Physical and Genetic Assays for the Study of DNA Joint Molecules Metabolism and Multi-invasion-Induced Rearrangements in S. cerevisiae

Rajput, Pallavi; Heyer, Wolf‐Dietrich

doi:10.1007/978-1-0716-0644-5_36

Cited by 5 publications

(15 citation statements)

References 21 publications

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“…MIs are readily formed in reconstituted in vitro reactions with ssDNA substrates of physiological length ( i.e. >100 nt) and yeast and human Rad51, Rad54, and RPA proteins, and in S. cerevisiae cells (Piazza et al 2017; Wright and Heyer 2014; Piazza et al 2021b). They are presumably byproducts of homology search by inter-segmental contact sampling demonstrated for RecA (Forget and Kowalczykowski 2012).…”

Section: Introductionmentioning

confidence: 99%

“…The translocation assay has been described previously (Piazza et al 2017(Piazza et al , 2021b. Briefly, the basal Lys + frequency was determined by plating yeast cells exponentially growing in YEP-lactate liquid culture on SD-LYS and YPD plates (control plating).…”

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confidence: 99%

“…The expression of the HO endonuclease was triggered in the remaining culture upon addition of 2% galactose. Two hours after galactose addition, when HO cutting is >99% (Piazza et al 2021b(Piazza et al , 2017, the induced Lys + frequency is determined by plating again on SD-LYS and YPD plates. Basal and induced Lys + frequencies as well as viability are determined after incubation of the plates at 30° C for 2-3 days.…”

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confidence: 99%

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Delineation of two multi-invasion-induced rearrangement pathways that differently affect genome stability

Reitz

Djeghmoum

Watson

et al. 2023

Preprint

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Punctuated bursts of structural genomic variations (SVs) have been described in various organisms, but their etiology remains incompletely understood. Homologous recombination (HR) is a template-guided mechanism of repair of DNA double-strand breaks and stalled or collapsed replication forks. We recently identified a DNA break amplification and genome rearrangement pathway originating from the endonucleolytic processing of a multi-invasion (MI) DNA joint molecule formed during HR. Genome-wide sequencing approaches confirmed that multi-invasion-induced rearrangement (MIR) frequently leads to several repeat-mediated SVs and aneuploidies. Using molecular and genetic analysis, and a novel, highly sensitive proximity ligation-based assay for chromosomal rearrangement quantification, we further delineate two MIR sub-pathways. MIR1 is a universal pathway occurring in any sequence context, which generates secondary breaks and frequently leads to additional SVs. MIR2 occurs only if recombining donors exhibit substantial homology, and results in sequence insertion without additional break or SV. The most detrimental MIR1 pathway occurs late on a subset of persisting DNA joint molecules in a PCNA/Poldelta-independent manner, unlike recombinational DNA synthesis. This work provides a refined mechanistic understanding of these HR-based SV formation pathways and shows that complex repeat-mediated SVs can occur without displacement DNA synthesis. Sequence signatures for inferring MIR1 from long-read data are proposed.

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Section: Introductionmentioning

confidence: 99%

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Delineation of two multi-invasion-induced rearrangement pathways that differently affect genome stability

Reitz

Djeghmoum

Watson

et al. 2023

Preprint

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“…DSB ends are resected first by the Mre11-Rad50-Xrs2 NBS1 (MRX) complex, but more extensive 5' to 3' resection depends on two other exonucleases, Exo1 or the Sgs1-Rmi1-Top3-Dna2 complex, to create long single-stranded 3'-ended tails (13). These strands are first bound by the single-strand DNA binding complex, RPA, and then by the Rad51 recombinase that initiates the search for homology and the repair of the DSB by homologous recombination (14)(15)(16).…”

Section: Introductionmentioning

confidence: 99%

Rad51 and Dmc1 have similar tolerance for mismatches in yeast meiosis

Choi

Xue

Cao

et al. 2022

Preprint

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In many eukaryotes, including both budding yeast and mammals, repair of double-strand breaks (DSBs) is carried out by different apparatus in somatic and meiotic cells. In mitotic cells, Rad51 recombinase, acting with Rad54, facilitates the search for homology and DNA strand exchange. In meiosis, Rad51 is inhibited by Hed1 and plays only an effector role, while strand exchange is driven by Rad51’s homolog, Dmc1, acting with Rad54’s homolog, Rdh54/Tid1. To directly compare the activities of Rad51 and Dmc1 and especially their tolerance for recombination between divergent sequences, we created diploids in which a site-specific DSB was created by HO endonuclease, either under control of a galactose-inducible promoter or a meiosis-specific SPO13 promoter. Homologous recombination was measured by an ectopic break-induced replication (BIR) assay in which a 108-bp homologous sequence shared between the DSB end and the donor sequence could be easily modified. As previously shown for a haploid mitotic strain, BIR efficiency decreased with increasing divergence between donor and recipient, but repair occurred even when every 6th base pair was mismatched. There was little difference in the tolerance of mismatches in mitotic haploids or meiotic diploids; however, there were notable differences in meiotic diploids when recombination was facilitated by Dmc1 or when Rad51 took over from Dmc1 in both hed1Δ and dmc1Δ hed1Δ mutants. We found that Dmc1 and Rad51 are similarly tolerant of mismatches during meiotic recombination in budding yeast. Surveillance of mismatches by the Msh2 mismatch repair protein proved to be Dmc1-specific. In all cases, assimilation of mismatches into the BIR product was dependent on the 3’ to 5’ exonuclease activity of DNA polymerase δ.Author SummaryIn many eukaryotes, including both budding yeast and mammals, repair of double-strand breaks (DSBs) is carried out by different apparatus in somatic and meiotic cells. In mitotic cells, Rad51 recombinase, acting with Rad54, facilitates the search for homology and DNA strand exchange. In budding yeast meiosis, Rad51 is inhibited by Hed1 and plays only an effector role, while strand exchange is driven by Rad51’s homolog, Dmc1, acting with Rad54’s homolog, Rdh54/Tid1. To directly compare the activities of Rad51 and Dmc1 and especially their tolerance for recombination between divergent sequences, we created diploids in which a site-specific DSB was created by HO endonuclease. Homologous recombination was measured by an ectopic break-induced replication (BIR) assay in which recombination occurred between a 108-bp homologous sequence shared between the DSB end and the donor sequence. The donor sequence could be easily modified to introduce different arrangements of mismatches. BIR efficiency decreased with increasing divergence between donor and recipient, but repair occurred even when every 6th base pair was mismatched. There was little difference in the tolerance of mismatches in mitotic or meiotic diploids; however, there were notable differences in meiotic diploids when recombination was facilitated by Dmc1 or when Dmc1 was deleted and Rad51 was activated. We found that Dmc1 and Rad51 are similarly tolerant of mismatches during meiotic recombination. Surveillance of mismatches by the Msh2 mismatch repair protein proved to be Dmc1-specific. As in mitotic cells, the assimilation of mismatches into the BIR product was dependent on the 3’ to 5’ exonuclease activity of DNA polymerase δ.

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“…During the homology-mediated strand invasion, it has been reported that the end of the single-strand DNA is not necessary for the strand exchange ( Wright and Heyer 2014 ; Piazza et al 2017 , 2021 ; Piazza and Heyer 2018 ). So the large 3’ overhang generated by DSB end resection might invade into multiple template DNA and form a multi-invasion joint molecule ( Piazza and Heyer 2019 ).…”

Section: Introductionmentioning

confidence: 99%

Double-strand breaks induce short-scale DNA replication and damage amplification in the fully grown mouse oocytes

Xie

et al. 2021

Genetics

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Break-induced replication (BIR) is essential for the repair of DNA double-strand breaks (DSBs) with single ends. DSBs-induced microhomology-mediated BIR (mmBIR) and template-switching can increase the risk of complex genome rearrangement. In addition, DSBs can also induce the multi-invasion-mediated DSB amplification. The mmBIR-induced genomic rearrangement has been identified in cancer cells and patients with rare diseases. However, when and how mmBIR are initiated haven’t been fully and deeply studied. Furthermore, it is not well understood about the conditions for initiation of multi-invasion-mediated DSB amplification. In the G2 phase oocyte of mouse, we identified a type of short scale BIR (ssBIR) using the DNA replication indicator 5-ethynyl-2´-deoxyuridine (EdU). These ssBIRs could only be induced in the fully-grown oocytes but not the growing oocytes. If the DSB oocytes were treated with Rad51 or Chek1/2 inhibitors, both EdU signals and DSB marker γH2A.X foci would decrease. In addition, the DNA polymerase inhibitor Aphidicolin could inhibit the ssBIR and another inhibitor ddATP could reduce the number of γH2A.X foci in the DSB oocytes. In conclusion, our results showed that DNA DSBs in the fully-grown oocytes can initiate ssBIR and be amplified by Rad51 or DNA replication.

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Physical and Genetic Assays for the Study of DNA Joint Molecules Metabolism and Multi-invasion-Induced Rearrangements in S. cerevisiae

Cited by 5 publications

References 21 publications

Delineation of two multi-invasion-induced rearrangement pathways that differently affect genome stability

Delineation of two multi-invasion-induced rearrangement pathways that differently affect genome stability

Rad51 and Dmc1 have similar tolerance for mismatches in yeast meiosis

Double-strand breaks induce short-scale DNA replication and damage amplification in the fully grown mouse oocytes

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