Distinct Regulation of Mlh1p Heterodimers in Meiosis and Mitosis in Saccharomyces cerevisiae

Mlh1p forms three heterodimers that are important for mismatch repair (Mlh1p/Pms1p), crossing over during meiosis (Mlh1p/Mlh3p), and channeling crossover events into a specific pathway (Mlh1p/Mlh2p). All four proteins contain highly conserved ATPase domains and Pms1p has endonuclease activity. Studies of the functional requirements for Mlh1p/Pms1p in Saccharomyces cerevisae revealed an asymmetric contribution of the ATPase domains to repairing mismatches. Here we investigate the functional requirements of the Mlh1p and Mlh3p ATPase domains in meiosis by constructing separation of function mutations in Mlh3p. These mutations are analogous to mutations of Mlh1p that have been shown to lead to loss of ATP binding and/or ATP hydrolysis. Our data suggest that ATP binding by Mlh3p is required for meiotic crossing over while ATP hydrolysis is dispensable. This has been seen previously for Mlh1p. However, when mutations that affect ATP hydrolysis by both Mlh3p and Mlh1p are combined within a single cell, meiotic crossover frequencies are reduced. These observations suggest that the function of the Mlh1p/Mlh3p heterodimer requires both subunits to bind ATP but only one to efficiently hydrolyze it. Additionally, two different amino acid substitutions to the same residue (G97) in Mlh3p affect the minor mismatch repair function of Mlh3p while only one of them compromises its ability to promote crossing over. These studies thus reveal different functional requirements among the heterodimers formed by Mlh1p.CROSSING over during meiosis not only generates variation but is also important for providing the necessary interactions between homologous chromosomes that ensure correct segregation at division I of meiosis. Recombination is initiated by the production of programmed double-strand breaks (DSBs), catalyzed by the covalently attached Spo11p (Bergerat et al. 1997; Keeney et al. 1997), aided by a number of proteins (reviewed in Keeney and Neale 2006). DSBs are made at a much higher frequency than crossovers, and designation of only a subset to yield crossovers is thought to occur during early stages of DSB repair (Borner et al. 2004). At least two distinct pathways contribute to the production of crossover events in Saccharomyces cerevisiae. The major pathway is dependent on Msh4p/Msh5p and the mismatch repair proteins Mlh1p and Mlh3p (Ross-MacDonald and Roeder 1994; Hollingsworth et al. 1995; Hunter and Borts 1997; Wang et al. 1999; Abdullah et al. 2004) and the second pathway is dependent on Mus81p/Mms4p endonuclease (de los Santos et al. 2001, 2003).Mitotic mismatch repair (MMR) is the process by which mutations that arise during DNA replication and recombination are recognized and removed (reviewed in Kolodner 1996; Harfe and Jinks-Robertson 2000). Msh2p forms a heterodimer with Msh6p (MutSα) to repair base–base mismatches and small insertions and/or deletions and with Msh3p (MutSβ) to repair large insertions and/or deletions (reviewed in Jiricny 2006). Mlh1p forms heterodimers with Pms1p, Mlh2p, and Mlh3p to coordinate the removal of these mismatches (Prolla et al. 1994; Wang et al. 1999). Mlh1p/Pms1p (MutLα) are involved in the repair of all types of mismatches in combination with MutSα and MutSβ, and in the absence of either protein a mutator phenotype is observed (Habraken et al. 1997, 1998). Mlh1p/Mlh2p (MutLβ) and Mlh1p/Mlh3p (MutLγ) are involved in the MutSβ pathway only, which repairs frameshift mutations caused by insertions or deletions. Consequently mlh3Δ mutants only exhibit a weak mutator phenotype, due to a lesser involvement in mismatch repair and a partial overlap in function with Pms1p (Flores-Rozas and Kolodner 1998; Harfe et al. 2000).Although the MutL homologs interact primarily through their C-terminal domains (Pang et al. 1997; Ban and Yang 1998), it is thought that the N-terminal domains must also interact for the complex to be fully functional (Ban and Yang 1998). Binding of ATP causes the proteins to undergo conformational changes, which are essential for the interaction between the N termini (Ban et al. 1999; Tran and Liskay 2000; Sacho et al. 2008). ATP hydrolysis and subsequent release of ADP is required to allow the protein complex to return to its initial state, completing the cycle so that the subunits are ready to bind ATP again if required. Using mutants of MLH1 and PMS1 that are presumed to be defective for ATP binding and/or ATP hydrolysis, it has been shown that both of these functions are essential for fully effective mismatch repair (Tran and Liskay 2000). However, the ATP binding and ATP hydrolysis mutants of PMS1 exhibited lower mitotic mutation rates than the corresponding MLH1 ATPase mutants, suggesting that there is functional asymmetry within the Mlh1p/Pms1p heterodimer (Tran and Liskay 2000; Hall et al. 2002). Another example of the asymmetry in the contributions of these subunits to function can be seen in assays that measure recombination between diverged sequences (homeologous recombination). The Mlh1p ATPase activity has been shown to be more important for the suppression of homeologous recombination than Pms1p ATPase activity (Welz-Voegele et al. 2002). This functional asymmetry is supported by in vitro biochemical analysis that demonstrated Pms1p has a lower ATP binding affinity than Mlh1p (Hall et al. 2002).As mentioned above, Mlh1p/Mlh3p function in the Msh4p/Msh5p pathway for meiotic recombination (Hunter and Borts 1997; Santucci-Darmanin et al. 2000). The Msh4p/Msh5p complex is thought to act in the stabilization of Holliday junction intermediates to allow their resolution in a crossover configuration (Snowden et al. 2004). The Mlh1p/Mlh3p complex has been suggested to act in the resolution of these structures, either directly or indirectly. Human Pms2 and its yeast homolog, Pms1p, have been shown to possess a latent endonuclease activity, conferred by a motif that is conserved among some of the MutL homologs, including Mlh3p (Kadyrov et al. 2006, 2007). Mutations in the DHQA(X)₂E(X)₄E motif in yeast MLH3 cause defects in both mismatch repair and meiotic recombination equivalent to mlh3Δ, suggesting that Mlh3p may also possess an endonuclease activity that is important for the generation of crossovers (Nishant et al. 2008).ATP binding by Mlh1p has been shown to be important for both of its meiotic functions (crossing over and repair of heteroduplex DNA) (Pang et al. 1997; Tran and Liskay 2000; Hoffmann et al. 2003). In contrast, the ATP hydrolysis mutant mlh1-E31A/mlh1-E31A appears to have no effect on meiotic recombination (Tran and Liskay 2000; Hoffmann et al. 2003). This may partly be explained by in vitro studies demonstrating that this mutant exhibits a low level of ATPase activity (Hall et al. 2002).The meiotic functions of MLH1 can be functionally separated as shown by mutating the same residue, G98, to different amino acids (Hoffmann et al. 2003). The residue G98 is situated in the ATPase motif in the GFRGEAL box (GYRGDAL in Mlh3p), which forms the lid of the ATP binding pocket. Mutations in this motif are predicted to affect ATP binding and/or heterodimerization with Pms1p (Ban and Yang 1998; Ban et al. 1999). Mutating the residue G98 in the ATP binding lid to alanine resulted in defective repair of heteroduplex DNA while crossing over was unaffected, but when the same residue was mutated to valine both mismatch repair and crossover functions were defective (Hoffmann et al. 2003). The mlh1-G98V mutant disrupts the interaction of Mlh1p with Pms1p, while mlh1-G98A does not (Pang et al. 1997). This may contribute to the difference observed in the effect on crossing over as Mlh1p is thought to interact with Pms1p and Mlh3p through the same residues (Wang et al. 1999; Kondo et al. 2001). Consequently if the interaction with Pms1p is affected then it is likely that the interaction with Mlh3p is also disrupted.We constructed mlh3 mutants corresponding to the ATP binding and ATP hydrolysis mutants of mlh1 to explore the role of Mlh3p in meiotic recombination. We also constructed mlh3-G97A and mlh3-G97V mutants, equivalent to the mlh1-G98A/V pair that has been shown to differentially affect the mitotic and meiotic functions of Mlh1p. All mutants were assayed for mitotic mismatch repair, meiotic heteroduplex repair, crossing over, and chromosome segregation.     

The roles of the Saccharomyces cerevisiae RecQ helicase SGS1 in meiotic genome surveillance

Background

The Saccharomyces cerevisiae RecQ helicase Sgs1 is essential for mitotic and meiotic genome stability. The stage at which Sgs1 acts during meiosis is subject to debate. Cytological experiments showed that a deletion of SGS1 leads to an increase in synapsis initiation complexes and axial associations leading to the proposal that it has an early role in unwinding surplus strand invasion events. Physical studies of recombination intermediates implicate it in the dissolution of double Holliday junctions between sister chromatids.

Methodology/Principal Findings

In this work, we observed an increase in meiotic recombination between diverged sequences (homeologous recombination) and an increase in unequal sister chromatid events when SGS1 is deleted. The first of these observations is most consistent with an early role of Sgs1 in unwinding inappropriate strand invasion events while the second is consistent with unwinding or dissolution of recombination intermediates in an Mlh1- and Top3-dependent manner. We also provide data that suggest that Sgs1 is involved in the rejection of ‘second strand capture’ when sequence divergence is present. Finally, we have identified a novel class of tetrads where non-sister spores (pairs of spores where each contains a centromere marker from a different parent) are inviable. We propose a model for this unusual pattern of viability based on the inability of sgs1 mutants to untangle intertwined chromosomes. Our data suggest that this role of Sgs1 is not dependent on its interaction with Top3. We propose that in the absence of SGS1 chromosomes may sometimes remain entangled at the end of pre-meiotic replication. This, combined with reciprocal crossing over, could lead to physical destruction of the recombined and entangled chromosomes. We hypothesise that Sgs1, acting in concert with the topoisomerase Top2, resolves these structures.

Conclusions

This work provides evidence that Sgs1 interacts with various partner proteins to maintain genome stability throughout meiosis.     

Epitope specificity of the anti-(B cell lymphoma) monoclonal antibody,LL2

LL2 is a murine monoclonal antibody IgG2a reactive with B cells and non-Hodgkin's B-cell lymphoma, which, in a radioiodinated form, induces responses in lymphoma patients [Goldenberg et al. (1991) J Clin Oncol 9:548–564]. In this report we identify LL2 as a member of the CD22 cluster. The molecular size of the antigen, its expression profile, and competitive blocking studies were used to establish this identification. By Western blot analysis and immunoprecipitation studies using the Raji Burkitt's lymphoma cell line metabolically labelled with [³H]leucine, the LL2 antigen was determined to correspond to a molecular mass of 140 kDa. The molecular mass of the LL2 antigen, and the B-cell-restricted reactivity of the LL2 antibody, were consistent with both the CD21 and CD22 clusters. To assess additional similarities and differences between LL2 and anti-CD22 and anti-CD21, the binding of these mAb to cultured cell lines. Nalm-6 and Molt-4, was compared by flow cytometry. The binding profile of LL2 on these cell lines was consistent with anti-CD22, but not anti-CD21. Sequential immunoprecipitation and cross-blocking studies with anti-CD22 monoclonal antibodies recognizing established CD22 epitopes were performed to confirm that LL2 reacts with CD22 and to determine which epitope LL2 recognizes. Binding of¹³¹I-LL2 to Raji cells is inhibited over 90% by prior incubation of the target cells with unlabelled RFB4, indicating that LL2 belongs to the same epitope group as RFB4, i.e., epitope B.This work was supported in part by USPHS grant CA39841 from the NIH     

Expression and regulation of α1β1 integrin in Schwann cells

The interaction of cells with the extracellular matrix plays a critical role in morphogenesis and cell differentiation. To define how Schwann cells might interact with the extracellular matrix, we chose to study the expression of the laminin/collagen receptor α1β1 integrin during nerve development in the rat from embryonic day 14 to maturity. We found that this integrin is expressed predominantly on mature non-myelin-forming cells and only at very low levels on myelin-forming cells. Significant levels of this integrin were not detected on Schwann cell precursors or embryonic Schwann cells in vivo. Experiments using transected and crushed sciatic nerve showed that α1β1 integrin expression is regulated at least in part by axonal contact. Furthermore, Schwann cell culture experiments showed that α1β1 integrin levels are strongly upregulated by transforming growth factor-βs and phorbol esters. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 914–928, 1997     

Trans events associated with crossovers are revealed in the absence of mismatch repair genes in Saccharomyces cerevisiae

Genetic analysis of recombination in Saccharomyces cerevisiae has revealed products with structures not predicted by the double-strand break repair model of meiotic recombination. A particular type of recombinant containing trans heteroduplex DNA has been observed at two loci. Trans events were originally identified only in tetrads in which the non-Mendelian segregations were not associated with a crossover. Because of this, these events were proposed to have arisen from the unwinding of double Holliday junctions. Previous studies used palindromes, refractory to mismatch repair, as genetic markers whereas we have used a complementary approach of deleting mismatch repair proteins to identify heteroduplex DNA. We found that the markers occurred in trans and were associated with crossovers. In both mlh1Delta and msh2Delta strains, the frequency of trans events associated with a crossover exceeded that predicted from the random association of crossovers with noncrossover trans events. We propose two different models to account for trans events associated with crossovers and discuss the relevance to wild-type DSB repair.     

Nutritional Requirements of Arizona, Citrobacter, and Providencia

The nutritional requirements of Arizona, Citrobacter, and Providencia are compared, and chemically defined media in which these organisms may be grown are reported.