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

Abstract

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.

Structural characterization of human RPA70N association with DNA damage response proteins

“…If we expand the dimer observed, we could get a string of RPA70N connected by BLMp1, BLMp2 or RMI1. Intriguingly, some of these partner proteins themselves (BTR complex, WRN, ATRIP, p53) are often dimers or oligomers (Cho et al , 1994; Compton et al , 2008; Deshpande et al ., 2017; Hodson et al , 2022). So there are indeed multiple copies of partner peptide in close range.…”

The MRN complex and topoisomerase IIIa–RMI1/2 synchronize DNA resection motor proteins

“…We propose that these physical interactions with BLM increase the processivity of BLM and suppress pausing by anchoring BLM to the ss/ds DNA junction. Similarly, the TRR complex also binds free DNA ends and interacts with BLM ( 34 ). Thus, TRR may also prevent BLM from engaging partially unwound DNA to promote processive helicase activity.…”

“…The human homologs of the S. cerevisiae Top3–Rmi1 are TOP3A (a type 1A topoisomerase) and the heterodimer RMI1–RMI2 (yeast do not encode an RMI2 homolog). These proteins interact with BLM to form the BTRR (BLM–TOP3A–RMI1/2) complex (also known as the BLM dissolvasome) ( 33 , 34 , 35 , 36 , 37 , 38 , 39 ). BTRR participates in DNA resection, double Holliday junction dissolution, resolution of ultrafine bridges, replication fork reversal, and interstrand crosslink repair ( 40 , 41 , 42 , 43 ).…”

MRN and Topoisomerase IIIα-RMI1/2 synchronize DNA resection motor proteins

“…The human homologs of the S. cerevisiae Top3-Rmi1 are TOP3A (a type 1A topoisomerase) and the heterodimer RMI1-RMI2 (yeast don't encode an RMI2 homolog). These proteins interact with BLM to form the BTRR complex (also known as the BLM dissolvasome) (33)(34)(35)(36)(37)(38)(39). BTRR participates in DNA resection, double Holliday junction dissolution, resolution of ultra-fine bridges, replication fork reversal, and interstrand crosslink repair (40)(41)(42)(43).…”