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

Huang, K N

doi:10.1128/mcb.13.6.3125

Cited by 33 publications

(26 citation statements)

References 35 publications

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“…The activity of the exonuclease to lead recombinant formation should be significantly lower in rate than that in the Mre11 complex but should be enough to attain a wild-type level at least. One of the candidate genes of the exonuclease may be the EXO1 gene (12,17), which is a multicopy suppressor of the MMS sensitivities of mre11 and rad50 deletions (45a). Why does another exonuclease not substitute for the meiotic recombination defect of the mre11-58 mutation?…”

Section: Discussionmentioning

confidence: 99%

A Novel mre11 Mutation Impairs Processing of Double-Strand Breaks of DNA during Both Mitosis and Meiosis

Tsubouchi

Ogawa

1998

Molecular and Cellular Biology

207

174

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Using complementation tests and nucleotide sequencing, we showed that the rad58-4 mutation was an allele of the MRE11 gene and have renamed the mutation mre11-58. Two amino acid changes from the wild-type sequence were identified; one is located at a conserved site of a phosphodiesterase motif, and the other is a homologous amino acid change at a nonconserved site. Unlike mre11 null mutations, the mre11-58 mutation allowed meiosis-specific double-strand DNA breaks (DSBs) to form at recombination hot spots but failed to process those breaks. DSB ends of this mutant were resistant to lambda exonuclease treatment. These phenotypes are similar to those of rad50S mutants. In contrast to rad50S, however, mre11-58 was highly sensitive to methyl methanesulfonate treatment. DSB end processing induced by HO endonuclease was suppressed in both mre11-58 and the mre11 disruption mutant. We constructed a new mre11 mutant that contains only the phosphodiesterase motif mutation of the Mre11-58 protein and named it mre11-58S. This mutant showed the same phenotypes observed in mre11-58, suggesting that the phosphodiesterase consensus sequence is important for nucleolytic processing of DSB ends during both mitosis and meiosis.The genes of the RAD52 epistasis group in Saccharomyces cerevisiae are necessary for repair of double-strand DNA breaks (DSBs) during mitosis and meiosis (35). Mutants resulting from mutations of these genes are classified into two subgroups according to their recombination abilities and meiotic DSB formation properties. One subgroup comprises rad51, -52, -54, -55, and -57 mutants. These are defective in mating-type switching and both mitotic and meiotic recombination (34,35,43). In these mutants, meiosis-specific DSBs form at recombination hot spots but are left unrepaired with extensive processing (34,42,47). Mutants resulting from mutations in these genes are also defective in viable spore formation, and spore inviability is not alleviated by introducing an additional spo13 mutation, which eliminates meiotic reductional division (24). The other subgroup consists of mre11, xrs2, and rad50 null mutants, which are proficient in mating-type switching and show spontaneous recombination at a high frequency during mitosis (1,14,18,30). In rad50 and xrs2 null mutants, processing of DSB ends is reduced and formation of recombinant is delayed (18,20,45). During meiosis, however, these three mutants are deficient in formation of meiosisspecific DSBs, induction of meiotic recombination, and viable spore formation (1,5,18,21), and their viable spore formation deficiency is alleviated by the introduction of a spo13 mutation (35). A mutant resulting from a non-null mutation of RAD50, called rad50S, accumulates unprocessed DSBs, and its spore inviability is not rescued by introducing a spo13 mutation (3). Therefore, MRE11, XRS2, and RAD50 appear to be involved in two distinct processes: (i) DSB repair during mitosis and (ii) DSB formation and processing from DSB ends during meiosis (5,18,21).Recently, a new mutation with ...

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Section: Discussionmentioning

confidence: 99%

A Novel mre11 Mutation Impairs Processing of Double-Strand Breaks of DNA during Both Mitosis and Meiosis

Tsubouchi

Ogawa

1998

Molecular and Cellular Biology

207

174

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“…This resection may be necessary for strand exchange to be initiated in homologous recombination, but results of others (3a) also suggest that the 3'-to-5' activity of HPP1 may be required for end joining. Recent evidence from reactions involving extracts of both Xenopus oocytes and yeast nuclei also shows that exonuclease digestion is an important component in the homologous recombination of linear DNA molecules with overlapping homology (10,15). Alternatively, it is not implausible that the opening up of the termini to allow single-strand annealing between the partners might be accomplished by helicases.…”

Section: Discussionmentioning

confidence: 99%

Characterization of DNA end joining in a mammalian cell nuclear extract: junction formation is accompanied by nucleotide loss, which is limited and uniform but not site specific.

Nicolás¹,

Young²

1994

Mol. Cell. Biol.

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Mammalian cells have a marked capacity to repair double-strand breaks in DNA, but the molecular and biochemical mechanisms underlying this process are largely unknown. A previous report has described an activity from mammalian cell nuclei that is capable of multimerizing blunt-ended DNA substrates (R. Fishel, M.K. Derbyshire, S.P. Moore, and C.S.H. Young, Biochimie 73:257-267, 1991). In this report, we show that nuclear extracts from HeLa cells contain activities which preferentially join linear plasmid substrates in either a head-to-head or tail-to-tail configuration, that the joining reaction is covalent, and that the joining is accompanied by loss of sequence at the junction. Sequencing revealed that there was a loss of a uniform number of nucleotides from junctions formed from any one type of substrate. The loss was not determined by any simple site-specific mechanism, but the number of nucleotides lost was affected by the precise terminal sequence. There was no major effect on the efficiency or outcome of the joining reaction with substrates containing blunt ends or 3' or 5' protruding ends. Using a pair of plasmid molecules with distinguishable restriction enzyme sites, we also observed that blunt-ended DNA substrates could join with those containing protruding 3' ends. As with the junctions formed between molecules with identical ends, there was uniform loss of nucleotides. Taken together, the data are consistent with two models for the joining reaction in which molecules are aligned either throughout most of their length or by using small sequence homologies located toward their ends. Although either model can explain the preferential formation of head-to-head and tail-to-tail products, the latter predicts the precise lossof nucleotides observed. These activities are found in all cell lines examined so far and most likely represent an important repair activity of the mammalian cell.

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“…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%

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

Fiorentini

Huang

Tishkoff

et al. 1997

Molecular and Cellular Biology

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183

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

Cited by 33 publications

References 35 publications

A Novel mre11 Mutation Impairs Processing of Double-Strand Breaks of DNA during Both Mitosis and Meiosis

A Novel mre11 Mutation Impairs Processing of Double-Strand Breaks of DNA during Both Mitosis and Meiosis

Characterization of DNA end joining in a mammalian cell nuclear extract: junction formation is accompanied by nucleotide loss, which is limited and uniform but not site specific.

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

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