Rmi1 stimulates decatenation of double Holliday junctions during dissolution by Sgs1-Top3

Abstract

A double Holliday junction (dHJ) is a central intermediate of homologous recombination that can be processed to yield crossover or non-crossover recombination products. To preserve genomic integrity, cells possess mechanisms to avoid crossing over. We show that Saccharomyces cerevisiae Sgs1 and Top3 proteins are sufficient to migrate and disentangle a dHJ to produce exclusively non-crossover recombination products, in a reaction termed "dissolution." We show that Rmi1 stimulates dHJ dissolution at low Sgs1-Top3 protein concentrations, although it has no effect on the initial rate of Holliday junction (HJ) migration. Rmi1 serves to stimulate DNA decatenation, removing the last linkages between the repaired and template DNA molecules. Dissolution of a dHJ is a highly efficient and concerted alternative to nucleolytic resolution that prevents crossing over of chromosomes during recombinational DNA repair in mitotic cells and thereby contributes to genomic integrity.

Figure 1

Sgs1 and Top3, stimulated by replication protein A (RPA), dissolve double Holliday junctions to yield non-crossover products. (a) A schematic representation of the double Holliday junction substrate (DHJS) showing dissolution by convergent branch migration or resolution by nucleolytic cleavage. Dissolution of the dHJ leads exclusively to monomeric non-crossover products (denoted as A and B). Resolution leads to both monomeric non-crossover products (A and B) and a dimeric crossover product (A/B dimer). The arrows indicate positions of a BamHI restriction site. (b) Sgs1 (6 nM) and/or Top3 (36 or 360 nM) and RPA (578 nM) were incubated with the DHJS as described in Methods. The expected reaction products, A and B markers, are loaded in lanes 7 and 8, respectively. To improve resolution, the assay products were digested with BamHI prior to electrophoretic analysis. (c) Processing of DHJS requires catalytically active Sgs1 and ATP. Wild type Sgs1 or ATPase-dead Sgs1 (K706A) mutant (both 6 nM) were incubated with Top3 (36 nM) with or without ATP, as indicated, in the presence of RPA. (d) DHJS dissolution catalyzed by Sgs1, Top3, and RPA leads exclusively to non-crossover products. The DHJS was incubated with Sgs1 (36 nM), Top3 (77 nM), and RPA. The reaction products were purified, and analyzed by electrophoresis without prior digestion by BamHI. (e) The dissolution of dHJs is optimal with the cognate Sgs1 and Top3 protein pair. Dissolution reactions were carried out with RPA (578 nM), Top3 (36 nM), and either yeast Sgs1 (6 nM, lane 2), human BLM (6 and 36 nM, lanes 3 and 4, respectively), E. coli RecQ (6 and 36 nM, lanes 5 and 6, respectively), or another S. cerevisiae helicase with 3′-5′ unwinding polarity, Srs2 (6 and 36 nM, lanes 7 and 8, respectively). The “CBM-intermediate” denotes the convergently branch-migrated intermediate with a reduced mobility relative to the DHJS. Some preparations of DHJS possess a small fraction of this intermediate after synthesis due to the action of reverse gyrase (which contains a type IA topoisomerase domain) in the final step of substrate synthesis. (f) Dissolution reactions were carried out with RPA (578 nM), Sgs1 (6 nM), and either yeast Top3 (36 nM, lane 2), human Topo IIIα (36 and 100 nM, lanes 3 and 4, respectively), E. coli Topo I (2.5 and 10 units, lanes 5 and 6, respectively), or wheat germ Topo I (2.5 and 10 units, lanes 7 and 8, respectively). The “CBM-intermediate” denotes the convergently branch-migrated intermediate with a reduced mobility relative to the DHJS.

Figure 2

Rmi1 stimulates a late step of dHJ dissolution catalyzed by Sgs1 and Top3. (a) Schematic representation for the progression of dissolution of the mismatched double Holliday junction substrate (MM-DHJS), showing that branch migration of 35 bp or more can be detected by digestion with restriction endonucleases. Cleavage of the substrate by BamHI results in a molecule that is resistant to further cleavage by SphI, and which migrates between the 800 and 900 bp linear markers. Branch migration of the left Holliday junction past the mismatch restores complementarity at the SphI site; digestion with BamHI produces a single DNA molecule migrating at approximately 900 bp (indicated as intermediate) and additional digestion with SphI cleaves this DNA to produce fragments A1 (265 bp) and A2 (200 bp), which are derived from A DNA, and also the linearized B DNA (416 bp), which lacks an SphI site. This structure is initially conjoined by the HJs; however, upon digestion the linked DNAs quickly dissociate due to spontaneous branch migration of the dHJ. Digestion of the final products of the dissolution reaction by BamHI yields linear bands 465 and 416 bp in length, corresponding to the linearized A and B markers, and subsequent digestion with SphI cleaves the A marker into A1 and A2 DNA fragments as above. (b) The helicase activity of Sgs1 is not sufficient for HJ migration in DHJS. MM-DHJS was incubated with Sgs1 (6 nM) and/or Top3 (36 nM), as indicated. All reactions contained RPA. The combined activity of Sgs1 and Top3 led to the full dissolution of the MM-DHJS (lanes 5 and 9). The helicase activity of Sgs1 alone did not produce dissolution products (lane 3), nor did it allow for HJ migration past the mismatch site (35 nucleotides distant from the nearest HJ position), as indicated by the resistance to SphI cleavage (lane 7). “CBM-intermediate” indicates the position of the DHJS with convergent branch of the HJs. (c) The MM-DHJS was incubated with Sgs1 (6 nM), wheat germ Topo I (10 U) and RPA (578 nM), as in Fig. 1f, to produce the reaction intermediate with a reduced electrophoretic mobility, denoted as a CBM-intermediate; this intermediate was then digested with BamHI and SphI, where indicated. The MM-DHJS is resistant to SphI (lane 3), whereas the Topo I-generated intermediate is sensitive to SphI (lane 5), showing that branch migration occurred. (d) Restriction analysis of the time course dissolution of MM-DHJS. The reactions were carried out with Sgs1 (1 nM), Top3 (1 nM), Rmi1, where indicated (10 nM, denoted as “+Rmi1”), and RPA, and terminated at the indicated times. Branch migration of the Holliday junction past the SphI site is initially identical in reactions without and with Rmi1, and is detected by the SphI-dependent B fragment (indicated by black arrows). Final dissolution products appear almost exclusively in reactions containing Rmi1 at later time points (indicated by white arrows). The contrast of this image was enhanced, compared to other images in this study, to enhance visualization of the faint product DNA bands. The “CBM-intermediate” denotes the convergently branch-migrated intermediate with a reduced mobility relative to the MM-DHJS. (e) Kinetics of MM-DHJS dissolution, based on 3 experiments such as that shown in panel d. Error bars, s.e.m.

Figure 3

Rmi1 greatly stimulates dissolution of dHJ-containing DNA that mimics the final steps of dHJ dissolution. (a) Sgs1 and Top3 can dissolve the oligonucleotide-based dHJ. The concentrations of Top3 in lanes 4-7 were 4, 8, 16 and 32 nM, and the Sgs1 concentrations in lanes 8-11 were 0.4, 0.9, 1.8 and 3.5 nM, respectively. The position of the oligonucleotide-based dHJ and the dissolution product are indicated on the right. The oligonucleotide-based dHJ and the respective dissolution products are schematically represented above the lanes. (b) Dissolution of the oligonucleotide-based dHJ is greatly stimulated by Rmi1 at low Top3 concentrations. Quantification of experiments such as in panel a, with Top3 concentration plotted on a logarithmic scale. The reaction contained Sgs1 (0.22 nM) and, where indicated, Rmi1 (2.7 nM). (c) Stimulation of dissolution by Rmi1. Quantification of experiments such as in panel a, with Rmi1 concentration plotted on a logarithmic scale. The reactions contained Sgs1 (0.56 nM) and Top3 (3.2 nM).

Figure 4

Rmi1 promotes decatenation of kinetoplast DNA (kDNA). (a) The reactions (lanes 4-8) were carried out with Sgs1, Top3, Rmi1 and Top3–Rmi1 heterodimer, as indicated, and RPA (340 nM); kDNA was decatenated with D. melanogaster Topo II as a positive control (lane 3). The positions of interlinked kDNA and the various monomer forms are indicated on the right. The kDNA, a network of dsDNA rings, is schematically represented above the lanes. (b) Decatenation of kDNA is dependent on Rmi1 concentration. The amount of monomeric kDNA (the sum of pixel intensity values, in arbitrary units, based on experiments as shown in Supplementary Fig. 7a) was plotted against Rmi1 concentration. The reactions contained RPA (340 nM), Sgs1 (2 nM), Top3 (2 nM), and the indicated amount of Rmi1. Error bars, s.e.m., based on 4 independent experiments. The lower dashed line indicates the amount of monomer kDNA present in the absence of Rmi1 (mainly monomer DNA present in the substrate DNA, and/or released by Sgs1 unwinding, see panel a), and the upper dashed line indicates the amount of monomer kDNA released by D. melanogaster Topo II. The stimulation of kDNA decatenation is saturated at 2 nM Rmi1, a concentration equimolar to that of Top3 and Sgs1.

Figure 5

Model of Sgs1–Top3–Rmi1 complex function in the dissolution of double Holliday junctions (dHJs). Sgs1 and Top3 catalyze convergent HJ migration. Rmi1 is likely an integral part of the Sgs1–Top3 complex, but its function is dispensable during this initial branch migration phase. Rmi1 stimulates dHJ dissolution at a later stage when both junctions are in close proximity. We cannot distinguish whether Rmi1 promotes dissolution of just the hemi-catenane, or of an intermediate that has several topological linkages. Rmi1 then stimulates the dissolution of a hemi-catenane, the anticipated last intermediate of dHJ dissolution.