Protective Effect of Vitamin C against Double-Strand Breaks in Reconstituted Chromatin Visualized by Single-Molecule Observation

Abstract Several functions have been proposed for the Escherichia coli DNA polymerase IV (pol IV). Although much research has focused on a potential role for pol IV in assisting pol III replisomes in the bypass of lesions, pol IV is rarely found at the replication fork in vivo. Pol IV is expressed at increased levels in E. coli cells exposed to exogenous DNA damaging agents, including many commonly used antibiotics. Here we present live-cell single-molecule microscopy measurements indicating that double-strand breaks induced by antibiotics strongly stimulate pol IV activity. Exposure to the antibiotics ciprofloxacin and trimethoprim leads to the formation of double strand breaks in E. coli cells. RecA and pol IV foci increase after treatment and exhibit strong colocalization. The induction of the SOS response, the appearance of RecA foci, the appearance of pol IV foci and RecA-pol IV colocalization are all dependent on RecB function. The positioning of pol IV foci likely reflects a physical interaction with the RecA* nucleoprotein filaments that has been detected previously in vitro. Our observations provide an in vivo substantiation of a direct role for pol IV in double strand break repair in cells treated with double strand break-inducing antibiotics.

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Evidence of protein-free homology recognition in magnetic bead force–extension experiments

Proceedings of The Royal Society A Mathematical Physical and Engineering Sciences ◽

10.1098/rspa.2016.0186 ◽

2016 ◽

Vol 472 (2191) ◽

pp. 20160186 ◽

Cited By ~ 7

Author(s):

D. J. (O’) Lee ◽

C. Danilowicz ◽

C. Rochester ◽

A. A. Kornyshev ◽

M. Prentiss

Keyword(s):

Single Molecule ◽

Magnetic Bead ◽

Double Strand Breaks ◽

Theoretical Studies ◽

Force Extension ◽

Strand Breaks ◽

Homologous Sequences ◽

Force Increase ◽

Physical Interactions ◽

Chromosome Regions

Earlier theoretical studies have proposed that the homology-dependent pairing of large tracts of dsDNA may be due to physical interactions between homologous regions. Such interactions could contribute to the sequence-dependent pairing of chromosome regions that may occur in the presence or the absence of double-strand breaks. Several experiments have indicated the recognition of homologous sequences in pure electrolytic solutions without proteins. Here, we report single-molecule force experiments with a designed 60 kb long dsDNA construct; one end attached to a solid surface and the other end to a magnetic bead. The 60 kb constructs contain two 10 kb long homologous tracts oriented head to head, so that their sequences match if the two tracts fold on each other. The distance between the bead and the surface is measured as a function of the force applied to the bead. At low forces, the construct molecules extend substantially less than normal, control dsDNA, indicating the existence of preferential interaction between the homologous regions. The force increase causes no abrupt but continuous unfolding of the paired homologous regions. Simple semi-phenomenological models of the unfolding mechanics are proposed, and their predictions are compared with the data.

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Single molecule microscopy reveals key physical features of repair foci in living cells

eLife ◽

10.7554/elife.60577 ◽

2021 ◽

Vol 10 ◽

Author(s):

Judith Miné-Hattab ◽

Mathias Heltberg ◽

Marie Villemeur ◽

Chloé Guedj ◽

Thierry Mora ◽

...

Keyword(s):

Single Molecule ◽

Liquid Droplet ◽

Physical Nature ◽

Living Cells ◽

Double Strand Breaks ◽

Single Molecule Microscopy ◽

Dsb Repair ◽

Strand Breaks ◽

Functional Homolog ◽

Physical Features

In response to double strand breaks (DSB), repair proteins accumulate at damaged sites, forming membrane-less sub-compartments or foci. Here we explored the physical nature of these foci, using single molecule microscopy in living cells. Rad52, the functional homolog of BRCA2 in yeast, accumulates at DSB sites and diffuses ~6 times faster within repair foci than the focus itself, exhibiting confined motion. The Rad52 confinement radius coincides with the focus size: foci resulting from 2 DSBs are twice larger in volume that the ones induced by a unique DSB and the Rad52 confinement radius scales accordingly. In contrast, molecules of the single strand binding protein Rfa1 follow anomalous diffusion similar to the focus itself or damaged chromatin. We conclude that while most Rfa1 molecules are bound to the ssDNA, Rad52 molecules are free to explore the entire focus reflecting the existence of a liquid droplet around damaged DNA.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2021963118 ◽

2021 ◽

Vol 118 (11) ◽

pp. e2021963118

Author(s):

Donna R. Whelan ◽

Eli Rothenberg

Keyword(s):

Homologous Recombination ◽

Single Molecule ◽

Protein A ◽

Super Resolution ◽

Initial Step ◽

Double Strand Breaks ◽

Homology Search ◽

Major Pathway ◽

Strand Breaks

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.

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Structural mechanism of DNA-end synapsis in the non-homologous end joining pathway for repairing double-strand breaks: bridge over troubled ends

Biochemical Society Transactions ◽

10.1042/bst20180518 ◽

2019 ◽

Vol 47 (6) ◽

pp. 1609-1619 ◽

Cited By ~ 5

Author(s):

Qian Wu

Keyword(s):

Single Molecule ◽

Genome Instability ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

End Joining ◽

Structural Mechanism ◽

Strand Breaks ◽

Single Molecule Studies ◽

Non Homologous End Joining ◽

Dna Ends

Non-homologous end joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), which is the most toxic DNA damage in cells. Unrepaired DSBs can cause genome instability, tumorigenesis or cell death. DNA end synapsis is the first and probably the most important step of the NHEJ pathway, aiming to bring two broken DNA ends close together and provide structural stability for end processing and ligation. This process is mediated through a group of NHEJ proteins forming higher-order complexes, to recognise and bridge two DNA ends. Spatial and temporal understanding of the structural mechanism of DNA-end synapsis has been largely advanced through recent structural and single-molecule studies of NHEJ proteins. This review focuses on core NHEJ proteins that mediate DNA end synapsis through their unique structures and interaction properties, as well as how they play roles as anchor and linker proteins during the process of ‘bridge over troubled ends'.

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