Super-Resolution Imaging of Homologous Recombination Repair at Collapsed Replication Forks

Whelan, Donna R.; Rothenberg, Eli

doi:10.1007/978-1-0716-0644-5_24

Cited by 2 publications

(3 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To induce RF stress and seDSB generation, cells were then treated with 100 nM CPT (Abcam; 120115) (11). Cells were preextracted and fixed as described previously (34,40,74) immediately following damage or released back into drug-free complete medium for a further 1, 2, 4, 8, 12, or 16 h. To inhibit MRE11 exo-/endonuclease ability, cells were cultured in the presence of either 25 μM Mirin (Fisher; 319010) or 25 μM PFM01 (Sigma; SML1735), respectively.…”

Section: Methodsmentioning

confidence: 99%

“…Previously, we established powerful assays that combined single-molecule super-resolution (SR) imaging with pulse labeling of DNA to visualize the recruitment and localization of DNA damage response proteins to DNA DSBs (27,(32)(33)(34). Using this approach, we were able to describe the extended timeline of HR repair at seDSBs as well as the specific roles and cross-talk of key HR proteins including BRCA2, RAD51, and RAD52 (27).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Super-resolution mapping of cellular double-strand break resection complexes during homologous recombination

Whelan

Rothenberg

2021

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Homologous recombination (HR) is a major pathway for repair of DNA double-strand breaks (DSBs). The initial step that drives the HR process is resection of DNA at the DSB, during which a multitude of nucleases, mediators, and signaling proteins accumulates at the damage foci in a manner that remains elusive. Using single-molecule localization super-resolution (SR) imaging assays, we specifically visualize the spatiotemporal behavior of key mediator and nuclease proteins as they resect DNA at single-ended double-strand breaks (seDSBs) formed at collapsed replication forks. By characterizing these associations, we reveal the in vivo dynamics of resection complexes involved in generating the long single-stranded DNA (ssDNA) overhang prior to homology search. We show that 53BP1, a protein known to antagonize HR, is recruited to seDSB foci during early resection but is spatially separated from repair activities. Contemporaneously, CtBP-interacting protein (CtIP) and MRN (MRE11-RAD51-NBS1) associate with seDSBs, interacting with each other and BRCA1. The HR nucleases EXO1 and DNA2 are also recruited and colocalize with each other and with the repair helicase Bloom syndrome protein (BLM), demonstrating multiple simultaneous resection events. Quantification of replication protein A (RPA) accumulation and ssDNA generation shows that resection is completed 2 to 4 h after break induction. However, both BRCA1 and BLM persist later into HR, demonstrating potential roles in homology search and repair resolution. Furthermore, we show that initial recruitment of BRCA1 and removal of Ku are largely independent of MRE11 exonuclease activity but dependent on MRE11 endonuclease activity. Combined, our observations provide a detailed description of resection during HR repair.

show abstract

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Super-resolution mapping of cellular double-strand break resection complexes during homologous recombination

Whelan

Rothenberg

2021

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…We and others have also developed novel SMLM assays and analyses, including nascent DNA pulse labelling and multi-colour immunolabelling, to map the spatiotemporal progression of different DSB repair pathways [76,[140][141][142][143][144]. Reid et al used both SMLM and single molecule Föster resonance energy transfer experiments to characterize the sequential steps involved in NHEJ, including the generation of XRCC4, XLF and DNA ligase IV filaments (figures 9(A)-(C)) [145].…”

Section: The Effects Of Dna Damage and Repair On Chromatin Structurementioning

confidence: 99%

Understanding DNA organization, damage, and repair with super-resolution fluorescence microscopy

Miriklis

Rozario

Rothenberg

et al. 2021

Methods Appl. Fluoresc.

Self Cite

View full text Add to dashboard Cite

Super-resolution microscopy (SRM) comprises a suite of techniques well-suited to probing the nanoscale landscape of genomic function and dysfunction. Offering the specificity and sensitivity that has made conventional fluorescence microscopy a cornerstone technique of biological research, SRM allows for spatial resolutions as good as 10 nanometers. Moreover, single molecule localization microscopies (SMLMs) enable examination of individual molecular targets and nanofoci allowing for the characterization of subpopulations within a single cell. This review describes how key advances in both SRM techniques and sample preparation have enabled unprecedented insights into DNA structure and function, and highlights many of these new discoveries. Ongoing development and application of these novel, highly interdisciplinary SRM assays will continue to expand the toolbox available for research into the nanoscale genomic landscape.

show abstract

Super-Resolution Imaging of Homologous Recombination Repair at Collapsed Replication Forks

Cited by 2 publications

References 19 publications

Super-resolution mapping of cellular double-strand break resection complexes during homologous recombination

Super-resolution mapping of cellular double-strand break resection complexes during homologous recombination

Understanding DNA organization, damage, and repair with super-resolution fluorescence microscopy

Contact Info

Product

Resources

About