Rad54 compete for potential binding sites within the Rad51 filament, where Rdh54/Tid1 acts as a physical roadblock to Rad54 translocation, limiting D-loop formation and D-loop length.

Homologous recombination (HR) uses a homologous template to accurately repair DNA double-strand breaks and stalled replication forks to maintain genome stability. During homology search, Rad51 nucleoprotein filaments probe and interact with dsDNA, forming the synaptic complex that is stabilized on a homologous sequence. Strand intertwining leads to the formation of a displacement-loop (D-loop). In yeast, Rad54 is essential for HR in vivo and required for D-loop formation in vitro, but its exact role remains to be fully elucidated. Using electron microscopy to visualize the DNA-protein complexes, here we find that Rad54 is crucial for Rad51-mediated synaptic complex formation and homology search. The Rad54−K341R ATPase-deficient mutant protein promotes formation of synaptic complexes but not D-loops and leads to the accumulation of stable heterologous associations, suggesting that the Rad54 ATPase is involved in preventing non-productive intermediates. We propose that Rad51/Rad54 form a functional unit operating in homology search, synaptic complex and D-loop formation.

Homologous recombination (HR) is a principal support pathway for DNA replication and for recovery from DNA breaks and interstrand crosslinks, making it a rational target for inhibition in cancer therapy. The ATPase RAD54 functions in molecular events that promote DNA sequence-preservation during HR-mediated damage repair, including homology search, DNA strand exchange, and transition to DNA repair synthesis within a displacement loop intermediate. We developed a high-throughput biochemical screen to identify small-molecule inhibitors of human RAD54, using a phosphate detection assay to monitor RAD54 ATPase activity in the presence of double-stranded DNA (dsDNA). After filtering potential DNA intercalators and ‘frequent hitters,’ we identified two chemotypes that reproducibly inhibited RAD54 ATPase in vitro . We evaluated these chemotypes for inhibition of RAD54-dsDNA binding and cancer cell survival. A halogenated carbazole/dihydroacridine scaffold inhibited a panel of SWI2/SNF2-related ATPases but not VCP/p97, an unrelated ATPase. Small molecules that interfere with key steps in HR— such as inhibitors of RAD54—may expose DNA repair-dependent vulnerabilities in cancer cells.

Displacement-loops (D-loops) are pivotal intermediates of homologous recombination (HR), a universal DNA double strand break (DSB) repair pathway. We developed a versatile assay for the physical detection of D-loops in vivo , which enabled studying the kinetics of their formation and defining the network of D-loop formation and reversal pathways. Nascent D-loops are detected within 2 hrs of DSB formation and extended over the next 2 hrs in a system allowing break-induced replication. The majority of D-loops are disrupted in wild type cells by two pathways: one supported by the Srs2 helicase and the other by the Mph1 helicase and the Sgs1-Top3-Rmi1 helicase-topoisomerase complex. Both pathways operate without significant overlap and are delineated by the Rad54 paralog Rdh54 in an ATPase-independent fashion. This study uncovers a novel layer of HR control in cells relying on nascent D-loop dynamics, revealing unsuspected complexities, and identifying a surprising role for a conserved Rad54 paralog.

Homologous recombination enables the cell to access and copy intact DNA sequence information in trans, particularly to repair DNA damage affecting both strands of the double helix. Here, we discuss the DNA transactions and enzymatic activities required for this elegantly orchestrated process in the context of the repair of DNA double-strand breaks in somatic cells. This includes homology search, DNA strand invasion, repair DNA synthesis, and restoration of intact chromosomes. Aspects of DNA topology affecting individual steps are highlighted. Overall, recombination is a dynamic pathway with multiple metastable and reversible intermediates designed to achieve DNA repair with high fidelity.

Synthesis-dependent strand annealing (SDSA) is the preferred mode of homologous recombination in somatic cells leading to an obligatory non-crossover outcome, thus avoiding the potential for chromosomal rearrangements and loss of heterozygosity. Genetic analysis identified the Srs2 helicase as a prime candidate to promote SDSA. Here, we demonstrate that Srs2 disrupts D-loops in an ATP-dependent fashion and with a distinct polarity. Specifically, we partly reconstitute the SDSA pathway using Rad51, Rad54, RPA, RFC, DNA Polymerase δ with different forms of PCNA. Consistent with genetic data showing the requirement for SUMO and PCNA binding for the SDSA role of Srs2, Srs2 displays a slight but significant preference to disrupt extending D-loops over unextended D-loops when SUMOylated PCNA is present, compared to unmodified PCNA or monoubiquitinated PCNA. Our data establish a biochemical mechanism for the role of Srs2 in crossover suppression by promoting SDSA through disruption of extended D-loops.

Inaccurate repair of broken chromosomes generates structural variants that can fuel evolution and inflict pathology. We describe a novel rearrangement mechanism in which translocation between intact chromosomes is induced by a lesion on a third chromosome. This multi-invasion-induced rearrangement (MIR) stems from a homologous recombination byproduct, where a broken DNA end simultaneously invades two intact donors. No homology is required between the donors, and the intervening sequence from the invading molecule is inserted at the translocation site. MIR is stimulated by increasing homology length and spatial proximity of the donors and depends on the overlapping activities of the structure-selective endonucleases Mus81-Mms4, Slx1-Slx4, and Yen1. Conversely, the 3'-flap nuclease Rad1-Rad10 and enzymes known to disrupt recombination intermediates (Sgs1-Top3-Rmi1, Srs2, and Mph1) inhibit MIR. Resolution of MIR intermediates propagates secondary chromosome breaks that frequently cause additional rearrangements. MIR features have implications for the formation of simple and complex rearrangements underlying human pathologies.

Never-in-mitosis A-related kinase 1 (Nek1) has established roles in apoptosis and cell cycle regulation. We show that human Nek1 regulates homologous recombination (HR) by phosphorylating Rad54 at Ser572 in late G2 phase. Nek1 deficiency as well as expression of unphosphorylatable Rad54 (Rad54-S572A) cause unresolved Rad51 foci and confer a defect in HR. Phospho-mimic Rad54 (Rad54-S572E), in contrast, promotes HR and rescues the HR defect associated with Nek1 loss. Although expression of phosphomimic Rad54 is beneficial for HR, it causes Rad51 removal from chromatin and degradation of stalled replication forks in S phase. Thus, G2-specific phosphorylation of Rad54 by Nek1 promotes Rad51 chromatin removal during HR in G2 phase, and its absence in S phase is required for replication fork stability. In summary, Nek1 regulates Rad51 removal to orchestrate HR and replication fork stability.

The RAD54 family DNA translocases have several biochemical activities. One activity, demonstrated previously for the budding yeast translocases, is ATPase-dependent disruption of RAD51-dsDNA binding. This activity is thought to promote dissociation of RAD51 from heteroduplex DNA following strand exchange during homologous recombination. In addition, previous experiments in budding yeast have shown that the same activity of Rad54 removes Rad51 from undamaged sites on chromosomes; mutants lacking Rad54 accumulate nonrepair-associated complexes that can block growth and lead to chromosome loss. Here, we show that human RAD54 also promotes the dissociation of RAD51 from dsDNA and not ssDNA. We also show that translocase depletion in tumor cell lines leads to the accumulation of RAD51 on chromosomes, forming complexes that are not associated with markers of DNA damage. We further show that combined depletion of RAD54L and RAD54B and/or artificial induction of RAD51 overexpression blocks replication and promotes chromosome segregation defects. These results support a model in which RAD54L and RAD54B counteract genome-destabilizing effects of direct binding of RAD51 to dsDNA in human tumor cells. Thus, in addition to having genome-stabilizing DNA repair activity, human RAD51 has genome-destabilizing activity when expressed at high levels, as is the case in many human tumors.

Mus81‐Mms4 is a structure‐selective endonuclease that cleaves joint DNA molecules that form during homologous recombination in mitotic and meiotic cells. Here, we demonstrate by kinetic analysis using physically tethered DNA substrates that budding yeast Mus81‐Mms4 requires inherent flexibility in DNA substrates for optimal catalysis. Förster Resonance Energy Transfer (FRET) experiments further reveal that recognition of 3’‐flap and nicked Holliday junction substrates by Mus81‐Mms4 involves induction of a sharp bend with a 100° angle between the two duplex DNA arms. In addition, thiol crosslinking of Mus81‐Mms4 bound to DNA substrates demonstrates that the heterodimer undergoes a conformational change induced by joint DNA molecules with preferred structural properties. The results from all three approaches support a model for catalysis by Mus81‐Mms4 in which initial DNA binding is based on minimal structural requirements and is followed by a rate‐limiting conformational transition of the substrate and protein, which leads to a sharply kinked substrate that may fray the DNA four bp away from the junction point to position the nuclease for cleavage between the fourth and fifth nucleotides. These data suggest that mutually compatible conformational changes of Mus81‐Mms4 and its substrates tailor its incision activity to nicked junction molecules, resulting in products that cannot be directly ligated.Grant Funding Source: Supported by NIH and TRDRP

The displacement loop (D loop) is the product of homology search and DNA strand invasion, constituting a central intermediate in homologous recombination (HR). In eukaryotes, the Rad51 DNA strand exchange protein is assisted in D loop formation by the Rad54 motor protein. Curiously, Rad54 also disrupts D loops. How these opposing activities are coordinated toward productive recombination is unknown. Moreover, a seemingly disparate function of Rad54 is removal of Rad51 from heteroduplex DNA (hDNA) to allow HR-associated DNA synthesis. Here, we uncover features of D loop formation/dissociation dynamics, employing Rad51 filaments formed on ssDNAs that mimic the physiological length and structure of in vivo substrates. The Rad54 motor is activated by Rad51 bound to synapsed DNAs and guided by a ssDNA-binding domain. We present a unified model wherein Rad54 acts as an hDNA pump that drives D loop formation while simultaneously removing Rad51 from hDNA, consolidating both ATP-dependent activities of Rad54 into a single mechanistic step.

Mus81-Mms4/EME1 is a DNA structure-selective endonuclease that cleaves joint DNA molecules that form during homologous recombination in mitotic and meiotic cells. Here, we demonstrate by kinetic analysis using physically tethered DNA substrates that budding yeast Mus81-Mms4 requires inherent rotational flexibility in DNA junctions for optimal catalysis. Forster Resonance Energy Transfer experiments further reveal that recognition of 3'-flap and nicked Holliday junction substrates by Mus81-Mms4 involves induction of a sharp bend with a 100. angle between two duplex DNA arms. In addition, thiol crosslinking of Mus81-Mms4 bound to DNA junctions demonstrates that the heterodimer undergoes a conformational change induced by joint DNA molecules with preferred structural properties. The results from all three approaches suggest a model for catalysis by Mus81-Mms4 in which initial DNA binding is based on minimal structural requirements followed by a rate-limiting conformational transition of the substrate and protein. This leads to a sharply kinked DNA molecule that may fray the DNA four base pairs away from the junction point to position the nuclease for cleavage between the fourth and fifth nucleotide. These data suggest that mutually compatible conformational changes of Mus81-Mms4 and its substrates tailor its incision activity to nicked junction molecules.

The formation of crossovers is a fundamental genetic process. The XPF-family endonuclease Mus81-Mms4 (Eme1) contributes significantly to crossing over in eukaryotes. A key question is whether Mus81-Mms4 can process Holliday junctions that contain four uninterrupted strands. Holliday junction cleavage requires the coordination of two active sites, necessitating the assembly of two Mus81-Mms4 heterodimers. Contrary to this expectation, we show that Saccharomyces cerevisiae Mus81-Mms4 exists as a single heterodimer both in solution and when bound to DNA substrates in vitro. Consistently, immunoprecipitation experiments demonstrate that Mus81-Mms4 does not multimerize in vivo. Moreover, chromatin-bound Mus81-Mms4 does not detectably form higher-order multimers. We show that Cdc5 kinase activates Mus81-Mms4 nuclease activity on 3' flaps and Holliday junctions in vitro but that activation does not induce a preference for Holliday junctions and does not induce multimerization of the Mus81-Mms4 heterodimer. These data support a model in which Mus81-Mms4 cleaves nicked recombination intermediates such as displacement loops (D-loops), nicked Holliday junctions, or 3' flaps but not intact Holliday junctions with four uninterrupted strands. We infer that Mus81-dependent crossing over occurs in a noncanonical manner that does not involve the coordinated cleavage of classic Holliday junctions.

Structure-selective nucleases perform DNA strand incisions crucial to the repair/resolution of branched DNA molecules arising during DNA replication, recombination, and repair. From a combination of genetics and in vitro nuclease assay studies, we are just beginning to understand how these enzymes recognize their substrates and to identify their in vivo DNA structure targets. By performing nuclease assays on a variety of substrates meant to mimic cellular intermediates, structural features of branched DNA molecules that are important for robust catalysis can be defined. However, since these enzymes often are capable of cleaving a range of DNA structures, caution must be taken not to overemphasize the significance of incision of a certain structure before a careful and detailed kinetic analysis of a variety of DNA substrates with different polarities and structural features has been completed. Here, we provide protocols for the production of a variety of oligo-based DNA joint molecules and their use in endonuclease assays, which can be used to derive the kinetic parameters KM and kcat. Determination of these values for a variety of substrates provides meaningful comparisons that allow inferences to be made regarding in vivo DNA structure target(s).