Tn10 transposition requires IS10 transposase and essential sequences at the two ends of the element. Mutations in terminal basepairs 6-13 confer particularly strong transposition defects. We describe here the identification of transposase mutations that suppress the transposition defects of such terminus mutations. These mutations are named "SEM" for suppression of ends mutations. All of the SEM mutations suppress more than a single terminus mutation and thus are not simple alterations of transposase/end recognition specificity. The mutations identified fall into two classes on the basis of genetic tests, location within the protein and nature of the amino acid substitution. Class I mutations, which are somewhat allele specific, appear to define a small structural and functional domain of transposase in which hydrophobic interactions are important at an intermediate stage of the transposition reaction, after an effective interaction between the ends but before transposon excision. Class II mutations, which are more general in their effects, occur at a single residue in a small noncritical amino-terminal proteolytic domain of transposase and exert their affects by altering a charge interaction; these mutations may affect act early in the reaction, before or during establishment of an effective interaction between the ends.

During meiosis in Saccharomyces cerevisiae, the first chemical step in homologous recombination is the occurrence of site-specific DNA double-strand breaks (DSBs). In wild-type cells, these breaks undergo resection of their 5' strand termini to yield molecules with 3' single-stranded tails. We have further characterized the breaks that accumulate in rad50S mutant stains defective in DSB resection. We find that these DSBs are tightly associated with protein via what appears to be a covalent linkage. When genomic DNA is prepared from meiotic rad50S cultures without protease treatment steps, the restriction fragments diagnostic of DSBs selectively partition to the organic-aqueous interphase in phenol extractions and band at lower than normal density in CsCl density gradients. Selective partitioning and decreased buoyant density are abolished if the DNA is treated with proteinase K prior to analysis. Similar results are obtained with sae2-1 mutant strains, which have phenotypes identical to rad50S mutants. The protein is bound specifically to the 5' strand termini of DSBs and is present at both 5' ends in at least a fraction of breaks. The stability of the complex to various protein denaturants and the strand specificity of the attachment are most consistent with a covalent linkage to DSB termini. We propose that the DSB-associated protein is the catalytic subunit of the meiotic recombination initiation nuclease and that it cleaves DNA via a covalent protein-DNA intermediate.

During meiosis, crossovers occur at a high level, but the level of noncrossover recombinants is even higher. The biological rationale for the existence of the latter events is not known. It has been suggested that a noncrossover-specific pathway exists specifically to mediate chromosome pairing. Using a physical assay that monitors both crossovers and noncrossovers in cultures of yeast undergoing synchronous meiosis, we find that both types of products appear at essentially the same time, after chromosomes are fully synapsed at pachytene. We have also analyzed a situation in which commitment to meiotic recombination and formation of the synaptonemal complex are coordinately suppressed (mer1 versus mer1 MER2++). We find that suppression is due primarily to restoration of meiosis-specific double-strand breaks, a characteristic of the major meiotic recombination pathway. Taken together, the observations presented suggest that there probably is no noncrossover-specific pathway and that restoration of intermediate events in a single pairing/recombination pathway promotes synaptonemal complex formation. The biological significant of noncrossover recombination remains to be determined, however.

The seqA gene negatively modulates replication initiation at the E. coli origin, oriC. seqA is also essential for sequestration, which acts at oriC and the dnaA promoter to ensure that replication initiation occurs exactly once per chromosome per cell cycle. Initiation is promoted by full methylation of GATC sites clustered in oriC; sequestration is specific to the hemimethylated forms generated by replication. SeqA protein purification and DNA binding are described. SeqA interacts with fully methylated oriC strongly and specifically. This reaction requires multiple molecules of SeqA and determinants throughout oriC, including segments involved in open complex formation. SeqA interacts more strongly with hemimethylated DNA; in this case, oriC and non-oriC sequences are bound similarly. Also, binding of hemimethylated oriC by membrane fractions is due to SeqA. Direct interaction of SeqA protein with the replication origin is likely to be involved in both replication initiation and sequestration.

The Tn10 transposition reaction has been reconstituted in vitro on short linear substrate fragments encoding transposon ends. This permits the direct detection of protein-DNA complexes formed during transposition by gel retardation analysis. We demonstrate that a stable synaptic complex containing transposase and a pair of transposon ends forms rapidly and efficiently, prior and prerequisite to the double-strand cleavages involved in transposon excision. These observations extend the general analogies between the Tn10 and Mu transposition reactions, and also reveal significant differences between the two cases. The speed and simplicity of synaptic complex formation in the Tn10/IS10 reaction is suitable for a modular insertion sequence. In contrast, the relative slowness and complexity of this process in the Mu is necessary to permit transposition immunity and control of transposition by Mu repressor protein, two features specifically important for a temperate bacteriophage. Further dissection of the reaction leads to a tentative working model for events preceding the first double-strand cleavage.

We describe the identification of a new meiosis-specific gene of Saccharomyces cerevisiae, NDT80. The ndt80 null and point mutants arrest at the pachytene stage of meiosis, with homologs connected by full-length synaptonemal complexes and spindle pole bodies duplicated but unseparated. Meiotic recombination in an ndt80 delta mutant is relatively normal, although commitment to heteroallelic recombination is elevated two- to threefold and crossing over is decreased twofold compared with those of the wild type. ndt80 arrest is not alleviated by mutations in early recombination genes, e.g., SPO11 or RAD50, and thus cannot be attributed to an intermediate block in prophase chromosome metabolism like that observed in several other mutants. The ndt80 mutant phenotype during meiosis most closely resembles that of a cdc28 mutant, which contains a thermolabile p34, the catalytic subunit of maturation-promoting factor. Cloning and molecular analysis reveal that the NDT80 gene maps on the right arm of chromosome VIII between EPT1 and a Phe-tRNA gene, encodes a 627-amino-acid protein which exhibits no significant homology to other known proteins, and is transcribed specifically during middle meiotic prophase. The NDT80 gene product could be a component of the cell cycle regulatory machinery involved in the transition out of pachytene, a participant in an unknown aspect of meiosis sensed by a pachytene checkpoint, or a SPO11- and RAD50-independent component of meiotic chromosomes that is the target of cell cycle signaling.

Tn10 is a composite transposon; inverted repeats of insertion sequence IS10 flank a tetracycline-resistance determinant. Previous work has identified several regulatory processes that modulate the interaction between Tn10 and its host. Among these, host-specified DNA adenine methylation, an IS10-encoded antisense RNA and preferential cis action of transposase are particularly important. We now find that the accessory host protein IHF and the sequences that encode the IHF-binding site in IS10 are also important regulators of the Tn10 transposition reaction in vivo and that these determinants are involved in two distinguishable regulatory processes. First, IHF and the IHF-binding site of IS10, together with other host components (e.g., HU), negatively regulate the normal intermolecular transposition process. Such negative regulation is prominent only for elements present on multicopy plasmid replicons. This multicopy plasmid-specific regulation involves effects both on the transposition reaction per se and on transposase gene expression. Second, specific interaction of IHF with its binding site stimulates transposon-promoted chromosome rearrangements but not transposition of a short Tn10-length chromosomal element. However, additional considerations predict that IHF action should favor chromosomal transposition for very long composite elements. On the basis of these and other observations we propose that, for chromosomal events, the major role of IHF is to promote the evolution of new IS10-based composite transposons.

The HIS4LEU2 meiotic recombination hot spot specifies two double-strand break (DSB) sites, I and II. Results presented demonstrate that DSBs at site I occur at many positions throughout a region of approximately 150 bp; we infer that breaks occur in a sequence non-specific fashion. Single-strand nicks at sites I and II are not detectable. Analysis of the effects of a 36 bp linker insertion at site I reveals the existence of communication along and between homologs prior to DSB formation. In cis, the insertion allele causes an increase in DSBs at site I but a decrease in DSBs at site II. In trans, two effects are observed. One effect likely reflects very early pre-DSB interhomolog interactions; the second is suggestive of a later, more intimate interaction in which sites I and II on the two homologs all compete for DSBs. The existence of interhomolog interactions in early meiotic prophase can explain how the sites of crossovers come to lie between the homolog axes at pachytene.

All of the DNA cleavage and strand transfer events required for transposition of insertion sequence IS10 are carried out by a 46-kDa IS10-encoded transposase protein. Limited proteolysis demonstrates that transposase has two principal structural domains, a 28-kDa N-terminal domain (N alpha beta; aa 1-246) and a 17-kDa C-terminal domain (C; aa 256-402). The two domains are connected by a 1-kDa proteolytic-sensitive linker region (aa 247-255). The N-terminal domain N alpha beta can be further subdivided into domains N alpha and N beta by a weaker protease-sensitive site located 6 kDa (53 aa) from the N terminus. The N beta and N alpha beta fragments are capable of nonspecific DNA binding as determined by Southwestern blot analysis. None of the fragments alone is capable of carrying out the first step of transposition, assembly of a synaptic complex containing a pair of transposon ends. Remarkably, complete transposition activity can be reconstituted by mixing fragment N alpha beta and fragment C, with or without the intervening linker region. We infer that the structural integrity of transposase during the transitions involved in the chemical steps of the transposition reaction is maintained independent of the linker, presumably by direct contacts between and among the principal domains. Reconstitution of activity in the absence of the linker region is puzzling, however, because mutations that block strand transfer or affect insertion specificity alter linker region residues. Additional reconstitution experiments demonstrate that the N alpha region is dispensable for formation of a synaptic complex but is required for complexes to undergo cleavage.

During Tn10 transposition, the element is excised from the donor site by double-strand cleavages at the two transposon ends. Double-strand cleavage is a central step in the nonreplicative transposition reaction of many transposons in both prokaryotes and eukaryotes. Evidence is presented to show that the Tn10 double-strand cut is made by an ordered, sequential cleavage of the two strands. The transferred strand is cut first, and then the nontransferred strand is cleaved. The single-strand nicked intermediate is seen to accumulate when Mn2+ is substituted for Mg2+ in the reaction or when certain mutant transposases are used. The fact that the transferred strand is cleaved before the non-transferred strand implies that the order of strand cleavages is not the determining factor that precludes a replicative mechanism of transposition.

During meiosis, branched DNA molecules containing information from both parental chromosomes occur in vivo at loci where meiosis-specific double-stranded breaks occur. We demonstrate here that these joint molecules are recombination intermediates: they contain single strands that have undergone exchange of information. Moreover, these joint molecules are resolved into both parental and recombinant duplexes when treated in vitro with Holliday junction-resolving endonucleases RuvC or T4 endo VII. Taken together with previous observations, these results strongly suggest that joint molecules are double Holliday junctions.

Fluorescence in situ hybridization analysis reveals that homologous chromosomes are paired in yeast cells about to enter meiosis. Pairing involves multiple interstitial interactions, one per approximately 65 kb. These observations exclude several classes of models for somatic/premeiotic pairing. The number of t = 0 pairing interactions is about the same as the number of subsequent meiotic recombination events. As cells enter meiosis, pairing disappears concomitant with DNA replication and then reappears, independent of synaptonemal complex. Mutant phenotypes suggest that formation of an individual meiotic pairing connection does not require a meiosis-specific double-stranded break (DSB). Mutants defective in recombination before or after DSBs exhibit pairing defects. These and other observations can be united by a model in which premeiotic pairing and early meiotic pairing occur by closely related paranemic DNA-DNA interactions between intact duplexes, with early meiotic interactions subsequently converted directly to plectonemic recombination intermediates via DSBs.

We have investigated DNA interactions between homologs and between sister chromatids during meiosis in S. cerevisiae. We have detected a DNA species containing information from both parental chromosomes at a specific hotspot for meiotic recombination and double strand breaks (DSBs). These joint molecules are a prominent feature of meiotic prophase. They appear to be a major intermediate stage in DSB-promoted recombination, because they occur with appropriate timing and require known recombination functions. Other possibilities cannot be completely dismissed, however. Most or all joint molecules contain two full-length nonrecombinant strands from each parental duplex and thus do not consist of single Holliday junctions. Joint molecules form between sister chromatids at approximately 10% the interhomolog level. Also, joint molecule formation is aberrant in a mutant defective in the HOP1 gene, which encodes a meiotic chromosome structure component. General models for discrimination between homologs and sisters during meiosis are discussed.

We report here the identification and characterization of pgm, a gene in Escherichia coli that encodes the enzyme phosphoglucomutase, specifically required for the catalysis of the interconversion of glucose 1-phosphate and glucose 6-phosphate. The predicted amino acid sequence of the pgm gene is highly conserved in E. coli, Acetobacter xylinum, Saccharomyces cerevisiae, rabbits, and humans. pgm deletion mutant strains are deficient in phosphoglucomutase activity.

In E. coli, replication initiates at a genetically unique origin, oriC. Rapidly growing cells contain multiple oriC copies. Initiation occurs synchronously, once and only once per cell cycle at all origins present. Secondary initiations are prevented by a sequestration process that acts uniquely on newly replicated origins, which are marked because they are hemimethylated at GATC sites. We report the identification of a gene required for sequestration and demonstrate that this gene, seqA, also serves as a negative modulator of the primary initiation process. All previously identified in vivo initiation factors play positive roles. Thus, precise control of replication initiation may involve a balance between positive and negative elements. We suggest that SeqA might be a cooperativity factor, acting to make the replication initiation process dependent upon cooperative interactions among components.

During Tn10 transposition, the transposon is fully excised from the donor site by double strand cleavages at the two ends of the element prior to integration at a new target site. Results presented here demonstrate that an interaction between the two transposon ends is required for double strand cleavage at either end. Furthermore, despite this essential interaction of ends, subsequent cleavages at the two ends can occur at observably distinct times prior to occurrence of strand transfer at either end. Moreover, the time between cleavages at the two ends is exaggerated by the presence of an appropriate mutation at one end of the element. Biological rationales for this constellation of mechanistic features are suggested. Additional results demonstrate that mutations at the three terminal basepairs of Tn10 confer defects subsequent to interaction of ends, in confirmation of inferences from genetic analysis. More specifically, mutations in bp 1-3 confer strong defects during conversion of the full excision intermediate to a complete strand transfer product; mutations in bp 1 and 2 also confer more subtle defects subsequent to interaction of ends but prior to full excision. Such defects might reflect roles for these basepairs in the chemical steps of transposition per se, the positioning of terminal residues for those chemical steps, and/or the coupling of cleavage(s) to subsequent conformational changes.

We describe a method for the purification of Tn10/IS10 transposase that relies on the aggregation of the protein after overexpression in Escherichia coli. Aggregated transposase was solubilized before the final purification step, a gel-filtration column, using a combination of salt and detergent. This procedure is the first reported for the preparation of concentrated and active transposase from any IS element. The yield is 11 mg of purified protein at a concentration of 1 mg/ml from 2.5 g of cells. The procedure can be scaled up with ease. We also describe a treatment that activates transposase in either a crude or purified state. This involves dilution into a solution of salt plus organic solvent. In transposition reactions using supercoiled substrate plasmid, the activity was directly proportional to the amount of transposase added over a wide range of transposase/DNA ratios (0.2-2.0 molecules/DNA substrate molecule). In this range 8 transposase molecules were added per transposition event. Maximum conversion of substrate to product (40%) was with 18 transposase molecules/transposition event. At higher levels of transposase with a constant amount of substrate, activity was reduced but could be restored by addition of nonspecific DNA. Both the specific activity of transposase and the type of products generated can be altered by changing in vitro assay conditions. The effects of salts, solvents, and pH value on the reaction are described.

Many different aspects of chromosome pairing, meiotic and/or somatic, can be explained conveniently if the interactions between homologous chromosomes are unstable. Initial pairing interactions should involve very unstable contacts. Such interactions could go a long way toward bringing each pair of homologous chromosomes into a joint domain, free of ectopic associations and random entanglements with other chromosomes, and in a topologically acceptable relationship to the domains of other chromosome pairs. More generally, colocalization into topologically acceptable domains could be a useful way of defining the existence of "order" at early stages in pairing; this definition requires that chromosomes have in some way recognized and interacted with one another but does not require that they necessarily be in close apposition and/or that they be aligned along their entire lengths. At later stages, interactions between pairing chromosomes could be unstable but still reversible, either intrinsically or due to an active cell-directed process. Transient homologous interactions could also contribute to maintaining colocalization between homologous chromosomes through DNA replication.

RecA protein is essential in eubacteria for homologous recombination and promotes the homologous pairing and strand exchange of DNA molecules in vitro. Recombination proteins with weak sequence similarity to bacterial RecA proteins have been identified in bacteriophage T4, yeast, and other higher organisms. Analysis of the primary sequence relationships of DMC1 from Saccharomyces cerevisiae and UvsX of T4 relative to the three-dimensional structure of RecA from Escherichia coli suggests that both proteins are structural homologs of bacterial RecA proteins. This analysis argues that proteins in this group are members of a single family that diverged from a common ancestor that existed prior to the divergence of prokaryotes and eukaryotes.