Guidelines for DNA recombination and repair studies: Cellular assays of DNA repair pathways

Klein, Hannah L.; Bačinskaja, Giedrė; Che, Jun; Cheblal, Anais; Elango, Rajula; Epshtein, Anastasiya; Fitzgerald, Devon M.; Gómez-González, Belén; Khan, Sharik R.; Kumar, Sandeep; Leland, Bryan A.; Marie, Léa; Mei, Qian; Miné-Hattab, Judith; Piotrowska, Alicja; Polleys, Erica J; Putnam, Christopher D.; Radchenko, Elina A.; Saada, Anissia Ait; Sakofsky, Cynthia J.; Shim, Eun Yong; Stracy, Mathew; Xia, Jun; Yan, Zhong; Yin, Yi; Aguilera, Andrés; Argueso, Juan Lucas; Freudenreich, Catherine H.; Gasser, Susan M.; Gordenin, Dmitry A.; Haber, James E.; Ira, Grzegorz; Jinks-Robertson, Sue; King, Megan C.; Kolodner, Richard D.; Kuzminov, Andrei; Lambert, Sarah; Lee, Sang Eun; Miller, Kyle M.; Mirkin, Sergei M.; Petes, Thomas D.; Rosenberg, Susan M.; Rothstein, Rodney; Symington, Lorraine S.; Zawadzki, Paweł; Kim, Nayun; Lisby, Michael; Malkova, Anna

doi:10.15698/mic2019.01.664

Cited by 50 publications

(43 citation statements)

References 366 publications

(508 reference statements)

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“…To estimate the apparent diffusion coefficient of Rad52 in the absence of DSB, we calculated their displacement histogram [32][33][34] . This method allows us to test whether Rad52 molecules exhibit a single diffusive regime across the nucleus or if they exist in several subpopulations characterized by distinct diffusion coefficients (see Methods).…”

Section: Rad52 Exhibits 2 Diffusive Behaviors In the Absence Of Dsbmentioning

confidence: 99%

“…In response to a DSB, Rad52 accumulates at the damaged site forming highly concentrated foci 8 .The mobility of Rad52 within these foci shows an apparent diffusion coefficient of DRad52,inside = 0.032 ± 0.006 m 2 /s. The characteristic equilibration time after which the effect of boundaries appears is defined by the time at which the MSD curve starts bending and is given by = 2 (6 • ) ⁄ , where Rc is the confinement radius and D the diffusion coefficient inside foci 33 . Taken the measured diffusion coefficient and confinement radius, Rad52 molecules start feeling the effect of the focus boundary at only tc = 80 ± 3 ms (representing 4 timesteps of 20 ms).…”

Section: Dynamic Behavior Of Individual Rad52 In Living Cellsmentioning

confidence: 99%

“…When the motion appeared confined, we fit the MSD with , where d is the dimension of the motion, and the diffusion coefficient D 33,47 .…”

Section: Mean Square Displacementmentioning

confidence: 99%

“…Displacement histogram: Probability Density Functions (PDF) are obtained after normalization of the displacement histograms and fitted with a 1, 2 or 3-population model33,34 : , where D is the diffusion coefficient and r is the position at time t. , where D1 and D2 are the diffusion coefficients of sub-population 1 and 2 respectively, and f is the fraction of sub-…”

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confidence: 99%

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

Miné-Hattab

Heltberg

Villemeur

et al. 2020

Preprint

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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 possibly reflecting the existence of a liquid droplet around damaged DNA.

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Section: Rad52 Exhibits 2 Diffusive Behaviors In the Absence Of Dsbmentioning

confidence: 99%

Section: Dynamic Behavior Of Individual Rad52 In Living Cellsmentioning

confidence: 99%

“…When the motion appeared confined, we fit the MSD with , where d is the dimension of the motion, and the diffusion coefficient D 33,47 .…”

Section: Mean Square Displacementmentioning

confidence: 99%

mentioning

confidence: 99%

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

Miné-Hattab

Heltberg

Villemeur

et al. 2020

Preprint

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“…We then used a PCR-mediated targeted deletion approach (Goldstein and McCusker, 1999) to knock out the centromere-proximal (left side) portion of the proximal duplicated segment in FCR8, resulting in a Chr5 configuration with two identical 35 kb directly-oriented engineered LCRs separated by a 12 kb single copy interstitial spacer (IS) region containing the SFA1 V208I -CUP1-KanMX4 copy number reporter cassette ( Fig. 2A-B) (Zhang et al, 2013;Klein et al, 2019). These 35 kb LCRs correspond to regions 2-5 in Fig.…”

Section: Construction Of the Recurrent Cnv Reporter Locusmentioning

confidence: 99%

A new experimental system to study meiotic recurrent non-allelic homologous recombination in budding yeast

Sedam

Argueso

2020

Preprint

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In humans, de novo recurrent copy number variations (CNVs) often arise during meiosis from non-allelic homologous recombination (NAHR) between low copy repeat elements (LCRs).These chromosomal rearrangements are responsible for a wide variety of genomic disorders involving duplication or deletion of dose-sensitive genes. The precise factors that steer meiotic cells toward this detrimental recombination pathway are not fully understood. To create a model for the investigation of LCR-mediated CNV mechanisms, we developed a diploid experimental system in Saccharomyces cerevisiae. We modified the right arm of chromosome V through the introduction of engineered LCRs: duplicated 5 to 35 kb segments of yeast DNA flanking single copy interstitial spacers, analogously to the meiotic NAHR substrates that exist in humans.Phenotypic markers, including a copy number reporter, were inserted within the interstitial spacer. Their segregation in the haploid meiotic progeny was used to phenotypically identity and classify recurrent CNV events. This system allowed us to measure the effects of LCR size on the frequency of meiotic de novo recurrent CNV formation, and to determine the relative proportions of each of the three main NAHR classes: interhomolog, intersister, and intrachromatid. The frequency of CNV increased as the LCRs became larger, and interhomolog NAHR was overrepresented relative to the two other classes. We showed that this experimental system directly mimics the features of de novo recurrent CNVs reported in human disease, thus it represents a promising tool for the discovery and characterization of conserved cellular factors and environmental exposures that can modulate meiotic NAHR.

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Eukaryotic Recombination: Initiation by Double‐Strand Breaks

Lalwani¹,

Goodenow²,

Richardson³

2020

Encyclopedia of Life Sciences

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A double‐strand break (DSB) in one deoxyribonucleic acid (DNA) double helix, caused by DNA damaging agents such as ionising radiation or normal metabolic processes, can stimulate repair through multiple pathways. Homologous recombination (HR) utilises a second unbroken DNA double helix containing homologous sequences as a donor of genetic information to restore the intact DNA structure, while nonhomologous end joining (NHEJ) relegates broken DNA ends together with little to no homology. Deficiency in these repair pathways can cause genome instability can drive carcinogenesis. DSB stimulated repair can cause illegitimate HR between heterologous chromosome of the ligation of ends of DNA from heterologous chromosomes. Exogenous and endogenous agents can cause deoxyribonucleic acid (DNA) breaks leading to genome instability. Repair of double‐strand breaks (DSB) in eukaryotes occurs by either homology‐dependent or homology‐independent mechanisms. Homologous recombination (HR) facilitates high‐fidelity repair of DSBs and other plausible DNA damage. Site‐specific DSBs within the genome are used to study the explicit mechanism involved in the DSB repair. Unlike other model organisms, HR in higher eukaryotes requires additional protein factors. Mutations in HR proteins can increase predisposition to cancer.

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Guidelines for DNA recombination and repair studies: Cellular assays of DNA repair pathways

Cited by 50 publications

References 366 publications

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

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

A new experimental system to study meiotic recurrent non-allelic homologous recombination in budding yeast

Eukaryotic Recombination: Initiation by Double‐Strand Breaks

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