A Uve1p-Mediated Mismatch Repair Pathway in Schizosaccharomyces pombe

UV damage endonuclease (Uve1p) from Schizosaccharomyces pombe was initially described as a DNA repair enzyme specific for the repair of UV light-induced photoproducts and proposed as the initial step in an alternative excision repair pathway. Here we present biochemical and genetic evidence demonstrating that Uve1p is also a mismatch repair endonuclease which recognizes and cleaves DNA 5' to the mispaired base in a strand-specific manner. The biochemical properties of the Uve1p-mediated mismatch endonuclease activity are similar to those of the Uve1p-mediated UV photoproduct endonuclease. Mutants lacking Uve1p display a spontaneous mutator phenotype, further confirming the notion that Uve1p plays a role in mismatch repair. These results suggest that Uve1p has a surprisingly broad substrate specificity and may function as a general type of DNA repair protein with the capacity to initiate mismatch repair in certain organisms.     

The Role of Topoisomerase II in Meiotic Chromosome Condensation and Segregation in Schizosaccharomyces pombe

Topoisomerase II is able to break and rejoin double-strand DNA. It controls the topological state and forms and resolves knots and catenanes. Not much is known about the relation between the chromosome segregation and condensation defects as found in yeast top2 mutants and the role of topoisomerase II in meiosis. We studied meiosis in a heat-sensitive top2 mutant of Schizosaccharomyces pombe. Topoisomerase II is not required until shortly before meiosis I. The enzyme is necessary for condensation shortly before the first meiotic division but not for early meiotic prophase condensation. DNA replication, prophase morphology, and dynamics of the linear elements are normal in the top2 mutant. The top2 cells are not able to perform meiosis I. Arrested cells have four spindle pole bodies and two spindles but only one nucleus, suggesting that the arrest is nonregulatory. Finally, we show that the arrest is partly solved in a top2 rec7 double mutant, indicating that topoisomerase II functions in the segregation of recombined chromosomes. We suggest that the inability to decatenate the replicated DNA is the primary defect in top2. This leads to a loss of chromatin condensation shortly before meiosis I, failure of sister chromatid separation, and a nonregulatory arrest.     

std1, a Gene Involved in Glucose Transport in Schizosaccharomyces pombe

A wild-type strain, Sp972 h⁻, of Schizosaccharomyces pombe was mutagenized with ethylmethanesulfonate (EMS), and 2-deoxyglucose (2-DOG)-resistant mutants were isolated. Out of 300 independent 2-DOG-resistant mutants, 2 failed to grow on glucose and fructose (mutants 3/8 and 3/23); however, their hexokinase activity was normal. They have been characterized as defective in their sugar transport properties, and the mutations have been designated as std1-8 and std1-23 (sugar transport defective). The mutations are allelic and segregate as part of a single gene when the mutants carrying them are crossed to a wild-type strain. We confirmed the transport deficiency of these mutants by [¹⁴C]glucose uptake. They also fail to grow on other monosaccharides, such as fructose, mannose, and xylulose, as well as disaccharides, such as sucrose and maltose, unlike the wild-type strain. Lack of growth of the glucose transport-deficient mutants on maltose revealed the extracellular breakdown of maltose in S. pombe, unlike in Saccharomyces cerevisiae. Both of the mutants are unable to grow on low concentrations of glucose (10 to 20 mM), while one of them, 3/23, grows on high concentrations (50 to 100 mM) as if altered in its affinity for glucose. This mutant (3/23) shows a lag period of 12 to 18 h when grown on high concentrations of glucose. The lag disappears when the culture is transferred from the log phase of its growth on high concentrations. These mutants complement phenotypically similar sugar transport mutants (YGS4 and YGS5) reported earlier by Milbradt and Hoefer (Microbiology 140:2617–2623, 1994), and the clone complementing YGS4 and YGS5 was identified as the only glucose transporter in fission yeast having 12 transmembrane domains. These mutants also demonstrate two other defects: lack of induction and repression of shunt pathway enzymes and defective mating.     

Meiotic Mismatch Repair Quantified on the Basis of Segregation Patterns in Schizosaccharomyces Pombe

Hybrid DNA with mismatched base pairs is a central intermediate of meiotic recombination. Mismatch repair leads either to restoration or conversion, while failure of repair results in post-meiotic segregation (PMS). The behavior of three G to C transversions in one-factor crosses with the wild-type alleles is studied in Schizosaccharomyces pombe. They lead to C/C and G/G mismatches and are compared with closely linked mutations yielding other mismatches. A method is presented for the detection of PMS in random spores. The procedure yields accurate PMS frequencies as shown by comparison with tetrad data. A scheme is presented for the calculation of the frequency of hybrid DNA formation and the efficiency of mismatch repair. The efficiency of C/C repair in S. pombe is calculated to be about 70%. Other mismatches are repaired with close to 100% efficiency. These results are compared with data published on mutations in Saccharomyces cerevisiae and Ascobolus immersus. This study forms the basis for the detailed analysis of the marker effects caused by G to C transversions in two-factor crosses.     

The Rec8 Gene of Schizosaccharomyces Pombe Is Involved in Linear Element Formation, Chromosome Pairing and Sister-Chromatid Cohesion during Meiosis

The fission yeast Schizosaccharomyces pombe does not form tripartite synaptonemal complexes during meiotic prophase, but axial core-like structures (linear elements). To probe the relationship between meiotic recombination and the structure, pairing, and segregation of meiotic chromosomes, we genetically and cytologically characterized the rec8-110 mutant, which is partially deficient in meiotic recombination. The pattern of spore viability indicates that chromosome segregation is affected in the mutant. A detailed segregational analysis in the rec8-110 mutant revealed more spores disomic for chromosome III than in a wild-type strain. Aberrant segregations are caused by precocious segregation of sister chromatids at meiosis I, rather than by nondisjunction as a consequence of lack of crossovers. In situ hybridization further showed that the sister chromatids are separated prematurely during meiotic prophase. Moreover, the mutant forms aberrant linear elements and shows a shortened meiotic prophase. Meiotic chromosome pairing in interstitial and centromeric regions is strongly impaired in rec8-110, whereas the chromosome ends are less deficient in pairing. We propose that the rec8 gene encodes a protein required for linear element formation and that the different phenotypes of rec8-110 reflect direct and indirect consequences of the absence of regular linear elements.     

The mutK Gene of Vibrio cholerae: a New Gene Involved in DNA Mismatch Repair

A new gene, mutK, of Vibrio cholerae, encoding a 19-kDa protein which is involved in repairing mismatches in DNA via a presumably methyl-independent pathway, has been identified. The product of the mutK gene cloned in either high- or low-copy-number vectors can reduce the spontaneous mutation frequency of Escherichia coli mutS, mutL, mutU, and dam mutants. The spontaneous mutation frequency of a chromosomal mutK knockout mutant was almost identical to that of wild-type V. cholerae cells, indicating that when the methyl-directed mismatch repair is blocked, the repair potential of MutK becomes apparent. The complete nucleotide sequence of the mutK gene has been determined, and the deduced amino acid sequence showed three open reading frames (ORFs), of which the ORF3 represents the mutK gene product. The mutK gene product has no significant homology with any of the proteins deposited in the EMBL data bank. ORF2, located upstream of mutK, encodes a 14-kDa protein which has more than 70% homology with a hypothetical protein found only downstream of the E. coli vsr gene. ORF1, located farther upstream of mutK, has more than 80% homology with a major cold shock protein found in several bacteria. Downstream of mutK, a partial ORF having 60% homology with an RNA methyltransferase has been identified. The mutK gene has recently been positioned in the ordered cloned DNA map of the genome of the V. cholerae strain from which the gene was isolated (10).     

Roles of Hop1 and Mek1 in Meiotic Chromosome Pairing and Recombination Partner Choice in Schizosaccharomyces pombe

Synaptonemal complex (SC) proteins Hop1 and Mek1 have been proposed to promote homologous recombination in meiosis of Saccharomyces cerevisiae by establishment of a barrier against sister chromatid recombination. Therefore, it is interesting to know whether the homologous proteins play a similar role in Schizosaccharomyces pombe. Unequal sister chromatid recombination (USCR) was found to be increased in hop1 and mek1 single and double deletion mutants in assays for intrachromosomal recombination (ICR). Meiotic intergenic (crossover) and intragenic (conversion) recombination between homologous chromosomes was reduced. Double-strand break (DSB) levels were also lowered. Notably, deletion of hop1 restored DSB repair in rad50S meiosis. This may indicate altered DSB repair kinetics in hop1 and mek1 deletion strains. A hypothesis is advanced proposing transient inhibition of DSB processing by Hop1 and Mek1 and thus providing more time for repair by interaction with the homologous chromosome. Loss of Hop1 and Mek1 would then result in faster repair and more interaction with the sister chromatid. Thus, in S. pombe meiosis, where an excess of sister Holliday junction over homologous Holliday junction formation has been demonstrated, Hop1 and Mek1 possibly enhance homolog interactions to ensure wild-type level of crossover formation rather than inhibiting sister chromatid interactions.Sexual reproduction in eukaryotes involves formation of haploid gametes from diploid cells by one round of DNA replication, pairing of the homologous chromosomes, and recombination and then by the two meiotic divisions (53). In fungi the gametes differentiate into haploid spores, which germinate to form vegetative cells. Crossover (CO) formation between homologous chromosomes and DNA repair processes between sister chromatids are required for spore viability (10, 55, 58).In vegetative cells homologous recombination (HR) is important for repair of DNA damage and stalled replication forks, with the sister chromatid as the preferred partner (28). Many of the enzymes involved in mitotic HR also contribute to meiotic recombination. In addition, meiosis-specific cytological structures and enzymes enhance recombination frequency (meiotic induction) and shift partner preference from sister chromatids to homologous chromosomes (3, 47, 64, 74). In detail the steps of HR vary between different types of sequence organization (allelic versus sister versus ectopic), between different types of DNA damage, between meiotic and mitotic cells, and between species (10, 55, 58).Meiotic recombination, including CO formation, is initiated by DNA double-strand breaks (DSBs). In Saccharomyces cerevisiae and other eukaryotes, DSBs are formed by Spo11. Many cofactors are required (29). The Schizosaccharomyces pombe homolog is Rec12, also requiring auxiliary factors whose elimination leads to loss of meiotic DSB formation (12). The 5′ single-strand ends at DSBs are processed by nucleases. In S. cerevisiae the MRX complex made up by the proteins Rad50, Mre11, and Xrs2 is required for this resection, as well as for DSB formation. The corresponding MRN complex of S. pombe (Rad50, Rad32, and Nbs1) is not required for DSB formation but is essential for DSB repair (43, 72). Deletion of rad50, rad32, or ctp1 (homologous to SAE2/COM1 in S. cerevisiae and CtIP in humans) leads to very low spore viability. These proteins are also essential for DSB processing (23, 24, 32, 43, 60, 62).Free DNA 3′ ends at DSBs are recruited for invasion of a sister or homologous chromatid by the strand transfer proteins Rad51 and Dmc1, again involving many accessory proteins (16). This results in the central intermediates of HR: heteroduplex DNA consisting of single strands originating from different chromatids and Holliday junctions (HJs). In S. cerevisiae HJs form preferably between homologs with a two- to sixfold excess over intersister HJs (64). Surprisingly, meiotic HJs form with about a fourfold excess between sisters in S. pombe (11). Eventually the intermediates are resolved into crossover (CO) and noncrossover (NCO) events. COs show exchange of the flanking sequences of the two chromatids involved and usually carry a patch of conversion (unilateral transfer of DNA sequences from one chromatid to its interacting partner) near the DSB site. NCOs are conversion events without associated COs (22). In S. pombe loss of core HR functions leads to very low spore viability: deletion of rad51 but not of dmc1 (20), double mutation of rad54 and rdh54 (7), inactivation of the endonuclease activity encoded by mus81 and eme1 (5, 52), and combined deletion of rad22 and rti1 (homologs of RAD52 of S. cerevisiae). But, differently from the other core functions, Rad22 and Rti1 are not required for CO and NCO (50).Early in meiotic prophase of many eukaryotes, axial elements (called lateral elements in later stages) form along sister chromatids, and pairing of homologous chromosomes is initiated, leading to juxtaposition of the homologous chromosomes along their whole length in the synaptonemal complex (SC) (54). In S. pombe no SC is formed, but linear elements (LEs), resembling axial elements of other eukaryotes, are formed. LEs do not form continuously along the chromosomes (1) but load the proteins Rec10, Hop1, and Mek1 (36, 44, 57), which are homologs of, or at least related, to the S. cerevisiae proteins Red1, Hop1, and Mek1, respectively, localizing to axial/lateral elements (2, 67). Hop1 carries a HORMA domain, also present in proteins associating with axial elements and regulating the progress of recombination in higher eukaryotes: Arabidopsis thaliana (61), Caenorhabditis elegans (9, 41), and mammals (18).In S. cerevisiae localization of Hop1 and Mek1 (meiosis-specific protein kinase) to axial elements is dependent on Red1 (2, 67). Mutation of the three S. cerevisiae genes results in reduction of DSB formation, CO and conversion frequencies, and spore viability (26, 31, 59). Direct comparison of unequal sister chromatid recombination (USCR) frequencies in an assay excluding the scoring of intrachromatid recombination (ICR) revealed no increase in the hop1 null mutant but about fourfold increases in the red1 and mek1 null mutants (69). The S. cerevisiae Hop1, Red1, and Mek1 proteins are involved in biasing meiotic DSB repair to occur between homologous chromosomes rather than between sister chromatids (47). Activated Mek1 kinase is required for the inhibition of sister chromatid-mediated DSB repair by Rad51, when the DMC1 gene is deleted and the meiotic recombination checkpoint is activated (4, 27, 38, 47). For Mek1 activation, phosphorylation of Hop1 by the Mec1/Tel1 kinases is also required (6).Less is known about the S. pombe proteins. Hop1 of S. pombe was identified as a nonsignificant hit by sequence comparison with full-length S. cerevisiae Hop1 and contains an N-terminal HORMA domain and a central zinc finger motif like Hop1 in S. cerevisiae. In addition they share a short homology block toward the C terminus (36). The Mek1 protein of S. pombe shares 34% identity and 54% similarity with its S. cerevisiae counterpart along the whole sequence. It contains an FHA domain in the N-terminal part like the other members of its family of checkpoint kinases and is involved in regulation of the meiotic cell cycle (57). Hop1 and Mek1 are strongly expressed in meiosis but not expressed or only slightly expressed in vegetative cells (42, 57). In prophase both proteins localize to LEs as defined by colocalization with the LE component Rec10 (36). Deletion of the distant RED1 homolog rec10 abolishes LE formation (36, 44) and strongly reduces meiotic recombination (17, 70). Rec10, but not Hop1 and Mek1, is required for localization of Rec7 (a distant homolog of S. cerevisiae Rec114) to meiotic chromosomes (34). Rec7 and Rec10 are required for Rec12 activity (12, 29).Obtaining information on the functions of Hop1 and Mek1 in S. pombe was the aim of the work presented here, especially on their possible roles in homolog versus sister discrimination for DSB repair. Deletion mutants have been studied with respect to spore viability and the frequencies of CO and conversion. They have also been assessed for genetic recombination events between sister chromatids in the known PS1 assay (63) and the newly developed VL1 assay (for details, see Fig. ​Fig.3).3). Physical analysis of DSB formation and repair has been performed in meiotic time course experiments. It is proposed that S. pombe Hop1 and Mek1 are promoting interactions between homologous chromosomes rather than inhibiting interactions between sister chromatids.Open in a separate windowFIG. 3.PS1 and VL1 assay systems for intrachromosomal recombination. Strains with constructs carrying repeated DNA sequences have been assayed for prototroph formation either by intrachromatid recombination (ICR, yielding prototrophs only in PS1) or by unequal sister chromatid recombination (USCR, in PS1 and VL1). Crosses of the constructs were performed with strains carrying a deletion of the ade6 gene to exclude other homologous recombination events. (A) The PS1 assay involves copies of the ade6 gene inactivated by either the hot spot mutation M26 or the mutation 469. The repeated sequences are separated by the ura4⁺ marker (63). ICR (left) or USCR (right) between the repeated sequences can lead to formation of adenine prototrophs that have lost the ura4⁺ marker by crossover (CO) or single-strand annealing (SSA) events. Adenine prototrophs maintaining the ura4⁺ marker can derive from noncrossover (NCO) events. Both types of pairing may lead to CO or NCO products. (B) The newly constructed VL1 assay (see the supplemental material) involves different truncations of the ade6 gene separated by the hyg^R marker (also called hphMX6), conferring hygromycin resistance. The left truncation carries a 3′ portion of ade6; the right truncation carries a 5′ portion of ade6. While the gray parts of the truncations are not overlapping, the white sections of 500-bp length are of almost identical sequence, allowing for homologous pairing. CO and SSA products resulting from ICR retain only the central portion of ade6 and remain auxotrophic. Adenine prototrophic CO and NCO products resulting from USCR both retain hygromycin resistance. Note that NCO events may arise through loop formation of one sister chromatid and pairing with a single block (500 bp) of the repeated ade6 sequence (39).     

The Yeast Mer2 Gene Is Required for Chromosome Synapsis and the Initiation of Meiotic Recombination

Mutation of the MER2 gene of Saccharomyces cerevisiae confers meiotic lethality. To gain insight into the function of the Mer2 protein, we have carried out a detailed characterization of the mer2 null mutant. Genetic analysis indicates that mer2 completely eliminates meiotic interchromosomal gene conversion and crossing over. In addition, mer2 abolishes intrachromosomal meiotic recombination, both in the ribosomal DNA array and in an artificial duplication. The results of a physical assay demonstrate that the mer2 mutation prevents the formation of meiosis-specific, double-strand breaks, indicating that the Mer2 protein acts at or before the initiation of meiotic recombination. Electron microscopic analysis reveals that the mer2 mutant makes axial elements, which are precursors to the synaptonemal complex, but homologous chromosomes fail to synapse. Fluorescence in situ hybridization of chromosome-specific DNA probes to spread meiotic chromosomes demonstrates that homolog alignment is also significantly reduced in the mer2 mutant. Although the MER2 gene is transcribed during vegetative growth, deletion or overexpression of the MER2 gene has no apparent effect on mitotic recombination or DNA damage repair. We suggest that the primary defect in the mer2 mutant is in the initiation of meiotic genetic exchange.     

DNA错配修复、染色体不稳定和肿瘤的关系

DNA错配修复系统可以识别并纠正DNA复制过程中出现的错误.保证基因组的稳定性和完整性.错配修复系统缺陷可能导致遗传物质发生突变,引发恶性肿瘤.肿瘤患者经常表现出染色体不稳定,具体表现为微卫星不稳定性和杂合性缺失.本文就DNA错配修复、染色体不稳定和肿瘤之间的联系予以综述.     

Xrs2, a DNA Repair Gene of Saccharomyces Cerevisiae, Is Needed for Meiotic Recombination

The XRS2 gene of Saccharomyces cerevisiae has been previously identified as a DNA repair gene. In this communication, we show that XRS2 also encodes an essential meiotic function. Spore inviability of xrs2 strains is rescued by a spo13 mutation, but meiotic recombination (both gene conversion and crossing over) is highly depressed in spo13 xrs2 diploids. The xrs2 mutation suppresses spore inviability of a spo13 rad52 strain suggesting that XRS2 acts prior to RAD52 in the meiotic recombination pathway. In agreement with the genetic data, meiosis-specific double-strand breaks at the ARG4 meiotic recombination hotspot are not detected in xrs2 strains. Despite its effects on meiotic recombination, the xrs2 mutation does not prevent mitotic recombination events, including homologous integration of linear DNA, mating-type switching and radiation-induced gene conversion. Moreover, xrs2 strains display a mitotic hyper-rec phenotype. Haploid xrs2 cells fail to carry out G2-repair of gamma-induced lesions, whereas xrs2 diploids are able to perform some diploid-specific repair of these lesions. Meiotic and mitotic phenotypes of xrs2 cells are very similar to those of rad50 cells suggesting that XRS2 is involved in homologous recombination in a way analogous to that of RAD50.     

Swi6, a Gene Required for Mating-Type Switching, Prohibits Meiotic Recombination in the Mat2-Mat3 ``cold Spot'''' of Fission Yeast

Mitotic interconversion of the mating-type locus (mat1) of the fission yeast Schizosaccharomyces pombe is initiated by a double-strand break at mat1. The mat2 and mat3 loci act as nonrandom donors of genetic information for mat1 switching such that switches occur primarily (or only) to the opposite mat1 allele. Location of the mat1 "hot spot" for transposition should be contrasted with the "cold spot" of meiotic recombination located within the adjoining mat2-mat3 interval. That is, meiotic interchromosomal recombination in mat2, mat3 and the intervening 15-kilobase region does not occur at all. swi2 and swi6 switching-deficient mutants possess the normal level of double-strand break at mat1, yet they fail to switch efficiently. By testing for meiotic recombination in the cold spot, we found the usual lack of recombination in a swi2 mutant but a significant level of recombination in a swi6 mutant. Therefore, the swi6 gene function is required to keep the donor loci inert for interchromosomal recombination. This finding, combined with the additional result that switching primarily occurs intrachromosomally, suggests that the donor loci are made accessible for switching by folding them onto mat1, thus causing the cold spot of recombination.     

Characterization of Schizosaccharomyces pombe Copper Transporter Proteins in Meiotic and Sporulating Cells

Meiosis requires copper to undertake its program in which haploid gametes are produced from diploid precursor cells. In Schizosaccharomyces pombe, copper is transported by three members of the copper transporter (Ctr) family, namely Ctr4, Ctr5, and Ctr6. Although central for sexual differentiation, very little is known about the expression profile, cellular localization, and physiological contribution of the Ctr proteins during meiosis. Analysis of gene expression of ctr4⁺ and ctr5⁺ revealed that they are primarily expressed in early meiosis under low copper conditions. In the case of ctr6⁺, its expression is broader, being detected throughout the entire meiotic process with an increase during middle- and late-phase meiosis. Whereas the expression of ctr4⁺ and ctr5⁺ is exclusively dependent on the presence of Cuf1, ctr6⁺ gene expression relies on two distinct regulators, Cuf1 and Mei4. Ctr4 and Ctr5 proteins co-localize at the plasma membrane shortly after meiotic induction, whereas Ctr6 is located on the membrane of vacuoles. After meiotic divisions, Ctr4 and Ctr5 disappear from the cell surface, whereas Ctr6 undergoes an intracellular re-location to co-localize with the forespore membrane. Under copper-limiting conditions, disruption of ctr4⁺ and ctr6⁺ results in altered SOD1 activity, whereas these mutant cells exhibit substantially decreased levels of CAO activity mostly in early- and middle-phase meiosis. Collectively, these results emphasize the notion that Ctr proteins exhibit differential expression, localization, and contribution in delivering copper to SOD1 and Cao1 proteins during meiosis.     

A Guaninine Nucleotide Exchange Factor Is a Component of the Meiotic Spindle Pole Body in Schizosaccharomyces pombe

Spore morphogenesis in yeast is driven by the formation of membrane compartments that initiate growth at the spindle poles during meiosis II and grow to encapsulate daughter nuclei. Vesicle docking complexes, called meiosis II outer plaques (MOPs), form on each meiosis II spindle pole body (SPB) and serve as sites of membrane nucleation. How the MOP stimulates membrane assembly is not known. Here, we report that SpSpo13, a component of the MOP in Schizosaccharomyces pombe, shares homology with the guanine nucleotide exchange factor (GEF) domain of the Saccharomyces cerevisiae Sec2 protein. ScSec2 acts as a GEF for the small Rab GTPase ScSec4, which regulates vesicle trafficking from the late-Golgi to the plasma membrane. A chimeric protein in which the ScSec2-GEF domain is replaced with SpSpo13 is capable of supporting the growth of a sec2Δ mutant. SpSpo13 binds preferentially to the nucleotide-free form of ScSec4 and facilitates nucleotide exchange in vitro. In vivo, a Spspo13 mutant defective in GEF activity fails to support membrane assembly. In vitro specificity experiments suggest that SpYpt2 is the physiological substrate of SpSpo13. These results demonstrate that stimulation of Rab-GTPase activity is a property of the S. pombe MOP essential for the initiation of membrane formation.     

Mlh1-Mlh3, a Meiotic Crossover and DNA Mismatch Repair Factor,Is a Msh2-Msh3-stimulated Endonuclease

Crossing over between homologous chromosomes is initiated in meiotic prophase in most sexually reproducing organisms by the appearance of programmed double strand breaks throughout the genome. In Saccharomyces cerevisiae the double-strand breaks are resected to form three prime single-strand tails that primarily invade complementary sequences in unbroken homologs. These invasion intermediates are converted into double Holliday junctions and then resolved into crossovers that facilitate homolog segregation during Meiosis I. Work in yeast suggests that Msh4-Msh5 stabilizes invasion intermediates and double Holliday junctions, which are resolved into crossovers in steps requiring Sgs1 helicase, Exo1, and a putative endonuclease activity encoded by the DNA mismatch repair factor Mlh1-Mlh3. We purified Mlh1-Mlh3 and showed that it is a metal-dependent and Msh2-Msh3-stimulated endonuclease that makes single-strand breaks in supercoiled DNA. These observations support a direct role for an Mlh1-Mlh3 endonuclease activity in resolving recombination intermediates and in DNA mismatch repair.     

Vba2p,A Vacuolar Membrane Protein Involved in Basic Amino Acid Transport in Schizosaccharomyces pombe

A recent study filling the gap in the genome sequence in the left arm of chromosome 2 of Schizosaccharomyces pombe revealed a homolog of budding yeast Vba2p, a vacuolar transporter of basic amino acids. GFP-tagged Vba2p in fission yeast was localized to the vacuolar membrane. Upon disruption of vba2, the uptake of several amino acids, including lysine, histidine, and arginine, was impaired. A transient increase in lysine uptake under nitrogen starvation was lowered by this mutation. These findings suggest that Vba2p is involved in basic amino acid transport in S. pombe under diverse conditions.