Raymond WE, Kleckner N. Expression of the Saccharomyces cerevisiae RAD50 gene during meiosis: steady-state transcript levels rise and fall while steady-state protein levels remain constant. Mol Gen Genet. 1993;238 (3) :390-400.Abstract
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.
Jain C, Kleckner N. IS10 mRNA stability and steady state levels in Escherichia coli: indirect effects of translation and role of rne function. Mol Microbiol. 1993;9 (2) :233-47.Abstract
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.
Jain C, Kleckner N. Preferential cis action of IS10 transposase depends upon its mode of synthesis. Mol Microbiol. 1993;9 (2) :249-60.Abstract
A number of bacterial DNA-binding proteins, including IS element transposases, act preferentially in cis. We show below that the degree of preferential cis action by IS10 transposase depends upon its mode of synthesis at steps subsequent to transcription initiation. Cis preference is increased several fold by mutations that decrease translation initiation, by the presence of IS10-specific antisense RNA and by plasmids that increase the level of cellular RNases. Conversely, cis preference is decreased by mutations that increase translation initiation; in some cases, cis preference is nearly abolished. Mutations that alter the rate of transcription initiation have no effect. In light of other observations, we suggest that cis preference is strongly dependent upon the rate at which transcripts are released from their templates and/or the half-life of the transposase message. These observations provide further evidence that inefficient translation plays multiple roles in the biology of IS10.
Raymond WE, Kleckner N. RAD50 protein of S.cerevisiae exhibits ATP-dependent DNA binding. Nucleic Acids Res. 1993;21 (16) :3851-6.Abstract
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.
Bishop DK, Park D, Xu L, Kleckner N. DMC1: a meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression. Cell. 1992;69 (3) :439-56.Abstract
DMC1 is a new meiosis-specific yeast gene. Dmc1 protein is structurally similar to bacterial RecA proteins. dmc1 mutants are defective in reciprocal recombination, accumulate double-strand break (DSB) recombination intermediates, fail to form normal synaptonemal complex (SC), and arrest late in meiotic prophase. dmc1 phenotypes are consistent with a functional relationship between Dmc1 and RecA, and thus eukaryotic and prokaryotic mechanisms for homology recognition and strand exchange may be related. dmc1 phenotypes provide further evidence that recombination and SC formation are interrelated processes and are consistent with a requirement for DNA-DNA interactions during SC formation. dmc1 mutations confer prophase arrest. Additional evidence suggests that arrest occurs at a meiosis-specific cell cycle "checkpoint" in response to a primary defect in prophase chromosome metabolism. DMC1 is homologous to yeast's RAD51 gene, supporting the view that mitotic DSB repair has been recruited for use in meiotic chromosome metabolism.
Benjamin HW, Kleckner N. Excision of Tn10 from the donor site during transposition occurs by flush double-strand cleavages at the transposon termini. Proc Natl Acad Sci U S A. 1992;89 (10) :4648-52.Abstract
Tn10 transposition is accomplished without extensive replication of the transposon sequences. Replicative cointegrate formation is precluded by efficient separation of transposon sequences from flanking donor DNA at an early stage in the transposition reaction. We report here that excision of Tn10 from its donor site occurs by a pair of flush double-strand breaks. Breaks occur at each end of the element precisely between the terminal base pair of the element and the first base pair of flanking DNA. This observation provides definitive evidence that cleavage of both strands of the element occurs under the direct control of Tn10 transposase protein. It is highly likely that transposase itself is directly responsible for these cleavages. The implications of this possibility are discussed.
Bender J, Kleckner N. IS10 transposase mutations that specifically alter target site recognition. EMBO J. 1992;11 (2) :741-50.Abstract
IS10 inserts preferentially into particular hotspots. We describe here mutations of IS10 transposase, called 'ATS' that confer Altered Target Specificity. These mutations yield a general relaxation in target specificity but do not affect other aspects of transposition. Thus, the preference for specific nucleotide sequences at the target site can be cleanly separated from other steps of the transposition reaction. Eleven ATS mutations identified in a genetic screen occur at only two codons in transposase, one in each of two regions of the protein previously implicated in target site interactions (Patch I and Patch II). Genetic analysis suggests that mutations at the two ATS codons affect the same specific function of transposase, thus raising the possibility that Patch I and Patch II interact. For wild-type IS10, insertion specificity is determined in part by a specific 6 bp consensus sequence and in part by the immediately adjacent sequence context of the target DNA. The ATS mutations do not qualitatively alter the hierarchy with which base pairs are recognized in the consensus sequence; instead, sites selected by ATS transposase exhibit a reduction in the degree to which certain base pairs are preferred over others. Models for the basis of this phenotype are discussed.
Mahillon J, Kleckner N. New IS10 transposition vectors based on a gram-positive replication origin. Gene. 1992;116 (1) :69-74.Abstract
We describe below a set of plasmid-based vehicles which can be used for delivery of IS10-derived transposons into Gram- bacteria. These vehicles replicate via a Gram+ plasmid origin that is inactive in Escherichia coli; they are easily maintained in Bacillus subtilis. Transposons are introduced by electroporation or transformation with the plasmid, and as in previous delivery systems, transpositions are selected with the appropriate antibiotic. This system should be particularly useful in situations where the standard delivery vehicles, based on bacteriophage lambda, are inappropriate. The system described incorporates a number of useful features: a variety of antibiotic markers (Er, Cm, Km or Tc), a polylinker containing restriction sites for rare-cutting endonucleases to facilitate physical mapping of chromosomal insertions, a mutant transposase that confers a relaxation in insertion specificity and positioning of the transposase-encoding gene outside of the transposing segment to ensure the stability of insertions once isolated.
Bender J, Kleckner N. Tn10 insertion specificity is strongly dependent upon sequences immediately adjacent to the target-site consensus sequence. Proc Natl Acad Sci U S A. 1992;89 (17) :7996-8000.Abstract
Transposon Tn10 inserts preferentially into particular "hotspots" that have been shown by sequence analysis to contain the symmetrical consensus sequence 5'-GCTNAGC-3'. This consensus is necessary but not sufficient to determine insertion specificity. We have mutagenized a known hotspot to identify other determinants for insertion into this site. This genetic dissection of the sequence context of a protein binding site shows that a second major determinant for Tn10 insertion specificity is contributed by the 6-9 base pairs that flank each end of the consensus sequence. Variations in these context base pairs can confer variations of at least 1000-fold in insertion frequency. There is no discernible consensus sequence for the context determinant, suggesting that sequence-specific protein-DNA contacts are not playing a major role. Taken together with previous work, the observations presented suggest a model for the interaction of transposase with the insertion site: symmetrically disposed subunits bind with specific contacts to the major groove of consensus-sequence base pairs, while flanking sequences influence the interaction through effects on DNA helix structure. We also show that the determinants important for insertion into a site are not important for transposition out of that site.
Haniford DB, Benjamin HW, Kleckner N. Kinetic and structural analysis of a cleaved donor intermediate and a strand transfer intermediate in Tn10 transposition. Cell. 1991;64 (1) :171-9.Abstract
Tn10 transposes by a nonreplicative "cut and paste" mechanism. We describe here two protein-DNA complexes that are reaction intermediates in the Tn10 transposition process: a cleaved donor complex whose DNA component consists of transposon sequences cleanly excised from flanking donor DNA, and a strand transfer complex whose DNA component contains transposon termini specifically joined to a target site. The kinetic behavior of the first species suggests that it is an early intermediate in the transposition reaction. These two Tn10 complexes are closely analogous to complexes identified in the pathway for replicative "cointegrate" formation by bacteriophage Mu and thus represent intermediates that may be common to both nonreplicative and replicative transposition. These and other results suggest that the Tn10 and Mu reactions are fundamentally very similar despite their very different biological outcomes. The critical difference between the two reactions is the fate of the DNA strand that is not joined to target DNA.
Kleckner N, Padmore R, Bishop DK. Meiotic chromosome metabolism: one view. Cold Spring Harb Symp Quant Biol. 1991;56 :729-43.
Kleckner N, Bender J, Gottesman S. Uses of transposons with emphasis on Tn10. Methods Enzymol. 1991;204 :139-80.
Bender J, Kuo J, Kleckner N. Genetic evidence against intramolecular rejoining of the donor DNA molecule following IS10 transposition. Genetics. 1991;128 (4) :687-94.Abstract
Tn10 and IS10 transpose by a nonreplicative mechanism in which the transposon is excised from the donor molecule and integrated into a target DNA site, leaving behind a break at the original donor site. The fate of this broken donor DNA molecule is not known. We describe here two experiments that address this issue. One experiment demonstrates that a polar IS10 element gives rise to polarity-relief revertants at less than 1% the frequency of transposition of the same element in the same culture. In a second experiment, transpositions of an IS10 element from one site in the bacterial genome to another are selected and the resulting isolates examined for alterations at the donor site; none of 1088 such isolates exhibited a detectable change at the donor locus. These results are compatible with two possible fates of the transposon donor molecule: degradation ("donor suicide"), or restoration of the original information at the donor site by a recombinational repair mechanism analogous to double-strand break repair. These results argue against the possibility that the donor molecule gap is simply resealed by intramolecular rejoining.
Roberts DE, Ascherman D, Kleckner N. IS10 promotes adjacent deletions at low frequency. Genetics. 1991;128 (1) :37-43.Abstract
Some transposable elements move by a replicative mechanism involving cointegrate formation. Intramolecular cointegration can generate a product called an "adjacent deletion" in which a contiguous chromosomal segment adjacent to the transposon is deleted while the element responsible remains intact. Insertion sequence IS10 is thought to transpose by a nonreplicative mechanism. In the simplest models, nonreplicative transposition cannot give rise to an adjacent deletion because an intrinsic feature of such transposition is excision of the IS element from the donor location. We report here that IS10 can generate adjacent deletions, but at a frequency which is approximately 1/30th the frequency of transposition for the same element. We suggest that these deletions might arise either by nonreplicative transposition events that involve two IS10 elements located on sister chromosomes or by aberrant nonreplicative events involving cleavage and ligation at only one end of the element.
Padmore R, Cao L, Kleckner N. Temporal comparison of recombination and synaptonemal complex formation during meiosis in S. cerevisiae. Cell. 1991;66 (6) :1239-56.Abstract
In synchronous cultures of S. cerevisiae undergoing meiosis, an early event in the meiotic recombination pathway, site-specific double strand breaks (DSBs), occurs early in prophase, in some instances well before tripartite synaptonemal complex (SC) begins to form. This observation, together with previous results, supports the view that events involving DSBs are required for SC formation. We discuss the possibility that the mitotic pathway for recombinational repair of DSBs served as the primordial mechanism for connecting homologous chromosomes during the evolution of meiosis. DSBs disappear during the period when tripartite SC structure is forming and elongating (zygotene); presumably, they are converted to another type of recombination intermediate. Neither DSBs nor mature recombinant molecules are present when SCs are full length (pachytene). Mature reciprocally recombinant molecules arise at the end of or just after pachytene. We suggest that the SC might coordinate recombinant maturation with other events of meiosis.
Alani E, Padmore R, Kleckner N. Analysis of wild-type and rad50 mutants of yeast suggests an intimate relationship between meiotic chromosome synapsis and recombination. Cell. 1990;61 (3) :419-36.Abstract
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.
Campbell JL, Kleckner N. E. coli oriC and the dnaA gene promoter are sequestered from dam methyltransferase following the passage of the chromosomal replication fork. Cell. 1990;62 (5) :967-79.Abstract
We have examined individual GATC sites throughout the E. coli genome for their kinetics of remethylation by dam methyltransferase following the passage of the chromosomal replication fork. We present evidence for three major conclusions: that oriC is a single function unit that is specifically sequestered from dam methyltransferase for a significant period of time and then released; that the dnaA promoter region is subject to sequestration analogous to that observed at oriC and thus that hemimethylation-dependent sequestration is a general phenomenon; and that each round of replication initiation triggers a transient, temporally coordinate block in both reinitiation at oriC and expression of the dnaA gene. These and other observations are all consistent with the notion that hemimethylation in these two regions acts coordinately to ensure that every origin undergoes initiation once and only once per cell cycle; other possible roles for sequestration at dnaA are also considered.
Cao L, Alani E, Kleckner N. A pathway for generation and processing of double-strand breaks during meiotic recombination in S. cerevisiae. Cell. 1990;61 (6) :1089-101.Abstract
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.
Kleckner N. Regulating tn10 and is10 transposition. Genetics. 1990;124 (3) :449-54.
Kleckner N. Regulation of transposition in bacteria. Annu Rev Cell Biol. 1990;6 :297-327.Abstract
Bacterial transposons are subject to a variety of regulatory processes that affect the quantity, quality, and timing of transposition events. Many of these processes seem specifically designed to provide features that favor the evolutionary success of the element. The most important conclusion reached from the identification and characterization of these regulatory mechanisms is that transposable elements are not mechanistic accidents of recent origin, but instead are highly evolved entities that have adapted to their ecological niche with a degree of sophistication comparable to that exhibited by plasmids and bacterial viruses.