Transformation system for prototrophic industrial yeasts using the <i>AUR1</i> gene as a dominant selection marker

Hashida-Okado, Takashi; Ogawa, Atsuko; Kato, Ikunoshin; Takesako, Kazutoh

doi:10.1016/s0014-5793(98)00211-7

Cited by 50 publications

(32 citation statements)

References 18 publications

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“…The plasmid pAUR101 (TAKARA), bearing the dominant AUR1-C allele conferring resistance to Aureobasidin A (Aur R ), lacks a centromere (CEN) and an autonomous replication site (ARS) (Fig. S2) (28). SD plates with 0.4 μg/mL Aureobasidin A (TAKARA) were used for selection.…”

Section: Methodsmentioning

confidence: 99%

Putative antirecombinase Srs2 DNA helicase promotes noncrossover homologous recombination avoiding loss of heterozygosity

Miura

Shibata

Kusano

2013

Proc. Natl. Acad. Sci. U.S.A.

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DNA damage alone or DNA replication fork arrest at damaged sites may induce DNA double-strand breaks and initiate homologous recombination. This event can result in a crossover with a homologous chromosome, causing loss of heterozygosity along the chromosome. It is known that Srs2 acts as an antirecombinase at the replication fork: it is recruited by the SUMO (a small ubiquitin-related modifier)-conjugated DNA-polymerase sliding clamp (PCNA) and interferes with Rad51/Rad52-mediated homologous recombination. Here, we report that Srs2 promotes another type of homologous recombination that produces noncrossover products only, in collaboration with PCNA and Rad51. Srs2 proteins lacking the Rad51-binding domain, PCNA-SUMO-binding motifs, or ATP hydrolysis-dependent DNA helicase activity reduce this noncrossover recombination. However, the removal of either the Rad51-binding domain or the PCNA-binding motif strongly increases crossovers. Srs2 gene mutations are epistatic to mutations in the PCNA modification-related genes encoding PCNA, Siz1 (a SUMO ligase) and Rad6 (a ubiquitin-conjugating protein). Knocking out RAD51 blocked this recombination but enhanced nonhomologous end-joining. We hypothesize that, during DNA double-strand break repair, Srs2 mediates collaboration between the Rad51 nucleofilament and PCNA-SUMO and directs the heteroduplex intermediate to DNA synthesis in a moving bubble. This Rad51/Rad52/Srs2/PCNAmediated noncrossover pathway avoids both interchromosomal crossover and imprecise end-joining, two potential paths leading to loss of heterozygosity, and contributes to genome maintenance and human health.utations in the DNA helicase Srs2 gene cause a hyperrecombination phenotype (1) and increase mitotic crossovers (2). These findings suggest that Srs2 negatively regulates somatic homologous recombination, and thus Srs2 is regarded as an antirecombinase. The Srs2 DNA helicase has a recombinase Rad51-binding motif in its C-terminal region (3, 4), and in vitro analyses have demonstrated that it disrupts Rad51 nucleofilaments formed on single-stranded DNA and inhibits heteroduplex formation mediated by Rad51 recombinase (4, 5). In addition, synthetic heteroduplexes with a D-loop with Rad51 nucleofilaments are efficiently dissociated by Srs2 (6). These biochemical results appear to explain the negative regulation of heteroduplex formation by Srs2 during homologous recombination. Other motifs around the C-terminal tip allow the Srs2 helicase to bind to SUMO-conjugated DNA-polymerase sliding clamp (PCNA-SUMO) (3, 7). The interaction between Srs2 and PCNA-SUMO is essential to prevent the Rad51/Rad52-mediated sister-chromatid exchanges that occur in DNA replication forks stalled at DNA lesions, to channel to the translesion DNA synthesis initiated by Rad6/Rad18-ubiquitinated PCNA (3,8). This postreplication repair is induced by base-modification types of DNA lesions, caused by DNA scission reagents such as UV, 4NQO, and MMS.Radiation and reactive oxygen species cause DNA doublestrand breaks, which are repaire...

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Section: Methodsmentioning

confidence: 99%

Putative antirecombinase Srs2 DNA helicase promotes noncrossover homologous recombination avoiding loss of heterozygosity

Miura

Shibata

Kusano

2013

Proc. Natl. Acad. Sci. U.S.A.

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“…pAUR101 (6687 bp; Takara, Kyoto, Japan), which bears the dominant AUR1-C allele conferring resistance to Aureobasidin A (Aur R ), does not possess either a centromere (CEN) or an autonomous replication site (ARS) (HashidaOkado et al 1998). The base substitution producing the dominance is at 4522 within the pAUR101 sequence (AB012282; Hashida-Okado et al 1998). The blunt-ended cleavages by the MscI and StuI restriction enzymes occur at 3231 and 4143 of pAUR101, respectively, and each recognition site resides within the AUR1-C allele.…”

Section: Targeted Integration (Crossover) Assaymentioning

confidence: 99%

“…The base substitution producing the dominance is at 4522 within the pAUR101 sequence (AB012282; Hashida-Okado et al 1998). The blunt-ended cleavages by the MscI and StuI restriction enzymes occur at 3231 and 4143 of pAUR101, respectively, and each recognition site resides within the AUR1-C allele.…”

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confidence: 99%

Homologous Recombination via Synthesis-Dependent Strand Annealing in Yeast Requires the Irc20 and Srs2 DNA Helicases

et al. 2012

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Synthesis-dependent strand-annealing (SDSA)-mediated homologous recombination replaces the sequence around a DNA double-strand break (DSB) with a copy of a homologous DNA template, while maintaining the original configuration of the flanking regions. In somatic cells at the 4n stage, Holliday-junction-mediated homologous recombination and nonhomologous end joining (NHEJ) cause crossovers (CO) between homologous chromosomes and deletions, respectively, resulting in loss of heterozygosity (LOH) upon cell division. However, the SDSA pathway prevents DSB-induced LOH. We developed a novel yeast DSB-repair assay with two discontinuous templates, set on different chromosomes, to determine the genetic requirements for somatic SDSA and precise end joining. At first we used our in vivo assay to verify that the Srs2 helicase promotes SDSA and prevents imprecise end joining. Genetic analyses indicated that a new DNA/RNA helicase gene, IRC20, is in the SDSA pathway involving SRS2. An irc20 knockout inhibited both SDSA and CO and suppressed the srs2 knockout-induced crossover enhancement, the mre11 knockout-induced inhibition of SDSA, CO, and NHEJ, and the mre11-induced hypersensitivities to DNA scissions. We propose that Irc20 and Mre11 functionally interact in the early steps of DSB repair and that Srs2 acts on the D-loops to lead to SDSA and to prevent crossoverv. D OUBLE-strand DNA breaks (DSBs) are generated by exposure to ionizing radiation or chemical compounds, such as topoisomerase inhibitors and apoptosis inducers, reactive oxygen species (ROS) generated as by-products of oxidative respiration, transposition of transposable elements, and by the actions of restriction enzymes or meiosis-specific endonucleases, such as the Spo11 complex and intron-homing nucleases. DSBs are repaired through various recombination-dependent pathways (Figure 1), and recombination deficiencies cause genome instability and carcinogenesis.Holliday-junction-mediated homologous recombination (Figure 1A) is a type of homologous recombination that produces either crossover or noncrossover products. These products are generated on the basis of the resolution of the double-Holliday structure (Resnick and Martin 1976;Szostak et al. 1983). It was proposed that the noncrossover products (B5) are generated from a double-Holliday structure (A4), with reverse migration of the Holliday junctions without cleavage (Wu and Hickson 2003).Synthesis-dependent strand-annealing (SDSA)-mediated homologous recombination ( Figure 1B) is the other type of homologous recombination, which produces only noncrossover products that are not associated with a flanking crossover. In this pathway, a D-loop, formed with a single-strand tail from the terminus of a DSB (B2), migrates with associated DNA synthesis primed at the 39 termini of the invading single-strand tail, but without forming Holliday junctions. These reactions are followed by the annealing of the newly synthesized strands dissociated from the template DNA with the other termini of the DSB (Nassif et...

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“…were shown to function in the appropriate K. lactis auxotrophic backgrounds and vice versa; the K. lactis homologues could be identified in S. cerevisiae by means of intergeneric complementation (Shuster et al, 1987;Stark and Milner, 1989;Zhang et al, 1992;Bai et al, 1999). There are also dominant markers conferring resistance to geneticin (G418 R ), bleomycin (BLE R ), and aureobasidin A (AUR1) or enabling acetamide utilization (amdS) (Sreekrishna et al, 1984;Chen and Fukuhara, 1988;Hashida-Okado et al, 1998;Castrillo et al, 1999). Transformation techniques include PEG-mediated DNA transfer into protoplasts, chemical lithium acetate protocols, and electroporation (Das and Hollenberg, 1982;Klebe et al, 1983;Meilhoc et al, 1990).…”

Section: Tools For Genetic Manipulationmentioning

confidence: 99%

Genetics and Molecular Physiology of the Yeast Kluyveromyces lactis

Schaffrath

Breunig

2000

Fungal Genetics and Biology

144

101

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Transformation system for prototrophic industrial yeasts using the AUR1 gene as a dominant selection marker

Cited by 50 publications

References 18 publications

Putative antirecombinase Srs2 DNA helicase promotes noncrossover homologous recombination avoiding loss of heterozygosity

Putative antirecombinase Srs2 DNA helicase promotes noncrossover homologous recombination avoiding loss of heterozygosity

Homologous Recombination via Synthesis-Dependent Strand Annealing in Yeast Requires the Irc20 and Srs2 DNA Helicases

Genetics and Molecular Physiology of the Yeast Kluyveromyces lactis

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