BRCA2 chaperones RAD51 to single molecules of RPA-coated ssDNA

Contributed by Stephen C. Kowalczykowski; received December 28, 2022; accepted February 24, 2023; reviewed by William K. Holloman and Scott W. Morrical

March 28, 2023

120 (14) e2221971120

Significance

Despite decades of genetic and cell biological studies, mechanistic biochemical analyses of human BRCA2 function in recombinational DNA repair have only been possible since the purification of full-length BRCA2. These mechanistic studies crucially inform with respect to the molecular function of BRCA2 in genome maintenance. Here, we use single-molecule methods to visualize the assembly of RAD51 on individual molecules of single-stranded DNA (ssDNA) coated with replication protein-A (RPA) and to see how this process is regulated by the tumor suppressor protein, BRCA2. We show that BRCA2 serves as a chaperone to nucleate RAD51 and deliver it to RPA-coated ssDNA. This work advances understanding of the molecular functions of BRCA2 and, consequently, the molecular etiology of breast cancer in an important way.

Abstract

Mutations in the breast cancer susceptibility gene, BRCA2, greatly increase an individual’s lifetime risk of developing breast and ovarian cancers. BRCA2 suppresses tumor formation by potentiating DNA repair via homologous recombination. Central to recombination is the assembly of a RAD51 nucleoprotein filament, which forms on single-stranded DNA (ssDNA) generated at or near the site of chromosomal damage. However, replication protein-A (RPA) rapidly binds to and continuously sequesters this ssDNA, imposing a kinetic barrier to RAD51 filament assembly that suppresses unregulated recombination. Recombination mediator proteins—of which BRCA2 is the defining member in humans—alleviate this kinetic barrier to catalyze RAD51 filament formation. We combined microfluidics, microscopy, and micromanipulation to directly measure both the binding of full-length BRCA2 to—and the assembly of RAD51 filaments on—a region of RPA-coated ssDNA within individual DNA molecules designed to mimic a resected DNA lesion common in replication-coupled recombinational repair. We demonstrate that a dimer of RAD51 is minimally required for spontaneous nucleation; however, growth self-terminates below the diffraction limit. BRCA2 accelerates nucleation of RAD51 to a rate that approaches the rapid association of RAD51 to naked ssDNA, thereby overcoming the kinetic block imposed by RPA. Furthermore, BRCA2 eliminates the need for the rate-limiting nucleation of RAD51 by chaperoning a short preassembled RAD51 filament onto the ssDNA complexed with RPA. Therefore, BRCA2 regulates recombination by initiating RAD51 filament formation.

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Acknowledgments

We would like to thank all the members of the Kowalczykowski lab for insightful comments and discussion. J.C.B. was supported in part by the National Institutes of Health – University of California Davis (NIH-UCD) Training Fellowship in Molecular and Cell Biology (T32 GM007377). C.C.D. and J.L.P. were supported by the NIH-UCD T32 Training Program in Oncogenic Signals and Chromosome Biology (CA10052159). J.L.P. was also supported by the NIH F32 Ruth L. Kirschstein National Research Service Award (CA136103). R.B.J. was supported by the American Cancer Society (#IRG 58-012-55); Breast Cancer Alliance; Pilot Project Program grant from Women’s Health Research at Yale; the Yale Comprehensive Cancer Center; and a Liz Tilberis Early Career Award from the Ovarian Cancer Research Fund Alliance. S.C.K. was supported by NIH (GM62653, GM64745, and R35 GM131900) and DOD-CDMRP (BC171869).

Author contributions

J.C.B., C.C.D., J.L.P., R.B.J., and S.C.K. designed research; J.C.B. and C.C.D. performed research; J.C.B., C.C.D., J.L.P., and R.B.J. contributed new reagents/analytic tools; J.C.B., C.C.D., J.L.P., R.B.J., and S.C.K. analyzed data; and J.C.B. and S.C.K. wrote the paper.

Competing interests

The authors declare no competing interest.

Supporting Information

Movie 1.

Binding of BRCA2 on gapped λDNA. Movie of a single gapped λDNA molecule held between two optically trapped polystyrene beads in a 6-channel flow cell as described and shown in Supplementary Figure 1. The molecule was imaged in Channel 5 and iteratively dipped into Channel 6, containing BRCA2 (purple), between images in the montage. The BRCA2 appears to slide in the direction if flow (left to right) between 32 s and 64 s towards the junction between the dsDNA and RPA-coated ssDNA. At the end of the movie, the gapped λDNA was re-stained in Channel 3.

Movie 2.

RAD51 binding to ssDNA in the absence of RPA using TIRF microscopy. A single gapped λDNA molecule was initially visualized using SYTOX Orange (red), which was subsequently dissociated upon the addition of binding buffer and fluorescent RAD51 (green) in the absence of RPA. Movie was collected as described in Fig. 2A and corresponds to the montage shown in Fig. 2B.

Movie 3.

RAD51 binding to ssDNA in the presence of RPA using TIRF microscopy. A single gapped λDNA molecule was initially visualized using SYTOX Orange (red), which was subsequently dissociated upon the addition of binding buffer and fluorescent RAD51 (green) in the presence of RPA. Movie was collected as described in Fig. 2A and corresponds to the montage shown in Fig. 2C.

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Go to Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences

Vol. 120 | No. 14
April 4, 2023

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Data, Materials, and Software Availability

Submission history

Received: December 28, 2022

Accepted: February 24, 2023

Published online: March 28, 2023

Published in issue: April 4, 2023

Keywords

DNA recombination
DNA repair
breast cancer
RAD51
single-molecule visualization

Acknowledgments

Author Contributions

Competing Interests

The authors declare no competing interest.

Notes

Reviewers: W.K.H., Weill Cornell Medicine; and S.W.M., University of Vermont.