Mechanism of Bloom syndrome complex assembly required for double Holliday junction dissolution and genome stability

The RecQ-like helicase BLM cooperates with topoisomerase IIIα, RMI1, and RMI2 in a heterotetrameric complex (the “Bloom syndrome complex”) for dissolution of double Holliday junctions, key intermediates in homologous recombination. Mutations in any component of the Bloom syndrome complex can cause genome instability and a highly cancer-prone disorder called Bloom syndrome. Some heterozygous carriers are also predisposed to breast cancer. To understand how the activities of BLM helicase and topoisomerase IIIα are coupled, we purified the active four-subunit complex. Chemical cross-linking and mass spectrometry revealed a unique architecture that links the helicase and topoisomerase domains. Using biochemical experiments, we demonstrated dimerization mediated by the N terminus of BLM with a 2:2:2:2 stoichiometry within the Bloom syndrome complex. We identified mutations that independently abrogate dimerization or association of BLM with RMI1, and we show that both are dysfunctional for dissolution using in vitro assays and cause genome instability and synthetic lethal interactions with GEN1/MUS81 in cells. Truncated BLM can also inhibit the activity of full-length BLM in mixed dimers, suggesting a putative mechanism of dominant-negative action in carriers of BLM truncation alleles. Our results identify critical molecular determinants of Bloom syndrome complex assembly required for double Holliday junction dissolution and maintenance of genome stability.

Homologous recombination (HR) is an essential and highly regulated cellular process required for maintenance of genome stability during mitosis and sexual reproduction. A key intermediate in HR and recombinational DNA repair is a four-way DNA junction known as a Holliday junction (HJ). During meiosis, double Holliday junctions (dHJs) are cut by endonucleases (resolution) in order to create the cross-overs necessary for proper chromosome segregation (1, 2). In somatic cells, however, cross-overs are potentially deleterious, and their formation is suppressed by the BLM protein (3). Failure to suppress cross-over formation leads to an increased frequency of sister chromatid exchanges and has the potential to result in translocations and/or somatic cell loss of heterozygosity (4), key drivers of genome instability and cancer. As a likely consequence, individuals with homozygous BLM mutations exhibit Bloom syndrome and are highly cancer prone (5). Heterozygous BLM mutation carriers are also significantly overrepresented in breast cancer families (6, 7).The 1,417-amino acid BLM protein suppresses DNA cross-overs by promoting dHJ “dissolution,” a process that unlinks the recombination intermediates back to their prerecombination state (8). Critical to dHJ dissolution by BLM is a RecQ-type superfamily II helicase domain. The ATPase function of this domain is required for DNA unwinding, translocation, and strand exchange within the HJ (9, 10). Dissolution also requires the concerted action of three additional factors, topoisomerase IIIα (TopoIIIα), RMI1, and RMI2. Together, these four proteins form the Bloom syndrome complex (BS complex; also known as the “dissolvasome” or “BTRR [BLM-TopoIIIα-RMI1-RMI2]”) (11). TopoIIIα belongs to the type IA class of topoisomerases, padlock-shaped enzymes that effect changes in DNA topology through a “strand-passage” mechanism (12). When coupled with TopoIIIα, BLM promotes DNA decatenation under physiological conditions by catalyzing DNA unlinking in an ATPase-dependent manner.The ancillary factors RMI1 (625 amino acids) and RMI2 (147 amino acids) are Replication Protein A–like, OB (oligonucleotide binding) fold–containing proteins that stimulate the dissolution reaction and have been implicated in either nucleic acid engagement (13–15) or protein–protein interactions (16–19). In particular, in the yeast dissolvasome complex, RMI1 stabilizes the covalently bound open form of the TopoIIIα DNA gate in a configuration that favors DNA strand passage (10). The significance of these components to BLM function is underscored by recent discoveries of homozygous mutant TOP3Α, RMI1, or RMI2 individuals who have Bloom syndrome–like disorders (20, 21).X-ray crystal structures of the RecQ helicase domain of BLM [encompassing residues 636 to 1298, Protein Data Bank (PDB) ID codes 4CGZ, 4CDG, and 403M (22, 23)] revealed a unique position of the DNA duplex relative to the helicase core and visualization of both pre- and posttranslocation states that flip out at least three bases of single-strand DNA (ssDNA). An additional structure of TopoIIIα (residues 21 to 639) in complex with RMI1 [residues 2 to 216; PDB ID code 4CHT (12)] also revealed the characteristic toroidal structure of type I topoisomerases, with a gate mechanism that is regulated by an insertion loop within the first OB fold of RMI1. While these structures informed on key residues and the biophysical action of each protein, they did not explain how the BS complex assembles or point to an overall mechanism of dHJ dissolution. Key to understanding this mechanism is a molecular characterization of the number and relative position of helicases and topoisomerases within the complex. However, there are contrasting reports in the literature on the oligomeric state of BLM. Size exclusion chromatography and atomic force microscopy support monomeric, dimeric, and larger oligomeric states, while EM data reveal five- or six-lobed structures reminiscent of ring-shaped multimeric ATPases (24–27). The oligomeric state of BLM within the BS complex has never been investigated, although RMI1:RMI2 purified in isolation forms a 1:1 heterodimer in published reports (16, 18, 19).The primary goal of this study was to establish how the BS complex assembles and how this assembly promotes dHJ dissolution and maintenance of genome stability. To achieve this, we purified an active recombinant BS complex. Using a combination of biochemical, biophysical, and cross-linking mass spectrometry (XL-MS) experiments, we determined that recombinant BS complex exists predominantly in a 2:2:2:2 stoichiometry. We found that dimerization of the BS complex and interaction with TopoIIIα/RMI1 are mediated by the N terminus of BLM, and we define critical residues required for these interactions and dHJ dissolution in vitro. To validate our in vitro experiments, we used cell complementation experiments with mutant BLM proteins and showed that these interactions are also required for genome stability in cells.