Eric Alani

A pathway for generation and processing of double-strand breaks during meiotic recombination in S. cerevisiae

We have identified and analyzed a meiotic reciprocal recombination hot spot in S. cerevisiae. We find that double-strand breaks occur at two specific sites associated with the hot spot and that occurrence of these breaks depends upon meiotic recombination functions RAD50 and SPO11. Furthermore, these breaks occur in a processed form in wild-type cells and in a discrete, unprocessed form in certain nonnull rad50 mutants, rad50S, which block meiotic prophase at an intermediate stage. The breaks observed in wild-type cells are similar to those identified independently at another recombination hot spot, ARG4. We show here that the breaks at ARG4 also occur in discrete form in rad50S mutants. Occurrence of breaks in rad50S mutants is also dependent upon SPO11 function. These observations provide additional evidence that double-strand breaks are a prominent feature of meiotic recombination in yeast. More importantly, these observations begin to define a pathway for the physical changes in DNA that lead to recombination and to define the roles of meiotic recombination functions in that pathway.

Analysis of wild-type and rad50 mutants of yeast suggests an intimate relationship between meiotic chromosome synapsis and recombination.

The RAD50 gene of S. cerevisiae is required during meiosis for both recombination and chromosome synapsis and is also required for repair of double strand breaks during vegetative growth. We present below the isolation and analysis of several types of rad50 mutants. We show that null mutations block both meiotic recombination and formation of synaptonemal complex (SC) at early stages, while nonnull mutations block both processes at intermediate stages. These observations suggest that recombination and SC formation involve a series of intimately related events. Furthermore, all rad50 mutants block formation of tripartite SC structure but permit other aspects of SC development, i.e., formation of axial cores. In light of this and other observations, the meiotic and mitotic defects of rad50 mutants can be accounted for economically by the proposal that meiotic recombination, meiotic chromosome pairing, and vegetative DNA repair all use a common chromosomal homology search that involves RAD50 function.

Distinctly regulated tandem upstream activation sites mediate catabolite repression of the CYC1 gene of S. cerevisiae

The upstream activation site (UAS) of the yeast CYC1 gene is shown to contain two homologous subsites, UAS1 and UAS2. Each site, when placed upstream of the transcriptional initiation region of the yeast LEU2 gene, activates LEU2 transcription which is regulated by catabolite repression. UAS1 is responsible for most of the transcription under glucose repressed conditions, while UAS1 and UAS2 contribute equally to lactate derepressed transcription. A single point mutation in UAS2 increases its activity in glucose 10- to 20-fold. Several experiments indicate that UAS1 and UAS2 are regulated distinctly at the molecular level. First, UAS1 but not UAS2 is fully depressed in glucose by increasing the levels of intracellular heme. Second, trans-acting regulatory mutations, hap1-1 and hap2-1, selectively abolish the activity of UAS1 or UAS2. HAP1 appears to encode a protein that mediates catabolite repression of UAS1 by responding to intracellular heme levels.

The Saccharomyces cerevisiae Msh2 and Msh6 proteins form a complex that specifically binds to duplex oligonucleotides containing mismatched DNA base pairs.

The yeast Saccharomyces cerevisiae encodes six proteins, Msh1p to Msh6p, that show strong amino acid sequence similarity to MutS, a central component of the bacterial mutHLS mismatch repair system. Recent studies with humans and S. cerevisiae suggest that in eukaryotes, specific MutS homolog complexes that display unique DNA mismatch specificities exist. In this study, the S. cerevisiae 109-kDa Msh2 and 140-kDa Msh6 proteins were cooverexpressed in S. cerevisiae and shown to interact in an immunoprecipitation assay and by conventional chromatography. Deletion analysis of MSH2 indicated that the carboxy-terminal 114 amino acids of Msh2p are important for Msh6p interaction. Purified Msh2p-Msh6p selectively bound to duplex oligonucleotide substrates containing a G/T mismatch and a +1 insertion mismatch but did not show specific binding to +2 and +4 insertion mismatches. The mismatch binding specificity of the Msh2p-Msh6p complex, as measured by on-rate and off-rate binding studies, was abolished by ATP. Interestingly, palindromic substrates that are poorly repaired in vivo were specifically recognized by Msh2p-Msh6p; however, the binding of Msh2p-Msh6p to these substrates was not modulated by ATP. Taken together, these studies suggest that the repair of a base pair mismatch by the Msh2p-Msh6p complex is dependent on the ability of the Msh2p-Msh6p-DNA mismatch complex to use ATP hydrolysis to activate downstream events in mismatch repair.

DNA bending and unbending by MutS govern mismatch recognition and specificity

DNA mismatch repair is central to the maintenance of genomic stability. It is initiated by the recognition of base–base mismatches and insertion/deletion loops by the family of MutS proteins. Subsequently, ATP induces a unique conformational change in the MutS–mismatch complex but not in the MutS–homoduplex complex that sets off the cascade of events that leads to repair. To gain insight into the mechanism by which MutS discriminates between mismatch and homoduplex DNA, we have examined the conformations of specific and nonspecific MutS–DNA complexes by using atomic force microscopy. Interestingly, MutS–DNA complexes exhibit a single population of conformations, in which the DNA is bent at homoduplex sites, but two populations of conformations, bent and unbent, at mismatch sites. These results suggest that the specific recognition complex is one in which the DNA is unbent. Combining our results with existing biochemical and crystallographic data leads us to propose that MutS: (i) binds to DNA nonspecifically and bends it in search of a mismatch; (ii) on specific recognition of a mismatch, undergoes a conformational change to an initial recognition complex in which the DNA is kinked, with interactions similar to those in the published crystal structures; and (iii) finally undergoes a further conformational change to the ultimate recognition complex in which the DNA is unbent. Our results provide a structural explanation for the long-standing question of how MutS achieves mismatch repair specificity.

Visualizing one-dimensional diffusion of eukaryotic DNA repair factors along a chromatin lattice

DNA-binding proteins survey genomes for targets using facilitated diffusion, which typically includes a one-dimensional (1D) scanning component for sampling local regions. Eukaryotic proteins must accomplish this task while navigating through chromatin. Yet it is unknown whether nucleosomes disrupt 1D scanning or eukaryotic DNA-binding factors can circumnavigate nucleosomes without falling off DNA. Here we use single-molecule microscopy in conjunction with nanofabricated curtains of DNA to show that the postreplicative mismatch repair protein complex Mlh1–Pms1 diffuses in 1D along DNA via a hopping/stepping mechanism and readily bypasses nucleosomes. This is the first experimental demonstration that a passively diffusing protein can traverse stationary obstacles. In contrast, Msh2–Msh6, a mismatch repair protein complex that slides while maintaining continuous contact with DNA, experiences a boundary upon encountering nucleosomes. These differences reveal important mechanistic constraints affecting intranuclear trafficking of DNA-binding proteins.

Mismatch repair proteins: key regulators of genetic recombination

Mismatch repair (MMR) systems are central to maintaining genome stability in prokaryotes and eukaryotes. MMR proteins play a fundamental role in avoiding mutations, primarily by removing misincorporation errors that occur during DNA replication. MMR proteins also act during genetic recombination in steps that include repairing mismatches in heteroduplex DNA, modulating meiotic crossover control, removing 3′ non-homologous tails during double-strand break repair, and preventing recombination between divergent sequences. In this review we will, first, discuss roles for MMR proteins in repairing mismatches that occur during recombination, particularly during meiosis. We will also explore how studying this process has helped to refine models of double-strand break repair, and particularly to our understanding of gene conversion gradients. Second, we will examine the role of MMR proteins in repressing homeologous recombination, i.e. recombination between divergent sequences. We will also compare the requirements for MMR proteins in preventing homeologous recombination to the requirements for these proteins in mismatch repair.

Characterization of DNA-binding and strand-exchange stimulation properties of y-RPA, a yeast single-strand-DNA-binding protein

Single-stranded DNA binding proteins (SSBs) have been isolated from many organisms, including Escherichia coli, Saccharomyces cerevisiae and humans. Characterization of these proteins suggests they are required for DNA replication and are active in homologous recombination. As an initial step towards understanding the role of the eukaryotic SSBs in DNA replication and recombination, we examined the DNA binding and strand exchange stimulation properties of the S. cerevisiae single-strand binding protein y-RPA (yeast replication protein A). y-RPA was found to bind to single-stranded DNA (ssDNA) as a 115,000 M(r) heterotrimer containing 70,000, 36,000 and 14,000 M(r) subunits. It saturated ssDNA at a stoichiometry of one heterotrimer per 90 to 100 nucleotides and binding occurred with high affinity (K omega greater than 10(9) M-1) and co-operativity (omega = 10,000 to 100,000). Electron microscopic analysis revealed that y-RPA binding was highly co-operative and that the ssDNA present in y-RPA-ssDNA complexes was compacted fourfold, arranged into nucleosome-like structures, and was free of secondary structure. y-RPA was also tested for its ability to stimulate the yeast Sepl and E. coli RecA strand-exchange proteins. In an assay that measures the pairing of circular ssDNA with homologous linear duplex DNA, y-RPA stimulated the strand-exchange activity of Sepl approximately threefold and the activity of RecA protein to the same extent as did E. coli SSB. Maximal stimulation of Sepl occurred at a stoichiometry of one y-RPA heterotrimer per 95 nucleotides of ssDNA. y-RPA stimulated RecA and Sepl mediated strand exchange reactions in a manner similar to that observed for the stimulation of RecA by E. coli SSB; in both of these reactions, y-RPA inhibited the aggregation of ssDNA and promoted the co-aggregation of single-stranded and double-stranded linear DNA. These results demonstrate that the E. coli and yeast SSBs display similar DNA-binding properties and support a model in which y-RPA functions as an E. coli SSB-like protein in yeast.

Genetic and Biochemical Analysis of Msh2p-Msh6p: Role of ATP Hydrolysis and Msh2p-Msh6p Subunit Interactions in Mismatch Base Pair Recognition

Recent studies have shown that Saccharomyces cerevisiae Msh2p and Msh6p form a complex that specifically binds to DNA containing base pair mismatches. In this study, we performed a genetic and biochemical analysis of the Msh2p-Msh6p complex by introducing point mutations in the ATP binding and putative helix-turn-helix domains of MSH2. The effects of these mutations were analyzed genetically by measuring mutation frequency and biochemically by measuring the stability, mismatch binding activity, and ATPase activity of msh2p (mutant msh2p)-Msh6p complexes. A mutation in the ATP binding domain of MSH2 did not affect the mismatch binding specificity of the msh2p-Msh6p complex; however, this mutation conferred a dominant negative phenotype when the mutant gene was overexpressed in a wild-type strain, and the mutant protein displayed biochemical defects consistent with defects in mismatch repair downstream of mismatch recognition. Helix-turn-helix domain mutant proteins displayed two different properties. One class of mutant proteins was defective in forming complexes with Msh6p and also failed to recognize base pair mismatches. A second class of mutant proteins displayed properties similar to those observed for the ATP binding domain mutant protein. Taken together, these data suggested that the proposed helix-turn-helix domain of Msh2p was unlikely to be involved in mismatch recognition. We propose that the MSH2 helix-turn-helix domain mediates changes in Msh2p-Msh6p interactions that are induced by ATP hydrolysis; the net result of these changes is a modulation of mismatch recognition.

Roles for Mismatch Repair Factors in Regulating Genetic Recombination

Mismatch repair (MMR) systems are evolutionarily conserved and play a primary role in mutation avoidance by removing base-base and small insertion-deletion mismatches that arise during DNA replication (31). In addition, MMR factors are required for the repair of mismatches in heteroduplex DNA (hDNA) that form as a result of sequence heterologies between recombining sequences (6, 41, 43). MMR also acts to inhibit recombination between moderately divergent (homeologous) sequences (11, 42). The roles of MMR during recombination are believed to reflect the interaction of MMR factors with mismatches that arise in hDNA or possibly with other structures such as Holliday junctions (2, 33). The full range of effects that MMR can exert on mitotic and meiotic recombination have been discussed elsewhere (11) and will only be summarized briefly here. The purpose of this review is to highlight recent results that have furthered our understanding of interactions between MMR factors and mitotic recombination intermediates.