Gene tagging and gene replacement using recombinase-mediated cassette exchange in Schizosaccharomyces pombe

Watson, Adam T.; Garcia, Valérie; Bone, Neil; Carr, Antony; Armstrong, J. T.

doi:10.1016/j.gene.2007.09.024

Cited by 76 publications

(98 citation statements)

References 25 publications

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“…Sp mutants were introduced into a pso2-deleted ''base strain'' using recombinase-mediated cassette exchange (Watson et al 2008;Fontebasso et al 2013), and the cisplatin sensitivity of the strain harboring the fan1 mutant was examined. We used five different mutants that exhibited reduced nuclease activities (Figs.…”

Section: In Vitro and In Vivo Functional Analysis Of The Key Motifs Imentioning

confidence: 99%

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Crystal structure of a Fanconi anemia-associated nuclease homolog bound to 5′ flap DNA: basis of interstrand cross-link repair by FAN1

et al. 2014

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Fanconi anemia (FA) is an autosomal recessive genetic disorder caused by defects in any of 15 FA genes responsible for processing DNA interstrand cross-links (ICLs). The ultimate outcome of the FA pathway is resolution of crosslinks, which requires structure-selective nucleases. FA-associated nuclease 1 (FAN1) is believed to be recruited to lesions by a monoubiquitinated FANCI-FANCD2 (ID) complex and participates in ICL repair. Here, we determined the crystal structure of Pseudomonas aeruginosa FAN1 (PaFAN1) lacking the UBZ (ubiquitin-binding zinc) domain in complex with 59 flap DNA. All four domains of the right-hand-shaped PaFAN1 are involved in DNA recognition, with each domain playing a specific role in bending DNA at the nick. The six-helix bundle that binds the junction connects to the catalytic viral replication and repair (VRR) nuclease (VRR nuc) domain, enabling FAN1 to incise the scissile phosphate a few bases distant from the junction. The six-helix bundle also inhibits the cleavage of intact Holliday junctions. PaFAN1 shares several conserved features with other flap structure-selective nucleases despite structural differences. A clamping motion of the domains around the wedge helix, which acts as a pivot, facilitates nucleolytic cleavage. The PaFAN1 structure provides insights into how archaeal Holliday junction resolvases evolved to incise 59 flap substrates and how FAN1 integrates with the FA complex to participate in ICL repair.

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Section: In Vitro and In Vivo Functional Analysis Of The Key Motifs Imentioning

confidence: 99%

“…Cre-lox recombinase-mediated cassette exchange was used to integrate the mutant fan1 genes into the S. pombe genome at the endogenous fan1 locus (Watson et al 2008) using base strain YFS228 (fan1TloxP-ura4+-loxM3, pso2TkanMX6). Correct integration of the fan1 mutant alleles was confirmed by sequencing.…”

Section: In Vivo Survival Assaysmentioning

confidence: 99%

Crystal structure of a Fanconi anemia-associated nuclease homolog bound to 5′ flap DNA: basis of interstrand cross-link repair by FAN1

et al. 2014

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“…1D). Mutant alleles were generated by fusion PCR and integrated at the rad9 chromosomal locus using the Cre-lox cassette exchange technique (Watson et al, 2008). This technique allows for the targeted integration of rad9 genes at its chromosomal locus which was replaced by a marker gene (ura4+) flanked by the recognition sites (loxP, loxM) of the Cre recombinase.…”

Section: A Cryptic Translation Initiation Site Is Required For Heat Imentioning

confidence: 99%

“…pombe strains and Cre-Lox system As described by Watson and colleagues (Watson et al, 2008), the loxP sequence was integrated 181 nt upstream of the rad9 start condon at position 1,714,271 and the loxM sequence was placed 136 nt down-stream of the stop codon at position 1,715,672 on chromosome 1. All rad9 mutant strains described in this report are variants of this 'base' strain (h-ade6-M216 rad9::loxP-ura4+-loxM leu1-32 ura4-D18).…”

Section: Strain Listmentioning

confidence: 99%

Heat induction of a novel Rad9 variant from a cryptic translation initiation site reduces mitotic commitment

Janes

Schmidt

Garrido

et al. 2012

Journal of Cell Science

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SummaryExposure of human cells to heat switches the activating signal of the DNA damage checkpoint from genotoxic to temperature stress. This change reduces mitotic commitment at the expense of DNA break repair. The thermal alterations behind this switch remain elusive despite the successful use of heat to sensitise cancer cells to DNA breaks. Rad9 is a highly conserved subunit of the Rad9-Rad1-Hus1 (9-1-1) checkpointclamp that is loaded by Rad17 onto damaged chromatin. At the DNA, Rad9 activates the checkpoint kinases Rad3ATR and Chk1 to arrest cells in G2. Using Schizosaccharomyces pombe as a model eukaryote, we discovered a new variant of Rad9, Rad9-M50, whose expression is specifically induced by heat. High temperatures promote alternative translation from a cryptic initiation codon at methionine-50. This process is restricted to cycling cells and is independent of the temperature-sensing mitogen-activated protein kinase (MAPK) pathway. While fulllength Rad9 delays mitosis in the presence of DNA lesions, Rad9-M50 functions in a remodelled checkpoint pathway to reduce mitotic commitment at elevated temperatures. This remodelled pathway still relies on Rad1 and Hus1, but acts independently of Rad17. Heatinduction of Rad9-M50 ensures that the kinase Chk1 remains in a hypo-phosphorylated state. Elevated temperatures specifically reverse the DNA-damage-induced modification of Chk1 in a manner dependent on Rad9-M50. Taken together, heat reprogrammes the DNA damage checkpoint at the level of Chk1 by inducing a Rad9 variant that can act outside of the canonical 9-1-1 complex.

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“…cDNAs encoding FP tags can easily be fused to the 3 ′ terminus of a gene at its genomic locus in less than a week via polymerase chain reaction techniques (Wach et al 1997;Bähler et al 1998;Janke et al 2004), whereas recombinase-mediated cassette exchange (RMCE) and marker switch approaches that take marginally longer support tagging at the amino terminus (MacIver et al 2003;Watson et al 2008;Tang et al 2011). The aim is to support stable expression at wild-type levels, because the single integrated FP-tagged allele remains under the control of the endogenous promoter at the endogenous chromosomal location.…”

Section: Fluorescent Proteinsmentioning

confidence: 99%

Live Cell Imaging in Fission Yeast

Mulvihill

2017

Cold Spring Harb Protoc

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Live cell imaging complements the array of biochemical and molecular genetic approaches to provide a comprehensive insight into functional dependencies and molecular interactions in fission yeast. Fluorescent proteins and vital dyes reveal dynamic changes in the spatial distribution of organelles and the proteome and how each alters in response to changes in environmental and genetic composition. This introduction discusses key issues and basic image analysis for live cell imaging of fission yeast.

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Gene tagging and gene replacement using recombinase-mediated cassette exchange in Schizosaccharomyces pombe

Cited by 76 publications

References 25 publications

Crystal structure of a Fanconi anemia-associated nuclease homolog bound to 5′ flap DNA: basis of interstrand cross-link repair by FAN1

Crystal structure of a Fanconi anemia-associated nuclease homolog bound to 5′ flap DNA: basis of interstrand cross-link repair by FAN1

Heat induction of a novel Rad9 variant from a cryptic translation initiation site reduces mitotic commitment

Live Cell Imaging in Fission Yeast

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