Homologous recombination between single-stranded DNA and chromosomal genes in Saccharomyces cerevisiae.

Simon, Jay R.; Moore, Peter D.

doi:10.1128/mcb.7.7.2329

Cited by 56 publications

(29 citation statements)

References 25 publications

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“…These results strongly suggest that inactivation of unlinked duplicated met2 genes through crosses also is due to cytosine methylation and results from the same process as that operating on tandemly duplicated met2 sequences. (20). Using M13 constructs in opposite orientations, we have found that the two DNA strands behave equally in transformation.…”

Section: Resultsmentioning

confidence: 95%

Targeted transformation of Ascobolus immersus and de novo methylation of the resulting duplicated DNA sequences.

1989

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To develop a method to modify genomic sequences in Ascobolus immersus by precisely reintroducing defined DNA segments previously manipulated in vitro, we investigated the effect of transforming DNA conformation on recombination with chromosomal sequences. Circular single-stranded DNA carrying the met2 gene and double-stranded DNA linearized by cutting within the met gene both transformed protoplasts of a met2 mutant strain of A. immersus to prototrophy. In contrast to the equivalent circular double-stranded DNA, which chiefly integrated at nonhomologous chromosomal sites, single-stranded and double-stranded cut DNAs recombined primarily with the homologous chromosomal met2 sequence. Of the single-stranded DNA transformants, 65% resulted from replacement of the resident met2 mutation by the exogenous wild-type allele. In 70% of the double-stranded-cut DNA transformants, one or more copies of the transforming DNA had integrated at the me2 locus, leading to tandem duplications of the met2 target region separated by plasmid DNA. These duplicated sequences could recombine, leading to progeny containing only one copy of the me2 region. This resulted in a precise gene replacement if the wild-type allele had been retained. In addition, we show that newly duplicated sequences were most often de novo methylated at the cytosine residues during the sexual phase. Cytosine methylation was associated with inactivation of the integrated me2 gene(s) in segregants of crosses. However, methylation was not accurately maintained at each DNA replication cycle, so that Met-segregants recovered a wild-type phenotype through successive mitotic divisions. This finding indicated that met2 genes were silenced by methylation alone.A procedure has recently been developed in our laboratory for integrative transformation of the filamentous ascomycete Ascobolus immersus (2); this procedure is based on the complementation of an auxotrophic met2 mutation by the wild-type cloned gene (3). Transformation of a met2 mutant strain of A. immersus by circular double-stranded (ds) DNA carrying the met2 gene (2) results either in the integration of the transforming DNA at nonhomologous chromosomal sites or in the substitution of the endogenous met2 mutation by the transforming wild-type allele. The relative frequency of both events varies according to the vector sequences carrying the transforming met2 gene, although nonhomologous integrations are the most often obtained. Homologous integration of the transforming plasmid by a reciprocal exchange at the chromosomal met2 locus has never been observed.We determined whether changing the conformation of the transforming DNA might have an effect on the transformation modalities of A. immersus and might eventually force homologous recombination with chromosomal sequences. We report on the ability of circular single-stranded (ss) DNA and linear ds DNA carrying the met2 gene to transform protoplasts of a met2 mutant strain of A. immersus. In contrast to circular ds DNA, they both most often participate in homolog...

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Section: Resultsmentioning

confidence: 95%

Targeted transformation of Ascobolus immersus and de novo methylation of the resulting duplicated DNA sequences.

1989

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“…Gene targeting using single-stranded DNA (ssDNA) has successfully been carried out on Saccharomyces cerevisiae (33). The authors have not only shown that gene targeting with ssDNA is possible, they also provided some evidence that ssDNA is more efficient than targeting with double-stranded DNA (dsDNA).…”

Section: ϫ6mentioning

confidence: 90%

Nuclear-Gene Targeting by Using Single-Stranded DNA Avoids Illegitimate DNA Integration in Chlamydomonas reinhardtii

2005

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Homologous DNA recombination (HR) allows the deletion (knockout), repair (rescuing), and modification of a selected gene, thereby rendering a functional analysis of the gene product possible. However, targeting of nuclear genes has been an inefficient process in most eukaryotes, including algae, plants, and animals, due to the dominance of integration of the applied DNA into nonhomologous regions of the genome. We have shown for the green alga Chlamydomonas reinhardtii by repairing a previously introduced truncated aminoglycoside 3-phosphotransferase gene, aphVIII, that single-stranded DNA can recombine with a homologous endogenous DNA region of interest. Nonhomologous DNA integration appeared to be more than 100-fold reduced compared with the use of double-stranded DNA, thus allowing isolation of the homologous recombinants. We propose that this method will be applicable to direct targeting of nuclear C. reinhardtii genes.

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“…White and J. E. Haber, personal communication). (iii) Single-stranded DNA is recombinogenic in yeast following transformation (28). (iv) Mismatch correction is involved in mitotic and meiotic gene conversion in yeast (29)(30)(31).…”

Section: Discussionmentioning

confidence: 99%

A DNA double chain break stimulates triparental recombination in Saccharomyces cerevisiae.

Ray

Machin

Stahl

1989

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

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Mitotic recombination between his3 heteroalleles on heterologous chromosomes is stimulated by a DNA double chain break delivered in vivo at a site 8.6 kilobase pairs distant from one his3 allele and unlinked to the other. The induced recombination at his3 is accompanied by gap repair at the break site using the uncut homolog as a template. The DNA between the break site and his3 is not deleted in most ofthe His+ recombinants.In Saccharomyces cerevisiae a DNA double chain break is repaired by homologous recombination (1-4). It has been imagined that the ends, after some exonucleolytic degradation, invade the intact homolog, the 3'-OH ends prime synthesis that repairs the gap, and the resulting dimer is resolved, sometimes producing recombinant products (3). In this double chain break repair (DCBR) model the site of the double chain break is coincident with the site of recombination. In phage A Red recombination, a double chain end is thought to be resected on one chain to promote a long region of heteroduplex DNA (5-8). By contrast, in the major Escherichia coli recombination pathway the RecBCD enzyme apparently enters the DNA helix at the site of a double chain break, travels along the molecule, encounters a specific DNA sequence termed Chi, and stimulates recombination in the neighborhood of Chi (9, 10). Thus, in the RecBCD pathway the site ofthe exchange is arbitrarily distant from the site of the double chain break. In fungi no evidence exists for a mechanism analogous to that in the RecBCD pathway; however, there is evidence of spatially discrete but statistically correlated multiple events of recombination (11)(12)(13)(14), some of which are difficult to reconcile with the current models of recombination (11,13,14).In this communication we demonstrate that in vegetative cells of S. cerevisiae a double chain break-induced repair event is accompanied by ectopic recombination 8.6 kilobase pairs (kb) away. These coincident recombination events, involving three chromosomes, yield products that are not altered between the two sites of recombination. MATERIALS AND METHODSStrains and Methods. Yeast strains are listed in Table 1 and their construction is described in Fig. 1. Media and biochemical methods are as described in Ray et al. (17) and in ref. 19. YEPL is YEP broth with 3 M lactate (pH 6.5). The E. coli strain was DH1 (15).Induction of HO. A single colony of the appropriate strain growing on SD-ura was suspended in YEPL broth and incubated with shaking for 8 hr. Aliquots were plated on YEP-gal, and the plates were incubated for 18 hr. Cells were washed off the plates, washed and diluted in water, and RESULTSDouble Chain Break in cis to his3-D Stimulates Ectopic Recombination. A difference between the current view of DCBR recombination in yeast (3) and that for RecBCDcatalyzed recombination in E. coli (1, 9, 10) is that in the latter process there is no degradation of DNA between the site of double chain break and the site of initiation ofrecombination. In yeast, can recombination initiated by a doub...

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Homologous recombination between single-stranded DNA and chromosomal genes in Saccharomyces cerevisiae.

Cited by 56 publications

References 25 publications

Targeted transformation of Ascobolus immersus and de novo methylation of the resulting duplicated DNA sequences.

Targeted transformation of Ascobolus immersus and de novo methylation of the resulting duplicated DNA sequences.

Nuclear-Gene Targeting by Using Single-Stranded DNA Avoids Illegitimate DNA Integration in Chlamydomonas reinhardtii

A DNA double chain break stimulates triparental recombination in Saccharomyces cerevisiae.

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