Generation of double Holliday junction DNAs and their dissolution/resolution within a chromatin context

Four-way DNA intermediates, also known as Holliday junctions (HJs), are formed during homologous recombination and DNA repair, and their resolution is necessary for proper chromosome segregation. To facilitate the biochemical analysis of HJ processing, we developed a method involving DNAzyme self-cleavage to generate 1.8-kb DNA molecules containing either single (sHJ) or double Holliday junctions (dHJs). We show that dHJ DNAs (referred to as HoJo DNAs) are dissolved by the human BLM–TopIIIα–RMI1–RMI2 complex to form two noncrossover products. However, structure-selective endonucleases (human GEN1 and SMX complex) resolve DNA containing single or double HJs to yield a mixture of crossover and noncrossover products. Finally, we demonstrate that chromatin inhibits the resolution of the double HJ by GEN or SMX while allowing BTRR-mediated dissolution.

Homologous recombination (HR) provides an important mechanism for the repair of DNA double-stranded breaks and the restoration of broken replication forks (1). Recombination in mitotic cells generally occurs between sister chromatids and can lead to the formation of DNA intermediates in which the sisters are covalently linked by four-way DNA junctions, known as Holliday junctions (HJs) (2). Failure to process these DNA intermediates leads to improper chromosome segregation and cell death (3, 4). Recombination also plays an important role in meiotic cells, when interactions occur between homologous chromosomes, and is responsible for the generation of genetic diversity.In mitotic cells, HJs are primarily processed by “dissolution,” in which two adjacent HJs (double Holliday junctions dHJs]) converge in an adenosine triphosphate (ATP)-hydrolysis–dependent reaction to form a hemicatenane that is subsequently decatenated by topoisomerase action. In human cells, this two-step process involves the BLM–topoisomerase IIIα–RMI1–RMI2 (BTRR) complex (5–7). In yeast, similar reactions are driven by the Sgs1–Top3–Rmi1 (STR) complex (8, 9). Dissolution yields exclusively noncrossover products, which help to maintain the heterozygous state, as a loss of heterozygosity can cause cancer development (10). Mutations in the BLM gene are linked to a human inherited disorder known as Bloom syndrome, which is characterized by short stature, sensitivity to sunlight, and a greatly increased risk of a broad range of cancers (11, 12). In the clinic, patients with Bloom syndrome are diagnosed by a cytogenetic test that detects elevated levels of sister chromatid exchanges. Pathogenic mutations in the TOP3A and RMI1 genes also cause a Bloom syndrome–like disorder, consistent with the fact that these genes participate in the same molecular pathway (12).Persistent HJs that escape processing by BTRR, and single Holliday junctions (sHJs), are resolved by structure-selective endonucleases (SSEs), which specifically recognize and cleave HJs by mediating a nucleolytic attack on two opposing strands at the junction point (2). In humans, these endonucleases include GEN1 and the SMX trinuclease, comprising SLX1–SLX4–MUS81–EME1–XPF–ERCC1 (3, 13–15). Unlike dissolution, resolution gives rise to both crossover and noncrossover products, thereby elevating the frequency of sister chromatid exchanges and increasing the potential for loss of heterozygosity.In contrast to BTRR-mediated dHJ dissolution, which is active throughout the cell cycle, the actions of SMX and GEN1 are tightly regulated. Firstly, SMX complex formation is restricted to prometaphase, as it is dependent upon the phosphorylation of EME1 by CDK1 and PLK1, which stimulates its association with the SLX4 scaffold (15, 16). Secondly, GEN1 is mainly sequestered from the cell nucleus and gains access to DNA after the breakdown of the nuclear envelope during cell division (17).Despite the importance of the BTRR complex in maintaining genetic stability, a detailed picture of dissolution is lacking. Mechanistic studies, using protein complexes from various organisms, led to a model in which two HJs are converged by the branch migration activity of the BLM helicase (18–20). Convergent migration generates positive supercoiling that is relaxed by topoisomerase IIIα and generates a hemicatenane that is processed by topoisomerase IIIα with the aid of RMI1–RMI2 (6–9, 21).Studies of dHJ dissolution have utilized two model systems: 1) a small dHJ prepared by annealing two synthetic oligos (5–7), and 2) a larger plasmid-sized molecule in which two HJs are separated by 165 bp (20, 22). However, the small size of the synthetic DNA substrate eliminates any possibility for branch migration as the two HJs are separated by only 14 bp, raising concerns as to whether these substrates recapitulate the physiological aspects of dissolution (19). The plasmid-sized substrate has been utilized for the dissolution of dHJs by Saccharomyces cerevisiae STR, Drosophila melanogaster BTR, and more recently human BTRR complex (8, 20, 23). However, there is the significant drawback that generation of this substrate is laborious (taking several weeks), requires purified Cre recombinase and reverse gyrase, and leads to low yields of product.To facilitate mechanistic analysis of dissolution and resolution, we developed a rapid and scalable methodology to prepare a 1.8-kb DNA containing single or double HJs. In the dHJ molecules, the two HJs are separated by a maximum of 746 bp of homologous sequence, allowing the two HJs to migrate within the region of homology. We demonstrate that these dHJ molecules are efficiently dissolved by the human BTRR complex to generate noncrossover products. We also show that GEN1 or SMX resolves the single or double HJs to yield the expected mixture of crossover and noncrossover products. Finally, we find that GEN1/SMX are unable to resolve HJs on chromatinized templates, whereas BTRR-mediated dissolution events are unaffected by nucleosome assembly, potentially indicative of an additional level of regulatory control that favors dissolution over resolution.