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

Abstract

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 ...

Exo1 Roles for Repair of DNA Double-Strand Breaks and Meiotic Crossing Over inSaccharomyces cerevisiae

“…In each interval, map distance was reduced 1.5-to 2.0-fold in the exo1 mutant (Table 3); this reduction is statistically significant (CEN3-HIS4, p Ͻ 0.02; CAN1-URA3, p Ͻ Ͻ 0.0001; URA3-HOM3, p Ͻ Ͻ 0.0001; HOM3-TRP2, p Ͻ 0.004). There is a modest but significant reduction of spore viability in the exo1 mutant (84%, 216 tetrads dissected, p Ͻ Ͻ 0.0001), as reported previously (Fiorentini et al, 1997). Furthermore, the number of viable spores per ascus is not random: fractions of asci that contain zero, two, or four viable spores per ascus (7, 12, and 74%, respectively) are more frequent than asci containing one or three viable spores per ascus (2 and 4%, respectively) ( Figure 8).…”

“…Because EXO1 encodes a dsDNA 5Ј to 3Ј exonuclease (Fiorentini et al, 1997;Tishkoff et al, 1997), promotion of ssDNA formation at DSB ends by EXO1 overexpression could explain the suppression of the repair defect of mre11. Although MRE11 is involved in the NHEJ pathway as well, EXO1 overproduction does not suppress the reduction of NHEJ, suggesting that EXO1 suppression is independent of the NHEJ pathway.…”

“…The Exo1 protein of fission and budding yeasts has been isolated as a 5Ј to 3Ј dsDNA exonuclease (Szankasi and Smith, 1995;Fiorentini et al, 1997). This protein is conserved through higher eukaryotes and is implicated in recombination and mismatch correction in vivo (Digilio et al, 1996;Tishkoff et al, 1997Tishkoff et al, , 1998Qiu et al, 1999).…”

The role of DNA exonucleases in protecting genome stability and their impact on ageing

“…There are some reports that loss of EXO1 gives rise to one form of the MMR-deficient syndrome human nonpolyposis colon cancer, HNPCC (Wu et al 2001), but a direct association has been questioned (Thompson et al 2004). Eukaryotic EXO1 is involved in various DNA metabolic pathways other than MMR, including meiosis (Fiorentini et al 1997) and recombination (Tsubouchi and Ogawa 2000). EXO1 may also play a role in resecting ends at broken replication forks to prevent generation of recombinogenic intermediates.…”

HNPCC mutations in the human DNA mismatch repair gene hMLH1 influence assembly of hMutLα and hMLH1–hEXO1 complexes

“…While, it is not known whether hEXO1 is involved in MMR, yeast strains de®cient in exonuclease 1 activity show increased spontaneous mutation rates and a small increase in recombination when the heteroduplex intermediate contains base ± base mismatches or small loops (Nicholson et al, 2000;Tischko et al, 1997Tischko et al, , 1998. It is known that both exonuclease 1 (EXO1) and MMR proteins are involved in homologous recombination (Fiorentini et al, 1997;Saparbaev et al, 1996;Sugawara et al, 1997) and that yeast EXO1 plays a role in double-stranded break repair and meiotic crossing over (Tsubouchi and Ogawa, 2000).…”