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. 2023 Feb;299(2):102802.
doi: 10.1016/j.jbc.2022.102802. Epub 2022 Dec 16.

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

Michael M Soniat  1 , Giaochau Nguyen  2 , Hung-Che Kuo  2 , Ilya J Finkelstein  3
Affiliations

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

Michael M Soniat et al. J Biol Chem. 2023 Feb.

Abstract

DNA resection-the nucleolytic processing of broken DNA ends-is the first step of homologous recombination. Resection is catalyzed by the resectosome, a multienzyme complex that includes bloom syndrome helicase (BLM), DNA2 or exonuclease 1 nucleases, and additional DNA-binding proteins. Although the molecular players have been known for over a decade, how the individual proteins work together to regulate DNA resection remains unknown. Using single-molecule imaging, we characterized the roles of the MRE11-RAD50-NBS1 complex (MRN) and topoisomerase IIIa (TOP3A)-RMI1/2 during long-range DNA resection. BLM partners with TOP3A-RMI1/2 to form the BTRR (BLM-TOP3A-RMI1/2) complex (or BLM dissolvasome). We determined that TOP3A-RMI1/2 aids BLM in initiating DNA unwinding, and along with MRN, stimulates DNA2-mediated resection. Furthermore, we found that MRN promotes the association between BTRR and DNA and synchronizes BLM and DNA2 translocation to prevent BLM from pausing during resection. Together, this work provides direct observation of how MRN and DNA2 harness the BTRR complex to resect DNA efficiently and how TOP3A-RMI1/2 regulates the helicase activity of BLM to promote efficient DNA repair.

Keywords: BLM; DNA curtains; DNA resection; DNA2; double-strand break; single molecule.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
MRN and TOP3A–RMI1/2 stimulate DNA2-mediated resection.A, schematic of the human resectosome, consisting of MRE11–RAD50–NBS1 (MRN; orange), the nucleases DNA2 or EXO1 (yellow), BLM–TOP3A–RMI1/2 (blue), and replication protein A (RPA; brown). B, schematic of single-molecule resection assay (C) representative kymographs of MRN (magenta), BTRR (green), and DNA2 or EXO1 resecting DNA. D, velocities and (E) processivities of BLM–DNA2 with and without TOP3A–RMI1/2 or MRN complex (n > 50 for all experiments). Black bars show the interquartile range (thick bars) and 1.5× interquartile range (thin bars). The black dot in the middle is the median. F, velocities and (G) processivities of BLM–EXO1 with and without TOP3A–RMI1/2 or MRN complex (n > 50 for all experiments). (Not significant; ns, p > 0.05; ∗∗∗∗p < 0.0001). BTRR, BLM–TOP3A–RMI1/2; EXO1, exonuclease 1; MRN, MRE11–RAD50–NBS1; TOP3A, topoisomerase IIIa.
Figure 2
Figure 2
MRN and TOP3A–RMI1/2 recruit DNA2 to free DNA ends.A, ratio of MRN–BTRR–DNA2 velocities and (B) processivities with nuclease-deficient (nd) or helicase-deficient (hd) DNA2 mutants. Both velocity and processivity are compared with the ratios of MRN–BTRR–DNA2 wildtype complex from Figure 1, D and E. (ns, p > 0.05; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001). C, representative kymographs of colocalization of DNA2 (magenta) with MRN and TOP3A–RMI1/2 (TRR; green) at DNA ends. MRN and TRR were imaged in different experiments to guarantee an unambiguous fluorescent signal. D, Venn diagram that shows MRN and TOP3A–RMI1/2 colocalize DNA2 at free DNA ends. This interaction greatly increases the number of DNA-bound DNA2 molecules. E, representative kymograph showing helicase activity of nd DNA2 (D277A) mutant in the presence of RPA. F, velocities and (G) processivities of DNA2 (D277A) helicase activity with and without TOP3A–RMI1/2 or MRN complex. (ns, p > 0.05; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001). BTRR, BLM–TOP3A–RMI1/2; MRN, MRE11–RAD50–NBS1; ns, not significant; RPA, replication protein A; TOP3A, topoisomerase IIIa.
Figure 3
Figure 3
TOP3A–RMI1/2 complex promotes initiation of BLM helicase activity.A, representative kymographs showing helicase activity of BLM (magenta) with the TOP3A–RMI1/2 complex (green) along DNA. B, TOP3A–RMI1/2 stimulates BLM helicase initiation. Error bars represent SD as determined by bootstrap analysis. C, velocities and (D) processivities of the helicase activity of the BTRR complex. (ns, p > 0.05; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001). BLM, bloom syndrome helicase; BTRR, BLM–TOP3A–RMI1/2; ns, not significant; TOP3A, topoisomerase IIIa.
Figure 4
Figure 4
MRN prevents BTRR DNA dissociation.A, representative kymographs showing the helicase activity of BLM (magenta) with MRN (green) and the TOP3–RMI1/2 complex (dark) along DNA. B, helicase initiation by the BTRR complex with the indicated MRN subcomplexes in the presence of RPA. The MRN mutant (mut) encodes RAD50(S1202R). n > 40 molecules for all conditions measured across at least two flow cells. Error bars represent SD as determined by bootstrap analysis. C, MR is sufficient to retain the BTRR complex on DNA. In the presence of MR(N), BTRR rarely dissociates from the DNA during the 30 min experiment. D, velocities and (E) processivities of the BTRR complex with the indicated MRN variants. F, Representative particle tracking of BTRR/DNA2 resection with (purple) and without MRN (blue). G, MRN decreases pausing events during BTRR–DNA2 resection. H and I, relative resectosome velocities and processivities with the indicated MRN mutants. The velocity and processivity are normalized to wildtype resectosome complexes. n > 40 molecules for all conditions measured across at least two flow cells (ns, p > 0.05; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001). BLM, bloom syndrome helicase; BTRR, BLM–TOP3A–RMI1/2; MR, MRE11–RAD50; MRN, MRE11–RAD50–NBS1; ns, not significant; RPA, replication protein A; TOP3, topoisomerase IIIa.
Figure 5
Figure 5
Model of how MRN regulates DNA resections.
See this image and copyright information in PMC

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