Resection of a DNA Double-Strand Break by Alkaline Gel Electrophoresis and Southern Blotting

Casari, Erika; Gobbini, Elisa; Clerici, Michela; Longhese, Maria Pia

doi:10.1007/978-1-0716-0644-5_3

Cited by 7 publications

(3 citation statements)

References 15 publications

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“…YEPR exponentially growing cell cultures of JKM139 derivative strains, carrying the HO-cut site at the MAT locus, were transferred to YEPRG at time zero. Ssp I-digested genomic DNA was run on an alkaline agarose gel and visualized after hybridization with an RNA probe that anneals with the unresected strand on one side of the HO-induced DSB as previously described ( 63 ). This probe was obtained by in vitro transcription using Promega Riboprobe System-T7 and plasmid pML514 as a template.…”

Section: Methodsmentioning

confidence: 99%

Rif2 interaction with Rad50 counteracts Tel1 functions in checkpoint signalling and DNA tethering by releasing Tel1 from MRX binding

Pizzul,

Casari,

Rinaldi

et al. 2024

Nucleic Acids Research

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The yeast Rif2 protein is known to inhibit Mre11 nuclease and the activation of Tel1 kinase through a short motif termed MIN, which binds the Rad50 subunit and simulates its ATPase activity in vitro. The mechanism by which Rif2 restrains Tel1 activation and the consequences of this inhibition at DNA double-strand breaks (DSBs) are poorly understood. In this study, we employed AlphaFold-Multimer modelling to pinpoint and validate the interaction surface between Rif2 MIN and Rad50. We also engineered the rif2-S6E mutation that enhances the inhibitory effect of Rif2 by increasing Rif2-Rad50 interaction. Unlike rif2Δ, the rif2-S6E mutation impairs hairpin cleavage. Furthermore, it diminishes Tel1 activation by inhibiting Tel1 binding to DSBs while leaving MRX association unchanged, indicating that Rif2 can directly inhibit Tel1 recruitment to DSBs. Additionally, Rif2S6E reduces Tel1-MRX interaction and increases stimulation of ATPase by Rad50, indicating that Rif2 binding to Rad50 induces an ADP-bound MRX conformation that is not suitable for Tel1 binding. The decreased Tel1 recruitment to DSBs in rif2-S6E cells impairs DSB end-tethering and this bridging defect is suppressed by expressing a Tel1 mutant variant that increases Tel1 persistence at DSBs, suggesting a direct role for Tel1 in the bridging of DSB ends.

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Section: Methodsmentioning

confidence: 99%

Rif2 interaction with Rad50 counteracts Tel1 functions in checkpoint signalling and DNA tethering by releasing Tel1 from MRX binding

Pizzul,

Casari,

Rinaldi

et al. 2024

Nucleic Acids Research

Self Cite

View full text Add to dashboard Cite

show abstract

“…YEPR exponentially growing cell cultures of JKM139 derivative strains, carrying the HO cut site at the MAT locus, were transferred to YEPRG at time zero. Ssp I-digested genomic DNA was run on an alkaline agarose gel and visualized after hybridization with an RNA probe that anneals with the unresected strand at one side of the HO-induced DSB ( 64 ). This probe was obtained by in vitro transcription using Promega Riboprobe System-T7 and plasmid pML514 as a template.…”

Section: Methodsmentioning

confidence: 99%

The Ku complex promotes DNA end-bridging and this function is antagonized by Tel1/ATM kinase

Rinaldi

Pizzul

Casari

et al. 2023

Nucleic Acids Research

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DNA double-strand breaks (DSBs) can be repaired by either homologous recombination (HR) or non-homologous end-joining (NHEJ). NHEJ is induced by the binding to DSBs of the Ku70–Ku80 heterodimer, which acts as a hub for the recruitment of downstream NHEJ components. An important issue in DSB repair is the maintenance of the DSB ends in close proximity, a function that in yeast involves the MRX complex and Sae2. Here, we provide evidence that Ku contributes to keep the DNA ends tethered to each other. The ku70-C85Y mutation, which increases Ku affinity for DNA and its persistence very close to the DSB ends, enhances DSB end-tethering and suppresses the end-tethering defect of sae2Δ cells. Impairing histone removal around DSBs either by eliminating Tel1 kinase activity or nucleosome remodelers enhances Ku persistence at DSBs and DSB bridging, suggesting that Tel1 antagonizes the Ku function in supporting end-tethering by promoting nucleosome removal and possibly Ku sliding inwards. As Ku provides a block to DSB resection, this Tel1 function can be important to regulate the mode by which DSBs are repaired.

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“…DSB end resection at the MAT locus in JKM139 derivative strains was analyzed on alkaline agarose gels by using a single-stranded probe that anneals to the unresected DSB strand, as previously described [83]. Quantification of DSB resection was determined by calculating the ratio of band intensities for ssDNA to the total amount of DSB products.…”

Section: Dsb Resection At the Mat Locusmentioning

confidence: 99%

The chromatin remodeler Chd1 supports MRX and Exo1 functions in resection of DNA double-strand breaks

2021

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Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) requires that the 5’-terminated DNA strands are resected to generate single-stranded DNA overhangs. This process is initiated by a short-range resection catalyzed by the MRX (Mre11-Rad50-Xrs2) complex, which is followed by a long-range step involving the nuclease Exo1 or Dna2. Here we show that the Saccharomyces cerevisiae ATP-dependent chromatin-remodeling protein Chd1 participates in both short- and long-range resection by promoting MRX and Exo1 association with the DSB ends. Furthermore, Chd1 reduces histone occupancy near the DSB ends and promotes DSB repair by HR. All these functions require Chd1 ATPase activity, supporting a role for Chd1 in the opening of chromatin at the DSB site to facilitate MRX and Exo1 processing activities.

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Resection of a DNA Double-Strand Break by Alkaline Gel Electrophoresis and Southern Blotting

Cited by 7 publications

References 15 publications

Rif2 interaction with Rad50 counteracts Tel1 functions in checkpoint signalling and DNA tethering by releasing Tel1 from MRX binding

Rif2 interaction with Rad50 counteracts Tel1 functions in checkpoint signalling and DNA tethering by releasing Tel1 from MRX binding

The Ku complex promotes DNA end-bridging and this function is antagonized by Tel1/ATM kinase

The chromatin remodeler Chd1 supports MRX and Exo1 functions in resection of DNA double-strand breaks

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