Poster Presentation 50th Lorne Proteins Conference 2025

Structural and biochemical analysis of the Bloom Syndrome complex (#330)

Shraddha Kameshwar 1 2 3 , Rohan Bythell-Douglas 2 , Shabih Shakeel 3 , Andrew Deans 2
  1. University of Melbourne, Melbourne, VIC, Australia
  2. St. Vincent's Institute of Medical Research, Fitzroy, Melbourne, VIC, Australia
  3. Walter and Eliza Hall Institute , Parkville, Melbourne, VIC, Australia

 

BLM helicase associates with the topoisomerase TOP3A and genome instability proteins RMI1 and RMI2 to form the Bloom Syndrome (BTRR) complex. This complex plays a crucial role in DNA unwinding and dissolving double Holliday junctions (dHJs) which are key intermediates of homologous recombination, a DNA double-strand break repair pathway1. Mutations in subunits of this complex cause cancer-predisposing Bloom’s syndrome or Bloom syndrome-like disorders2,3. However, structurally how the complex assembles in cells is still a mystery. We have used single particle electron microscopy (EM) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to gain a better structural and functional understanding of the TOP3A-RMI1-RMI2 (TRR) subcomplex protein interaction with each other and in the presence of DNA.

Using negative-stain EM we generated a preliminary low-resolution 3D reconstruction of the TRR complex, which revealed structural features consistent with published crystal structures of individual subunit fragments. We then characterised the interaction of TRR with DNA by establishing conditions for a 1:1 complex with a DNA substrate and employing HDX-MS to identify regions undergoing conformational changes upon DNA binding. Our analysis revealed that all four domains of the TOP3A subunit showed significant conformational changes in terms of exposure and protection in the presence of DNA. Four such regions of significant protection were mapped to the poorly categorised C-terminus of TOP3A that harbours multiple predicted zinc-finger domains. These domains have been reported to direct topoisomerases to Holliday junction-like DNA structures in different species4,5. RMI1 also showed significant conformational changes, including in its decatenation loop, when bound to DNA however RMI2 does not appear to be involved in DNA interactions. These findings have helped identify specific peptide regions likely crucial for DNA binding. Future studies will examine BTRR using similar methods to understand how the presence of BLM contributes to structural changes in the complex.

  1. Bythell-Douglas R, Deans AJ. A Structural Guide to the Bloom Syndrome Complex. Structure. 2021;29(2):99-113. doi:10.1016/j.str.2020.11.020
  2. Shahrabani-Gargir L, Shomrat R, Yaron Y, Orr-Urtreger A, Groden J, Legum C. High Frequency of a Common Bloom Syndrome Ashkenazi Mutation Among Jews of Polish Origin. Genetic Testing. 1998;2(4):293-296. doi:10.1089/gte.1998.2.293
  3. Martin CA, Sarlós K, Logan CV, et al. Mutations in TOP3A Cause a Bloom Syndrome-like Disorder. The American Journal of Human Genetics. 2018;103(2):221-231. doi:10.1016/j.ajhg.2018.07.001
  4. Ahumada A, Tse-Dinh YC. The role of the Zn(II) binding domain in the mechanism of E. coli DNA topoisomerase I. BMC Biochem. 2002;3(1):13. doi:10.1186/1471-2091-3-13
  5. Dorn A, Röhrig S, Papp K, et al. The topoisomerase 3α zinc-finger domain T1 of Arabidopsis thaliana is required for targeting the enzyme activity to Holliday junction-like DNA repair intermediates. Copenhaver GP, ed. PLoS Genet. 2018;14(9):e1007674. doi:10.1371/journal.pgen.1007674