Biomedical subjects

N Kleckner

Publications and source records attributed to N Kleckner.

At least 37 records · Page 2Linked to original sources

PubMed1995

E. coli SeqA protein binds oriC in two different methyl-modulated reactions appropriate to its roles in DNA replication initiation and origin sequestration.

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.

Bacterial Outer Membrane Proteins↗

PubMed1995

Identification and characterization of a pre-cleavage synaptic complex that is an early intermediate in Tn10 transposition.

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.

Bacteriophage mu↗

PubMed1995

Structural domains of IS10 transposase and reconstitution of transposition activity from proteolytic fragments lacking an interdomain linker.

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.

Amino Acid Sequence↗

PubMed1995

Crossover and noncrossover recombination during meiosis: timing and pathway relationships.

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.

Crossing Over, Genetic↗

PubMed1995

The two single-strand cleavages at each end of Tn10 occur in a specific order during transposition.

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.

DNA Replication↗

PubMed1995

Negative and positive regulation of Tn10/IS10-promoted recombination by IHF: two distinguishable processes inhibit transposition off of multicopy plasmid replicons and activate chromosomal events that favor evolution of new transposons.

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.

Bacterial Proteins↗

PubMed1995

NDT80, a meiosis-specific gene required for exit from pachytene in Saccharomyces cerevisiae.

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.

Amino Acid Sequence↗

PubMed1994

Tn 10 transposition in vivo: temporal separation of cleavages at the two transposon ends and roles of terminal basepairs subsequent to interaction of ends.

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.

DNA Mutational Analysis↗

PubMed1994

Chromosome pairing via multiple interstitial interactions before and during meiosis in yeast.

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.

Cell Nucleus↗

PubMed1994

SeqA: a negative modulator of replication initiation in E. coli.

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.

Amino Acid Sequence↗

PubMed1994

Tn10/IS10 transposase purification, activation, and in vitro reaction.

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.

Blotting, Southern↗

PubMed1994

Identification of joint molecules that form frequently between homologs but rarely between sister chromatids during yeast meiosis.

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.

Blotting, Southern↗

PubMed1994

Molecular cloning and characterization of the pgm gene encoding phosphoglucomutase of Escherichia coli.

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.

Amino Acid Sequence↗

PubMed1993

RAD50 protein of S.cerevisiae exhibits ATP-dependent DNA binding.

RAD50 function of Saccharomyces cerevisiae is required during vegetative growth for recombinational repair of DNA double strand breaks, and during meiosis for initiation of meiotic recombination and formation of synaptonemal complex. RAD50 encodes a 153 kDa polypeptide which includes an amino-terminal ATP binding domain essential for function and two long heptad repeat regions. We show below that RAD50 protein purified from yeast exhibits ATP-dependent binding to double stranded DNA. Physical properties of the purified protein are also described. Models for RAD50 function in vivo are discussed.

Adenosine Triphosphate↗

PubMed1993

Structural relationship of bacterial RecA proteins to recombination proteins from bacteriophage T4 and yeast.

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.

Adenosine Triphosphatases↗

PubMed1993

Expression of the Saccharomyces cerevisiae RAD50 gene during meiosis: steady-state transcript levels rise and fall while steady-state protein levels remain constant.

In Saccharomyces cerevisiae, the RAD50 gene is required for repair of X-ray and MMS-induced DNA damage during vegetative growth, and for synaptonemal complex formation and genetic recombination during meiosis. We show below that the RAD50 gene encodes major and minor transcripts of 4.2 and 4.6 kb in length which differ primarily at their 5' ends. Steady-state levels of both RAD50 transcripts increase coordinately during meiosis, reaching maximal levels midway through meiotic prophase, about 3 or 4 h after transfer of cells to sporulation medium. The 5' ends of the major RAD50 transcript in both meiotic and vegetative cells map to the same cluster of sites approximately 20 bp upstream of the amino-terminal ATG of the RAD50 coding sequence. We conclude that the increased RAD50 transcript level observed during meiosis does not reflect utilization of a new promoter. In contrast, steady-state levels of Rad50 protein do not increase during meiosis. Thus, changes in RAD50 transcript levels are not necessarily accompanied by commensurate changes in Rad50 protein levels. Possible explanations are considered.

Base Sequence↗

PubMed1993

Potential advantages of unstable interactions for pairing of chromosomes in meiotic, somatic, and premeiotic cells.

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.

Animals↗

PubMed1993

IS10 mRNA stability and steady state levels in Escherichia coli: indirect effects of translation and role of rne function.

Translation of the IS10 transposase gene is known to be very infrequent. We have identified mutations whose genetic properties suggest that they act directly to increase or decrease the intrinsic level of translation initiation. Also, we have analysed in detail the effects of these mutations on IS10 mRNA using one particular IS10 derivative. In this case, increases or decreases in translation are accompanied by increases or decreases in both the steady state level and the half-life of transposase mRNA; effects on steady state levels are much more dramatic than effects on message half-life. At wild-type levels of translation initiation, the rate-limiting step in physical decay of full length IS10 message for a particular IS10 derivative is shown to be rne-dependent endonucleolytic cleavage; 3' exonucleases appear to play a secondary role, degrading primary cleavage products. Analysis of interplay between translation mutations and rne function, together with the above observations, suggests that translation stabilizes messages in a general way against rne-dependent endonucleolytic cleavage, and that significant protection may be conferred by one or a few ribosomes. However, dramatic effects of translation on steady state message levels are still observed in an rne mutant and involve the 3' end of the transcript; we propose that these additional effects reflect translation-mediated stimulation of transcript release.

Bacterial Proteins↗