Alteration of N-Terminal Phosphoesterase Signature Motifs Inactivates Saccharomyces cerevisiae Mre11

Corresponding author: John H. J. Petrini, Laboratory of Genetics, University of Wisconsin Medical School, 445 Henry Mall, Madison, WI 53706., jpetrini{at}facstaff.wisc.edu (E-mail).

Communicating editor: M. LICHTEN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Saccharomyces cerevisiae Mre11, Rad50, and Xrs2 function in a protein complex that is important for nonhomologous recombination. Null mutants of MRE11, RAD50, and XRS2 are characterized by ionizing radiation sensitivity and mitotic interhomologue hyperrecombination. We mutagenized the four highly conserved phosphoesterase signature motifs of Mre11 to create mre11-11, mre11-2, mre11-3, and mre11-4 and assessed the functional consequences of these mutant alleles with respect to mitotic interhomologue recombination, chromosome loss, ionizing radiation sensitivity, double-strand break repair, and protein interaction. We found that mre11 mutants that behaved as the null were sensitive to ionizing radiation and deficient in double-strand break repair. We also observed that these null mutants exhibited a hyperrecombination phenotype in mitotic cells, consistent with previous reports, but did not exhibit an increased frequency of chromosome loss. Differential ionizing radiation sensitivities among the hypomorphic mre11 alleles correlated with the trends observed in the other phenotypes examined. Two-hybrid interaction testing showed that all but one of the mre11 mutations disrupted the Mre11-Rad50 interaction. Mutagenesis of the phosphoesterase signatures in Mre11 thus demonstrated the importance of these conserved motifs for recombinational DNA repair.

RECOMBINATIONAL DNA repair in Saccharomyces cerevisiae is mediated by the gene products of the RAD52 epistasis group (RAD50-57, RAD59, MRE11, and XRS2; IVANOV et al. 1992 ; AJIMURA et al. 1993 ; GAME 1993 ; BAI and SYMINGTON 1996 ). Mutants exhibit recombination defects and ionizing radiation (IR) sensitivity and can be subdivided into two subgroups according to their mitotic and meiotic recombination phenotypes. One subgroup, composed of the gene products of RAD51, RAD52, RAD54, RAD55, and RAD57, mediates homologous recombination (GAME 1993 ), whereas Mre11, Rad50, and Xrs2 function in a protein complex that is important for nonhomologous end-joining (SCHIESTL et al. 1994 ; JOHZUKA and OGAWA 1995 ; MOORE and HABER 1996 ; TSUKAMOTO et al. 1996 ).

S. cerevisiae mre11, rad50, and xrs2 null mutants are proficient at homologous recombination-based repair and exhibit a hyperrecombination phenotype in mitotic cells (MALONE et al. 1990 ; IVANOV et al. 1992 ; AJIMURA et al. 1993 ; SCHIESTL et al. 1994 ). However, double-strand breaks (DSBs) to initiate meiotic recombination do not form in these null mutants (ALANI et al. 1990 ; IVANOV et al. 1992 ), resulting in inviable spores (GAME and MORTIMER 1974 ; AJIMURA et al. 1993 ). The sporulation defect in mre11, rad50, and xrs2 null mutants is rescued in a spo13 background through bypass of meiosis I, confirming a role for these proteins at an early stage of meiosis (MALONE et al. 1991 ; IVANOV et al. 1992 ; AJIMURA et al. 1993 ).

The specific biochemical functions of the S. cerevisiae Mre11/Rad50/Xrs2 protein complex in recombinational DNA repair have not been elucidated. One model for the function of this protein complex is suggested by the similarity to the Escherichia coli exonuclease SbcCD (SHARPLES and LEACH 1995 ), which mediates single-strand endonuclease and ATP-dependent double-strand exonuclease activities (CONNELLY and LEACH 1996 ; CONNELLY et al. 1997 ). The N-terminal portion of Mre11 contains four motifs that are similar to SbcD, a component of the SbcCD nuclease complex (Figure 1). Three of these motifs are conserved in a functionally diverse spectrum of proteins that cleave phosphoester bonds, including serine/threonine protein phosphatases, whereas the fourth is unique to exonucleases such as SbcD. Hence, the S. cerevisiae Mre11/Rad50/Xrs2 protein complex may function as a nuclease in recombinational DNA repair (KOONIN 1994 ; ZHUO et al. 1994 ; SHARPLES and LEACH 1995 ).

View larger version (42K): In this window In a new window Download PPT slide   Figure 1. Mutagenesis of conserved Mre11 motifs. Identical or similar amino acids are shaded in black. The following amino acids are considered similar: D, E, N, and Q; F, W, and Y; K and R; A and G; I, V, and L; M; S, and T; C; H; and P. Scmre11 alleles are indicated above the sequence alignment; the wild-type and mutant residues are left and right of the symbol, respectively. Motifs are indicated below the sequence alignment. Residue numbers at the left indicate the number of the first residue in each species. Numbers in parentheses indicate the number of amino acids between each motif. Organisms, GenBank/SwissProt accession numbers, and references are as follows: Sc, Saccharomyces cerevisiae (D11463; JOHZUKA and OGAWA 1995 ); Sp, Schizosaccharomyces pombe (X82322; TAVASSOLI et al. 1995 ); Hs, Homo sapiens (U37359; PETRINI et al. 1995 ); Ec, Escherichia coli (P13457; NAOM et al. 1989 ); Ce, Caenorhabditis elegans (Z73978; WILSON et al. 1994 ); Af, Archaeoglobus fulgidus (AF1031; KLENK et al. 1997 ); and PPase, bacteriophage lambda protein phosphatase (P03772; COHEN et al. 1988 ).

The phenotypic features of mre11 mutants in both mitotic and meiotic cells support this hypothesis. In mitotic cells, the processing of double-stranded ends in mating-type switching and the resection of DSB ends during repair by single-strand annealing are delayed in mre11, rad50, and xrs2 mutants (IVANOV et al. 1994 , IVANOV et al. 1996 ; NAIRZ and KLEIN 1997 ; TSUBOUCHI and OGAWA 1998 ). The hypomorphic mre11S and rad50S alleles are proficient in the formation of DSBs in meiotic recombination, but are deficient in the resection of those breaks (ALANI et al. 1990 ; NAIRZ and KLEIN 1997 ; TSUBOUCHI and OGAWA 1998 ). In addition, the apparent mitotic cell cycle specificity of recombination defects (MOORE and HABER 1996 ), as well as the hyperrecombination phenotypes associated with the mre11, rad50, and xrs2 mutants, may indicate additional in vivo roles of the S. cerevisiae Mre11/Rad50/Xrs2 protein complex.

We mutagenized each of the four conserved motifs in Mre11 to create the mre11 alleles, mre11-11, mre11-2, mre11-3, and mre11-4. Phenotypic characterizations of these mutants were carried out with respect to mitotic recombination, chromosome loss, IR sensitivity, DSB repair, and association with Rad50. We found that all mre11 mutants exhibited increased IR sensitivity and explicit defects in DSB repair. These mutants also displayed a hyperrecombination phenotype in mitotic cells, the severity of which correlated with the degree of IR sensitivity and DSB repair deficiency. Finally, two-hybrid interaction testing showed that all but one of the mre11 mutations disrupted the ability of Mre11 to interact with Rad50.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Yeast strains and growth media:
The genotypes of the S. cerevisiae strains used in this study are described in Table 1. The mre11::hisG disruption strains, JPY33 and JPY44, were created by two-step gene replacement (ALANI et al. 1987 ) in strains 4053-5-2a and JPY40, respectively. The mre11::HIS3 disruption strains, JPY35 and JPY36, were created by one-step gene replacement (MORROW et al. 1995 ) in strains YPH500 (SIKORSKI and HIETER 1989 ) and YPH1124, respectively. YPH1124 is isogenic to YPH1015 (CONNELLY and HIETER 1996 ), except the chromosome fragment in YPH1015 is marked with HIS3. The mre11::HIS3 disruption deleted the entire coding region; the mre11::hisG disruption deleted all but the N-terminal 105 amino acids. Both alleles conferred a similar degree of IR sensitivity. Disruptions were confirmed by Southern blotting. All strains were cultured at 30°. Yeast media and manipulations were carried out according to standard procedures (AUSUBEL et al. 1989 ; GUTHRIE and FINK 1991 ). Additional reagents were used at the following concentrations: 5-bromo-4-chloro-3-indolyl ß-D-galactopyranoside (X-Gal, 40 µg/ml; Sigma Chemical, St. Louis, MO), L-canavanine (60 mg/liter; Sigma), and 5-fluoroorotic acid (5-FOA, 1 mg/ml; U. S. Biologicals).

Molecular biology:
Plasmid construction, Southern blotting, DNA sequencing, PCR, and DNA isolation were carried out according to standard procedures (AUSUBEL et al. 1989 ). The pSK-Scmre11 disruption plasmid was constructed by insertion of the hisG-URA3-hisG cassette (ALANI et al. 1987 ) at the HpaI sites in pSK-ScMRE11 [MRE11, cloned from a YCp50 genomic library (ROSE et al. 1987 ) by complementation of the IR sensitivity phenotype of the mre11 mutant, inserted into pBlue-script (Stratagene, La Jolla, CA) as a BamHI restriction fragment].

For construction of MRE11 expression constructs, the ADH1 promoter and terminator from pDB20 (BECKER et al. 1991 ) were inserted in pRS314 (SIKORSKI and HIETER 1989 ) as a BamHI restriction fragment. A 3X HA epitope was fused to the C terminus of Mre11 on pRS314-derived plasmids by gap repair using a PCR product derived from pMPY-3X-HA (SCHNEIDER et al. 1995 ). The 3X HA tag did not affect the ability of the construct to complement the IR sensitivity phenotype of the mre11 strain (data not shown).

For construction of two-hybrid N-terminal fusion expression constructs, EcoRI/XhoI fragments of MRE11, RAD50, hMRE11, and hRAD50 were cloned into the EcoRI/XhoI restriction sites of pEG202 or pJG4-5 (ESTOJAK et al. 1995 ). All constructs included the entire coding sequence, except the hMre11 fusions deleted the first four amino acids. Details of their constructions are available upon request.

Site-specific mutations in MRE11 were generated by linear amplification using complementary 35-mer primers. Restriction fragments bearing the desired mutation(s) were confirmed by DNA sequencing. Western blotting confirmed that all four mre11 alleles were expressed at similar levels. The allele bearing the mutation in motif I was named mre11-11, as mre11-1 had been previously described and characterized (AJIMURA et al. 1993 ).

Irradiation studies:
Strains were grown to mid-log phase (typical OD = 0.5; 1 x 10⁷ cells/ml), sonicated, and diluted to 4 x 10⁴ cells/ml in sterile ddH₂O. Cells were -irradiated in a Mark I ¹³⁷Cs source at a dose rate of ~2.5 Gy/min and plated at a density of 2000 cells/plate. Plates were incubated at 30° and scored for colony formation for 5 days after irradiation.

Pulsed-field gel electrophoresis:
Haploid JPY33 transformants were grown in liquid media lacking tryptophan (Trp) to maintain mre11 expression constructs to mid-log phase (typical OD = 0.5; 1 x 10⁷ cells/ml) and labeled with ³²P as described (JONG et al. 1995 ). Cells were -irradiated in 1x PBS [10 mM phosphate buffer (pH 7.4), 150 mM NaCl] and processed for pulsed-field gel electrophoresis (PFGE) immediately or after 3 hr postirradiation recovery in YPD as described (FRIEDL et al. 1995 ). Briefly, cells were washed in 50 mM EDTA (pH 8) and resuspended in 50 mM EDTA (pH 8), 10 mM Tris-Cl (pH 7.5), 1 mg/ml Zymolase 100T (U. S. Biologicals) buffer. An equal volume of 2% low-melting-point agarose (GIBCO, Grand Island, NY) at 55° was added to the cell suspension, and plugs were cast in plug molds (90 µl per plug). Plugs were transferred to 0.5 M EDTA (pH 8) and 10 mM Tris-Cl (pH 9) buffer; incubated at 37° for 8–16 hr; transferred to 0.5 M EDTA (pH 9), 10 mM Tris-Cl (pH 7.5), 20 mg/ml Sarkosyl, and 2 mg/ml proteinase K; and incubated at 50° for 24 hr. The plugs were washed once with cold rinsing buffer [10 mM EDTA (pH 8), 10 mM Tris-Cl (pH 7)] and rotated at 4° with three subsequent washing steps over the next 40 hr. Half of each plug was subjected to PFGE, and the other half counted in a scintillation counter. PFGE was carried out in 1% agarose gels (0.5 x TBE) on a Bio-Rad CHEF-DR II system (Bio-Rad Laboratories, Hercules, CA) at 14° for 22 hr at 6 V/cm, with an included angle of 120° and a switch time of 60–120 sec. After separation by PFGE, radiolabeled DNA was visualized and quantitated by phosphorimaging (Molecular Dynamics).

Mitotic recombination and chromosome loss:
The rates of mitotic interhomologue recombination and endogenous chromosome loss were determined as described (MEEKS-WAGNER and HARTWELL 1986 ), with the following exceptions: Single colonies were picked from plates lacking methionine (Met) and tryptophan (Trp), diluted to less than 200 cells/ml culture, grown to late-log phase (~20 doublings) in liquid media lacking Met and Trp, and plated onto solid canavanine-containing media lacking Met and/or Trp for analysis. A visual assay for ploidy of a nonessential chromosome fragment was performed essentially as described (HIETER et al. 1985 ). Telomeric sequences and the entire left arm of chromosome III were added onto the ends of a linear centromere-containing DNA fragment by homologous recombination, with endogenous chromosomes to generate the nonessential chromosome fragment (SPENCER et al. 1990 ). Single colonies were picked from a Trp dropout plate into sterile water, sonicated, diluted, and plated onto solid Trp dropout media containing 5-FOA and limiting amounts of adenine (6 µg/ml) to score loss rate of the nonessential URA3-marked chromosome fragment. Dilutions were plated onto solid Trp dropout media to control for cell number in each assay. Colonies were scored after 2–5 days of incubation at 30°.

Data analysis:
Calculation of mitotic recombination and chromosome loss rates using the method of the median and the maximum likelihood method (LEA and COULSON 1947 ) do not take into account differences in colony sizes, which may introduce error into the calculation of rates. We performed data analysis using a computer program written by B. E. Nelms (nelms@cms-stl.com). This program calculates mitotic recombination and chromosome loss rates using the method of the median and the maximum likelihood method, but it additionally uses a corrected maximum likelihood method that takes into account the variability introduced by unequal colony size. The maximum likelihood method is first used to approximate the mitotic recombination or chromosome loss rate based on the average colony size. This estimated rate is then used in a scaling procedure to account for additional mutations (if scaling up) or to subtract out recent mutations (if scaling down), based on the difference in individual colony sizes compared with the average. The corrected data represented by mutant and colony counts for colonies of equal size are then used to recalculate the mitotic recombination or chromosome loss rate by the maximum likelihood method. The corrected values thus obtained generally fell within 10% of those derived by the method of the median (LEA and COULSON 1947 ).

The statistical significance of differences observed in mitotic interhomologue recombination rate was determined using Wilcoxon Rank Sum and chi-square analyses. Pairwise comparisons of mutant fractions (corrected number of mutants divided by average colony size) for wild-type and mre11 mutant strains were carried out using data from all five independent transformants per strain.

Two-hybrid analysis:
The EGY48 strain, containing either a pJK103 or pSH18-34 lacZ reporter plasmid, was cotransformed with pEG202-DNA-binding domain (LexA) and pJG4-5-activation domain hybrid constructs (ESTOJAK et al. 1995 ) in the configurations shown in Figure 5. Transformants were examined for protein interaction by galactose-dependent ß-galactosidase production and leucine prototrophy, as described (AUSUBEL et al. 1989 ).

View larger version (69K): In this window In a new window Download PPT slide   Figure 2. Expression of mre11. Immunoblot analysis of yeast protein extracts (10 µg of total protein per lane) was performed using the 12CA5 mAb (A and B, top rows) or with polyclonal antiserum directed against hMre11 (C, top row). Loading controls shown for each filter (bottom rows) were performed as described in MATERIALS AND METHODS. (A) JPY33 strains transformed with wild-type or mre11 mutant expression constructs or empty vector. (B) EGY48 strains transformed with AD-ScMre11 or AD-Scmre11 fusion constructs. (C) EGY48 strains transformed with AD-hMre11 or AD-hmre11 fusion constructs.

View larger version (16K): In this window In a new window Download PPT slide   Figure 3. Radiation sensitivity of mre11 mutants. Strains were -irradiated at the doses indicated and scored for colony formation 5 days after irradiation. Plotted values indicate average of triplicate platings. Error bars represent standard deviation. (A) Haploid JPY33 transformants. (B) Haploid vs. diploid wild-type and mre11 strains. Open circles, JPY41 (MRE11/MRE11); solid circles, JPY23 (MRE11); open squares, JPY45 (mre11/mre11); solid squares, JPY44 (mre11).

View larger version (121K): In this window In a new window Download PPT slide   Figure 4. Detection of DSB repair in mre11 strains by pulsed-field gel electrophoresis. (A) Scanned image of pulsed-field agarose gel with 32P-labeled DNA from JPY33 transformants: MRE11 (lanes 1–3); mre11 (lanes 4–6). Unirradiated samples (lanes 1 and 4); irradiated samples (600 Gy) prepared at t = 0 hr (lanes 2 and 5) and at t = 3 hr (lanes 3 and 6) of recovery incubation. (B) Quantitative analysis of band intensities, normalized to the total number of radioactive counts loaded per lane, for MRE11 (graphs 1–3), mre11-11 (graphs 4–6), and mre11 (graphs 7–9) strains. Unirradiated samples (graphs 1, 4, and 7); irradiated samples (600 Gy) prepared at t = 0 hr (graphs 2, 5, and 8) and at t = 3 hr (graphs 3, 6, and 9) of recovery incubation. Normalized band intensity scale for the mre11-11 strain is half that of the MRE11 and mre11 strains due to differences in the total number of radioactive counts loaded per lane.

View larger version (50K): In this window In a new window Download PPT slide   Figure 5. Two-hybrid interaction between Mre11 and Rad50. Ten independent transformants per strain were picked onto solid media lacking uracil, histidine, and tryptophan. Cell patches were replica plated onto solid dropout media containing X-Gal and scored after 7 days of incubation at 30°. (A) EGY48 strains cotransformed with the pSH18-34 lacZ reporter plasmid, LexA-ScRad50 and AD-ScMre11, or AD-Scmre11 mutants. (B) EGY48 strains cotransformed with the JK103 lacZ reporter plasmid, LexA-hRad50 and AD-hMre11, or AD-hmre11 mutants. The mutations generated in hMRE11 are identical to those made in ScMRE11, except hmre11-2 changes DLF to GLS in motif II. (C) Configuration of AD-Mre11 or AD-mre11 fusion construct transformants.

Immunoblot analysis:
Protein extracts were prepared as described (NASMYTH 1977 ), and protein concentration was quantitated using the Bio-Rad Protein Assay. Western blotting was carried out according to standard procedures (HARLOW and LANE 1988 ) using the 12CA5 mAb (Boehringer Mannheim, Indianapolis, IN), -LexA antibody (gift from R. Brent), or with polyclonal antiserum directed against hMre11 or hRad50 (DOLGANOV et al. 1996 ). Chemiluminescent detection of antigen-antibody complexes was carried out with horseradish peroxidase-conjugated protein G, plus protein A (Pierce Chemical, Rockford, IL), or HRP-conjugated -mouse (Santa Cruz) secondary antibodies in conjunction with Super Signal Luminol reagent (Pierce). A portion of each filter was stained using the Bio-Rad Biotin-Blot Protein Detection Kit to control for protein loading according to manufacturer's instructions.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

mre11 alleles:
The four conserved N-terminal phosphoesterase motifs of Mre11 contain a number of residues that are invariant over vast phylogenetic distances (Figure 1). Our mutagenesis to create mre11-11, mre11-2, mre11-3, and mre11-4 was focused on those residues in motifs I–IV, respectively. The invariant histidine residues in motifs I and III (altered in mre11-11 and mre11-3) and the invariant aspartate in motif II (altered in mre11-2) occupy positions analogous to critical active site residues in the catalytic subunit of mammalian and bacteriophage serine/threonine phosphatases (ZHUO et al. 1994 ; GOLDBERG et al. 1995 ; GRIFFITH et al. 1995 ). Motif IV, which does not appear to be present in those protein phosphatases, similarly contains histidine residues (altered in mre11-4) that are invariant in the Mre11 homologues from archaea to humans (Figure 1). Phenotypic analysis of the engineered mre11 mutants was carried out in an mre11 deletion strain transformed with each of the four mre11 expression constructs. Control strains consisted of an mre11 deletion strain transformed with either the wild-type MRE11 expression construct or an empty vector. Expression was confirmed by Western blotting with the 12CA5 mAb (Figure 2).

Spontaneous mitotic interhomologue recombination is increased in mre11 mutants:
We assessed the impact of alterations in the Mre11 phosphoesterase motifs on the rates of mitotic interhomologue recombination and endogenous chromosome loss in JPY45. This strain is heterozygous for linked positive selection (HOM3 by methionine) and negative selection (can1 by canavanine) markers on opposite arms of chromosome V. Acquisition of canavanine resistance by chromosome loss results in methionine auxotrophy through concomitant loss of the HOM3+ allele, whereas interhomologue recombinants at CAN1 retain the HOM3+ allele, and the strain remains a methionine prototroph (HARTWELL and SMITH 1985 ). Recombination and loss events were scored as HOM3+ can1 and hom3- CAN1, respectively, and frequencies were then calculated based on the total number of cells plated, determined from control platings.

The engineered mre11 alleles fell into two general classes with respect to these phenotypic parameters; one class behaved essentially as the null mre11 mutant, and the other displayed an intermediate phenotype (Table 2). mre11-2 and mre11-4 transformants exhibited mitotic interhomologue recombination rates of 4.4 x 10^-3 and 3.4 x 10^-3, respectively. These values were increased about sevenfold relative to the wild-type transformant (6.4 x 10^-4) and were not significantly different from the mre11 mutant (4.0 x 10^-3). In contrast, mre11-11 was somewhat less affected, with a rate of 2.5 x 10^-3 (a fourfold increase over wild type). The increases in mitotic interhomologue recombination rates observed between these mre11 transformants and the wild-type transformant are statistically significant at a confidence level >99%. The recombination frequency in mre11-3 transformants was 1.1 x 10^-3, a twofold increase over the wild-type transformant. The rate in the heterozygous MRE11/mre11 strain JPY67 (6.4 x 10^-4) was indistinguishable from the wild-type rate (Table 2).

The frequency of endogenous chromosome loss measured in the chromosome V system was not increased in any of the mre11 transformants (data not shown). This result was unexpected given the dramatic impact of the Sprad32 mutation (S. pombe MRE11 homologue) on chromosome loss. In that context, spontaneous loss of a nonessential minichromosome was elevated 300-fold over the wild-type rate (TAVASSOLI et al. 1995 ). To ascertain whether loss of a nonessential chromosome fragment would be similarly increased in S. cerevisiae, we used the system developed by SPENCER et al. 1990 in JPY37 (GERRING et al. 1990 ; SPENCER et al. 1990 ).

JPY37 harbors a nonessential chromosome fragment marked with the SUP11 and URA3 genes. We plated JPY37 transformants on solid media containing 5-FOA to select for cells that had undergone chromosome fragment loss events and then scored 5-FOA-resistant colonies to calculate loss rate based on control platings. As above, the mre11 mutants did not dramatically increase the rate of chromosome loss in this assay system (Table 2). The mre11 strain exhibited a loss rate of 1.64 x 10^-3, a fourfold increase over the wild type (3.87 x 10^-4), and mre11-11, mre11-2, and mre11-4 were each threefold higher than wild type. Although we consistently observed at least a twofold increase in chromosome fragment loss rate in these mre11 transformants, this increase was not striking considering the relative impact of the Sprad32 mutation on chromosome loss described above. The nonessential chromosome fragment was as stable in mre11-3 as in the wild-type transformant, with a loss rate of 3.07 x 10^-4 (Table 2).

This system also permits distinction between chromosome fragment loss and nondisjunction. JPY37 is homozygous for the ade2-101 mutation, which results in the accumulation of red pigment in cells plated on solid media containing limiting amounts of adenine. This pigmented phenotype is suppressed by SUP11 in a dosage-dependent manner. The presence of one copy of the SUP11 gene results in pink colonies, and that of two copies results in white colonies. When grown under conditions that allow for loss of the chromosome fragment, nondisjunction (2:0) produces colonies with red and white sectors, whereas loss of the nonessential chromosome fragment (1:0) results in red- and pink-sectored colonies. All of the mre11 transformants displayed a bias toward chromosome fragment loss events over nondisjunction in a ratio of about 9:1, similar to the wild-type strain (data not shown).

In addition to the mitotic interhomologue recombination phenotypes observed, the null class of mre11 transformants also exhibited decreased plating efficiency of at least 2.5- to 3-fold relative to wild type. The relative plating efficiency of untransformed wild-type and mre11 strains plated on YPD was 2.3-fold, similar to that observed for the transformants plated on solid Trp dropout media (Table 2, footnote d).

mre11 mutants are sensitive to ionizing radiation:
Having established the phenotypes of mre11-11, mre11-2, mre11-3, and mre11-4 under normal growth conditions, we next assessed the impact of these mutations in cells treated with IR. mre11 transformants were -irradiated in liquid suspension at 0, 100, 200, and 300 Gy and plated in triplicate for scoring of colony formation. As we observed in normally growing cells, the mre11 mutants fell into two phenotypic classes, in this case according to severity of their IR sensitivity phenotypes relative to the wild-type transformant. The LD37 of the wild-type strain was >300 Gy. In contrast, the mre11-2 and mre11-4 mutants exhibited LD37s of 35 and 50 Gy, respectively (Figure 3A), indistinguishable from the mre11 strain (LD37 = 40 Gy). mre11-11 and mre11-3 transformants were more resistant to IR, exhibiting LD37s of 105 and 285 Gy, respectively.

Previous studies have shown that diploid rad50 and xrs2 strains exhibit increased radiation resistance in comparison to the corresponding mutant haploids (IVANOV et al. 1992 ). This diploid effect is not seen in rad52 mutants, presumably because homologous recombination is blocked in these strains. Consistent with the observation that the mre11 mutant is competent to undergo mitotic recombination (AJIMURA et al. 1993 ) (Table 2), we observed the same diploid-specific increase in radiation resistance upon comparison of haploid and diploid mre11 strains (Figure 3B).

mre11 mutants are defective in DSB repair:
We used PFGE to assay DSB repair directly. Chromosomal DNA of mre11 transformants was metabolically labeled by growth in the presence of ³²P-containing inorganic phosphate (JONG et al. 1995 ). ³²P-labeled DNA from unirradiated and -irradiated cells was separated into distinct chromosome bands by PFGE, and DSB repair was monitored by autoradiography (Figure 4A). Immediately after -irradiation with 600 Gy (t = 0 hr), a dose at which a wild-type MRE11 strain exhibits 27% survival (data not shown), bands corresponding to individual chromosomes diminished in intensity, and a heterogeneous population of lower molecular weight DNA became evident (Figure 4A, lanes 2 and 5).

Quantitation of radiolabeled chromosomal DNA in irradiated mre11 transformants was effected by phosphorimaging analysis (Figure 4B). In the wild-type transformant, we observed restoration of the three most prominent chromosome band intensities (migration distances of 25, 34, and 43 mm) to 42–49% of the unirradiated control by 3 hr postirradiation (t = 3 hr) and a decrease in the abundance of low molecular weight DNA (Figure 4A, lane 3, and Figure 4B, graphs 1–3). The mre11, mre11-2, and mre11-4 strains exhibited severe defects in the repair of IR-induced chromosome breaks. The intensities of the three most prominent bands diminished to 10% of the unirradiated control values during the 3-hr recovery period (Figure 4B, graphs 7 and 9). In contrast, the mre11-11 mutants exhibited an intermediate DSB repair capacity, with postrecovery peak intensities of 31–34% of the control values (Figure 4B, graphs 4 and 6). The mre11-3 transformants were essentially indistinguishable from the wild-type transformants by this criterion (data not shown).

mre11 mutants affect interaction with Rad50:
Epistasis and two-hybrid analyses have demonstrated that Mre11, Rad50, and Xrs2 function as a protein complex (JOHZUKA and OGAWA 1995 ; PETRINI et al. 1997 ). To address whether null and hypomorphic alleles impair protein function by disrupting complex formation, we examined the ability of mre11 mutant proteins to interact with Rad50 by two-hybrid interaction testing. Analysis of EGY48 transformants revealed that only the Mre11 (WT) and mre11-3 proteins were capable of interacting with Rad50, as determined by galactose-dependent lacZ activation (Figure 5A). The mutants that behaved as the null (mre11-2 and mre11-4) or had an intermediate phenotype (mre11-11) according to previous analyses did not exhibit this interaction.

Conservation of the Mre11/Rad50 protein complex in human cells (DOLGANOV et al. 1996 ) prompted us to generate corresponding mutations in hMRE11 (PETRINI et al. 1995 ). We tested the ability of the hmre11 mutant proteins to interact with hRad50 in EGY48 and found that hmre11 mutations that abolished or impaired interaction with hRad50 corresponded to null Scmre11 mutations (Figure 5B). Scmre11-11 did not interact with ScRad50, whereas hmre11-11 did interact with hRad50. These data were confirmed by galactose-dependent activation of the LEU2 reporter gene (data not shown), although the interaction between ScMre11 and ScRad50 detected by this method was relatively weak.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We mutagenized the conserved phosphoesterase signature motifs in the N terminus of Mre11 and assessed the impact of these mutations by several phenotypic parameters. The residues altered in this study correspond to those found at the catalytic center of proteins containing the canonical phosphoesterase motifs (ZHUO et al. 1994 ; GOLDBERG et al. 1995 ; GRIFFITH et al. 1995 ). Our findings demonstrate the importance of these motifs for recombination and DSB repair. The severity of spontaneous and radiation-induced phenotypes observed fell into two classes. One class behaved as the null mutant, exhibiting spontaneous interhomologue hyperrecombination, sensitivity to killing by IR, and DSB repair deficiency. The second class was less severe, but exhibited similar defects. Aside from the mutation in phosphoesterase motif III, mutations in this region also disrupted interaction with Rad50. The relative importance of impaired enzymatic function and reduced protein association are not distinguished in this study. Nonetheless, our results support the hypothesis that Mre11 functions require association with Rad50.

Because a sharply elevated frequency of minichromosome loss was observed in mutants of the MRE11 homologue in S. pombe, RAD32 (TAVASSOLI et al. 1995 ), we expected, but did not observe, increased chromosome loss in the mre11 mutants. Similarly, discordant phenotypic outcomes have been observed in mutants of the S. cerevisiae and S. pombe RAD52 homologues. Despite the relatively high degree of conservation between S. cerevisiae and S. pombe gene products, mutation of the S. pombe rad22 gene does not significantly impair homologous recombination, whereas S. cerevisiae rad52 mutations completely abolish that process (GAME 1993 ; FORTUNATO et al. 1996 ).

Mre11 is important for repair of spontaneous DSBs:
Colinearity in the trends exhibited by mre11 mutants with respect to mitotic interhomologue hyperrecombination, IR sensitivity, and DSB repair deficiency suggests that these phenotypic endpoints arose from similar DNA lesions. The role of the S. cerevisiae Mre11/Rad50/Xrs2 protein complex in the metabolism of DSBs induced by exogenous agents has been well established. Considerable evidence now exists to support the notion that DSBs occur spontaneously in bacterial, as well as eukaryotic cells (KUZMINOV 1995 ). In null mutants of HDF1, the S. cerevisiae Ku70 homologue, the presence of spontaneous DSBs is indicated by the induction of transcriptional and cell-cycle DNA damage responses when cells are shifted to growth conditions that inactivate residual Ku activity (BARNES and RIO 1997 ). Further, inducible inactivation of hRad51 in DT-40 chicken B lymphocyte cells revealed that DSBs form spontaneously in essentially all cells upon traversal through S phase (SONODA et al. 1998 ). Hence, it is reasonable to suggest the spontaneous mre11 phenotypes are attributable to spontaneously forming DSBs. Failure to efficiently repair such spontaneous lesions may account for the lethality of Mumre11 mutations in murine embryonic stem cells, where the persistence of DSBs may trigger apoptotic cell death or terminal cell cycle arrest (XIAO and WEAVER 1997 ).

Haploid mre11 mutants exhibit global defects in DSB repair:
The primary role of MRE11 appears to be in nonhomologous end-joining (SCHIESTL et al. 1994 ; MOORE and HABER 1996 ). The mitotic interhomologue hyperrecombination observed in mre11 mutants suggests that homologous recombination is not grossly affected in mre11 strains. However, the severity of DSB repair defects observed in PFGE analysis of the engineered mre11 mutants supports the hypothesis that the Mre11/Rad50/Xrs2 protein complex mediates sister chromatid cohesion in addition to its role in DSB repair (MOORE and HABER 1996 ). Haploid yeast strains were used in the PFGE experiments. Therefore, cells irradiated in G1 would lack a homologous template and would thus be restricted to DSB repair by nonhomologous end-joining, which is impaired by Mre11 deficiency. The profound DSB repair defect observed in mre11 mutants argues that both end-joining and sister chromatid recombination-based DSB repair in G2 cells are defective.

In wild-type cells, we found that the intensity of chromosomal bands was restored to <50% of control values at 3 hr following irradiation. Based on flow cytometric analyses of asynchronously growing cultures of this strain, ~60% of cells are in G1 during mid-log phase (data not shown). The commensurate reduction in intact chromosomes may reflect that even in wild-type haploid cells, nonhomologous end-joining-based repair of damage induced in G1 is inefficient. Consistent with this idea, we have observed that the ability of irradiated cells to reconstitute intact chromosomes is increased in diploid cells (data not shown). Because the PFGE assay we used relied upon measurement of intact chromosomes, misrepair events and degradation of unrepaired DNA do not appear in our calculations, although to some extent, the reduction in chromosome band intensity may be attributed to those causes. Because the values obtained for intact chromosomes are normalized to the amount of DNA loaded, restoration of intact chromosomes at 3 hr postirradiation indicates repair, rather than reduction in the background of intermediately sized broken DNA.

Scmre11 mutations do not appear to affect interhomologue recombination. This notion is supported by the increased IR resistance of diploid mre11 cells. The increased spontaneous mitotic interhomologue recombination observed in mre11 transformants may thus reflect increased reliance upon the homologue for repair of spontaneous damage. Genetic evidence for a distinct mitotic interhomologue recombination pathway has recently been obtained (KLEIN 1997 ).

Phosphoesterase signature motifs are important for Mre11 function in mitotic cells:
In previous mutational analyses of MRE11, intragenic complementation between N- and C-terminal mutations was observed (NAIRZ and KLEIN 1997 ). These data suggest that Mre11 contains two functionally distinct domains. The N-terminal mre11S mutations, which alter residues outside the conserved phosphoesterase motifs, affect DSB processing in meiotic recombination and confer relatively mild phenotypes in mitotic cells (NAIRZ and KLEIN 1997 ; TSUBOUCHI and OGAWA 1998 ). Mutations in the C terminus of Mre11 impair the creation of DSBs in meiotic cells. Hence, this region of the protein was hypothesized to control the assembly of a protein complex required for the initiation of meiotic recombination. C-terminal mutants also exhibit relatively severe mitotic phenotypes, suggesting that this Mre11 domain is important for function of the S. cerevisiae Mre11/Rad50/Xrs2 protein complex in mitotic cells as well.

The similarity between Mre11/Rad50 and SbcD/SbcC (SHARPLES and LEACH 1995 ) suggests that these proteins constitute the catalytic core of the S. cerevisiae Mre11/Rad50/Xrs2 protein complex. Our data show that alteration of conserved residues in the phosphoesterase motifs affects interaction with Rad50. If the Mre11 C terminus is indeed important for protein interaction, it is conceivable that it contains the Xrs2 interaction domain. The Mre11 homologues are not well conserved at the C termini. We have shown that the human hMre11/hRad50 protein complex does not contain a conserved Xrs2 homologue (CARNEY et al. 1998 ), suggesting that Xrs2 is conserved to a much lesser extent than Mre11 and Rad50. Based on the residues altered in the conserved phosphoesterase signature motifs, it is likely that the observed mre11 phenotypes may be explained by loss of enzymatic activity. However, these mutations also impair interaction between Mre11 and Rad50. Assessment of the relative contributions of impaired enzymatic activity and disrupted protein interactions to the phenotypes observed requires biochemical analysis of the mutant proteins.

	ACKNOWLEDGMENTS

We thank B. Garvik, L. Hartwell, C. Connelly, P. Hieter, our laboratory, and members of the Culbertson and Craig laboratories at the University of Wisconsin for materials, assistance, or support, and N. Drinkwater for helpful discussions. We also thank D. Bishop, D. Brow, M. Cox, and T. Prolla for critical reviews of the manuscript. This work was supported by grants from the March of Dimes, the Milwaukee Foundation, the American Cancer Society (grant NP-918), the Howard Hughes Medical Institute (J.H.J.P.), and the National Institutes of Health predoctoral training grant 5T32GM-07133 (D.A.B.). This is manuscript no. 3511 from the University of Wisconsin, Madison, Laboratory of Genetics.

Manuscript received March 5, 1998; Accepted for publication June 24, 1998.

	LITERATURE CITED

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