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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 119; no. 6; p. 1
• Main Authors: Hodson, Charlotte, Low, Jason K K, van Twest, Sylvie, Jones, Samuel E, Swuec, Paolo, Murphy, Vincent, Tsukada, Kaima, Fawkes, Matthew, Bythell-Douglas, Rohan, Davies, Adelina, Holien, Jessica K, O'Rourke, Julienne J, Parker, Benjamin L, Glaser, Astrid, Parker, Michael W, Mackay, Joel P, Blackford, Andrew N, Costa, Alessandro, Deans, Andrew J
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 08.02.2022
• Subjects: Alleles; Assembly; Biological Sciences; Bloom Syndrome - genetics; Bloom's syndrome; Breast cancer; Carrier Proteins - genetics; Cell Line; Crosslinking; Dimerization; Dimers; Dissolution; DNA helicase; DNA Topoisomerases, Type I - genetics; DNA, Cruciform - genetics; Genomes; Genomic instability; Genomic Instability - genetics; Holliday junctions; Homologous recombination; Homology; Humans; Intermediates; Mass spectrometry; Mass spectroscopy; Mutation; Mutation - genetics; Protein Binding - genetics; Recombination, Genetic - genetics; RecQ Helicases - genetics; RecQ protein; Solubility; Stability; Stoichiometry; genome stability; topoisomerase; helicase; cross-link mass spectrometry; Bloom’s complex
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2109093119

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.