Double-strand-break repair and recombination catalyzed by a nuclear extract of Saccharomyces cerevisiae.

Symington, Lorraine S.

doi:10.1002/j.1460-2075.1991.tb08033.x

Cited by 32 publications

(17 citation statements)

References 41 publications

(22 reference statements)

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“…The 11.5-kb product (P1) corresponds in size to the expected overlap recombination product. The high-molecular-weight product (P2) has been observed previously with crude extracts and has a branched structure, as determined by two-dimensional agarose gel electrophoresis (20,51). The kinetics of product formation observed with the purified exonuclease were similar to those observed with crude nuclear extracts.…”

Section: Purification Of Exo Isupporting

confidence: 61%

“…In previous work aimed at developing an in vitro system for recombination in S. cerevisiae, we identified a 5Ј-3Ј exonuclease that was required for an end-joining reaction and for the formation of joint molecules between two linear duplexes that had a region of overlapping terminal homology (20,51). Because this activity was undiminished in cell extracts prepared from a strain containing mutations in the NUC1 and SEP1 genes, we concluded that it was distinct from these previously characterized exonucleases.…”

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confidence: 89%

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Exonuclease I of Saccharomyces cerevisiae Functions in Mitotic Recombination In Vivo and In Vitro

Fiorentini

Huang

Tishkoff

et al. 1997

Molecular and Cellular Biology

182

134

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We previously described a 5-3 exonuclease required for recombination in vitro between linear DNA molecules with overlapping homologous ends. This exonuclease, referred to as exonuclease I (Exo I), has been purified more than 300-fold from vegetatively grown cells and copurifies with a 42-kDa polypeptide. The activity is nonprocessive and acts preferentially on double-stranded DNA. The biochemical properties are quite similar to those of Schizosaccharomyces pombe Exo I. Extracts prepared from cells containing a mutation of the Saccharomyces cerevisiae EXO1 gene, a homolog of S. pombe exo1, had decreased in vitro recombination activity and when fractionated were found to lack the peak of activity corresponding to the 5-3 exonuclease. The role of EXO1 on recombination in vivo was determined by measuring the rate of recombination in an exo1 strain containing a direct duplication of mutant ade2 genes and was reduced sixfold. These results indicate that EXO1 is required for recombination in vivo and in vitro in addition to its previously identified role in mismatch repair.The currently favored models for homologous recombination predict a requirement for nucleases at several steps in the recombination reaction (18,37,55). Endonucleases are envisioned to function as initiators of recombination and in the resolution of crossed-strand intermediates. Exonucleases are thought to process break sites to generate single-stranded DNA, the substrate for binding by homologous pairing proteins. Late steps in the reaction, such as the repair of heteroduplex DNA (mismatch correction), also require the activity of exonucleases. The importance of exonucleases in recombination has been clearly demonstrated in Escherichia coli, in which 5Ј-to-3Ј exonucleases are required for all homologous recombination pathways (30). In addition, the single-stranded exonucleases RecJ (5Ј), exonuclease VII (Exo VII) (5Ј and 3Ј), and Exo I (3Ј) are required for mismatch repair in vitro (8).In vivo studies of double-strand break (DSB) repair in Saccharomyces cerevisiae provide further support for the role of exonucleases in recombination. After HO endonuclease cleavage at the MAT locus during mating-type switching, the DNA is resected to produce a 3Ј tail on the distal side of the HO cut site (64). This single-strand tail is believed to invade the donor locus, thus initiating the transfer of information. Formation of the 3Ј single-stranded tail is thought to result from the activity of a double-stranded 5Ј-3Ј exonuclease, but it could arise from the combined activities of a helicase and single-stranded endonuclease or exonuclease (35). Although mutation of several known recombination genes prevents mating-type switching, none appears to block the exonucleolytic processing step (49, 64). However, the degradation of the 5Ј strand is slower in rad50 and xrs2 mutants, suggesting that these genes may encode or regulate the activity of a nuclease (21). Most meiotic recombination hot spots are cleaved by endonucleases during meiosis, and the resulting DSBs are ...

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Section: Purification Of Exo Isupporting

confidence: 61%

mentioning

confidence: 89%

Exonuclease I of Saccharomyces cerevisiae Functions in Mitotic Recombination In Vivo and In Vitro

Fiorentini

Huang

Tishkoff

et al. 1997

Molecular and Cellular Biology

182

134

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“…This pattern is reproducible and has been observed at least four times. Normally, a streak in this region is attributed to recombination intermediates (3,46). Determination of the significance of this observation will require further analysis.…”

Section: Methodsmentioning

confidence: 99%

Specific initiation at an origin of replication from Schizosaccharomyces pombe.

Caddle

Calos

1994

Mol. Cell. Biol.

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Using a genetic assay for efficient autonomous replication, we have isolated from Schizosaccharomyces pombe a 6.2-kb fragment which shows the properties expected of an origin of DNA replication in S. pombe. A 2.8-kb subclone of the fragment has the same replication properties. Two-dimensional gel analysis of replication intermediates throughout plasmids carrying the 6.2-or 2.8-kb fragments shows that replication initiates only in a specific region, which can be localized to within several hundred base pairs, in the fragments. This region is also a site of replication initiation in the S. pombe chromosome where the fragments normally reside. These results provide strong evidence that initiation of replication in S. pombe is localized and mediated by specific DNA sequence signals.Characterization of replication origins in fission yeasts has lagged behind studies of budding yeasts, and it has remained unclear whether specific DNA sequences are involved. Both budding and fission yeasts are characterized by small (14-Mb) genomes and facile genetic analysis, thereby serving as valuable eukaryotic model organisms for a wide variety of studies. In particular, an advanced degree of understanding of the cell cycle exists for both types of yeasts (reviewed in reference 12) and presents the most promising available opportunity to elucidate the control of eukaryotic DNA replication at the molecular level. The definition of replication origins in fission yeasts is a key element in such studies.Origins of replication in the budding yeast Saccharomyces cerevisiae were initially identified genetically as autonomously replicating sequences (19,44). Autonomously replicating sequences characterized for S. cerevisiae contain an 11-bp consensus sequence (reviewed in reference 35). Mutations at each base pair of this sequence inhibit replication (24, 49). Less well defined flanking regions are also critical for efficient replication (6,29,37,48,51). By two-dimensional replication mapping techniques, it has been possible to correlate these genetically defined autonomously replicating sequences with the physical sites of initiation on both plasmids and chromosomes (3,9,20,21). Proteins that bind to the consensus region have recently been identified and are candidates to represent proteins mediating the initiation of replication (2).Knowledge about replication origins in the fission yeast Schizosaccharomyces pombe is far less complete. Various fragments that mediate autonomous replication, as judged by high transformation frequency and plasmid instability, have been reported for S. pombe (1,14,18,23,32,33,40 The inability to identify specific sequence requirements for replication initiation and the paucity of physical mapping studies have led to an ambiguous picture of origins of replication in the fission yeast, in which it has not been clear whether or not initiation is sequence specific. The only physical mapping study employing two-dimensional (2-D) gels to characterize replication intermediates in S. pombe involved a survey across a...

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“…In vitro recombination and repair systems using yeast extracts have been described previously (49,50). We have shown that nuclear extracts catalyze recombination between homologous linear and circular DNA substrates (49).…”

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confidence: 99%

“…We have shown that nuclear extracts catalyze recombination between homologous linear and circular DNA substrates (49). In this study, we have used yeast nuclear extracts to stimulate recombination between two DNAs with overlapping, homologous ends.…”

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confidence: 99%

A 5'-3' exonuclease from Saccharomyces cerevisiae is required for in vitro recombination between linear DNA molecules with overlapping homology.

Huang¹

1993

Mol. Cell. Biol.

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When two linear DNA molecules with overlapping, homologous ends were incubated with a yeast nuclear extract, they recombined at the region of homology to produce a joint molecule. We have identified a 5'-3' exonuclease in the extract that is likely to be responsible for the formation of the observed product. We propose that the exonuclease degrades each substrate to reveal regions of complementary sequence which anneal to form a recombinant product. Consistent with this model, we have partially purified the activity that promotes joint molecule formation and found it to cofractionate with a 5'-3' exonuclease activity through three consecutive chromatography steps. We have further characterized the reaction to determine the optimal length of homology. Substrates with homologous terminal overlaps of 29 to 958 bp were capable of product formation, whereas substrates with longer overlaps were not. Extracts prepared from a number of recombinationdefective or nuclease-deficient strains revealed no defect in exonuclease activity, indicating that the reaction is likely to be dependent upon the product of an as yet unidentified gene.The importance of strand-specific exonucleases in genetic recombination has been well documented. In prokaryotes, the recE gene product (exonuclease VIII), the T7 gene 6 product, and X exonuclease are all 5'-3' double-stranded exonucleases proven to play a role in recombination (reviewed in reference 41). Several models for recombination in eukaryotes also predict the involvement of exonucleases. In the double-strand break repair (DSBR) model, it is proposed that the DNA ends created at the break are degraded by a strand-specific exonuclease to create single-stranded tails which are able to invade homologous DNA, thus initiating the recombination process (51). In vivo studies have documented 5'-3' degradation of the broken ends formed at several double-strand breaks associated with recombination hot spots in the yeast Saccharomyces cerevisiae. After HO endonuclease cleavage at the AMT locus during mating-type switching, the DNA undergoes exonucleolytic degradation to produce a 3' tail on the distal side of the HO cut (55). This strand is believed to invade the intact donor of mating-locus information, thus initiating transfer of information. A double-strand break induced at theARG4 locus during meiosis is processed to leave a 3' single-stranded tail (48). This site has been shown to be a recombination initiation site (34), indicating that a 3' tail may be involved in the initiation of meiotic recombination. Similarly, a meiotic recombination hot spot created by the insertion of the LEU2 gene at the HIS4 locus is associated with a meiosis-specific doublestrand break that is processed to generate 3' single-stranded tails (5, 7).An exonuclease is also envisioned to act in the singlestrand annealing (SSA) model of recombination, which was proposed to explain certain recombination events in mammalian systems (24)(25)(26). In this model, strand-specific degradation by an exonuclease is predicted to...

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Double-strand-break repair and recombination catalyzed by a nuclear extract of Saccharomyces cerevisiae.

Cited by 32 publications

References 41 publications

Exonuclease I of Saccharomyces cerevisiae Functions in Mitotic Recombination In Vivo and In Vitro

Exonuclease I of Saccharomyces cerevisiae Functions in Mitotic Recombination In Vivo and In Vitro

Specific initiation at an origin of replication from Schizosaccharomyces pombe.

A 5'-3' exonuclease from Saccharomyces cerevisiae is required for in vitro recombination between linear DNA molecules with overlapping homology.

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