A chromosome containing HOT1 preferentially receives information during mitotic interchromosomal gene conversion.

The recombination-stimulating sequence, HOT1, is derived from yeast ribosomal DNA and corresponds to the sequences required for promotion of transcription by RNA polymerase I. The effect of HOT1 on mitotic interchromosomal gene conversion was examined in diploid strains carrying his4 heteroalleles. When HOT1 is inserted adjacent to both copies of HIS4, the frequency of His+ recombinants is increased approximately 10-fold. When HOT1 is present on only one of the two homologs, recombination is enhanced and the his4 gene on the HOT1-containing chromosome is preferentially converted. In both pairs of his4 heteroalleles examined, HOT1 stimulates conversion of the his4 mutation which is further from the site of HOT1 insertion more than it stimulates conversion of the HOT1-proximal his4 allele. Compared to recombinants isolated from control strains that lack HOT1, HOT1-promoted His+ recombinants are more often homozygous for sequences distal to HIS4. The preferential conversion of sequences on the HOT1-containing chromosome is consistent with the double-strand-gap repair model of recombination and suggests that HOT1-promoted gene conversion initiates with a double-strand break in HOT1-adjacent sequences.

Gene conversion tracts stimulated by HOT1-promoted transcription are long and continuous.

The recombination-stimulating sequence, HOT1, corresponds to the promoter of transcription by yeast RNA polymerase I. The effect of HOT1 on mitotic interchromosomal recombination was examined in diploid strains carrying a heterozygous URA3 gene on chromosome III. The frequency of Ura- recombinants was increased 20-fold when HOT1 was inserted into the chromosome III copy marked with URA3, at a location 48 kbp centromere-proximal to URA3. Ura- recombinants were increased only 2-fold when HOT1 and URA3 were on opposite homologues. These results suggest that most HOT1-promoted Ura- recombinants result from gene conversion and that sequences on the HOT1-containing chromosome are preferentially converted. Characterization of Ura- recombinants isolated from strains carrying multiple markers on chromosome III indicates that HOT1-promoted gene conversion tracts are unusually long (often greater than 75 kbp) and almost always continuous. Furthermore, conversion tracts frequently extend to both sides of HOT1. We suggest that HOT1 promotes the formation of a double-strand break which is often followed by exonucleolytic digestion. Repair of the broken chromosome could then result from gap repair or from replicative repair primed only by the centromere-containing chromosomal fragment.

Molecular cloning and genetic mapping of the DNA topoisomerase II gene of Saccharomyces cerevisiae.

The structural gene for DNA topoisomerase II from the yeast Saccharomyces cerevisiae has been cloned. The clones were selected from a YEp13 plasmid bank of yeast DNA by complementing a temperature-sensitive mutation (top2-1) in the topoisomerase II gene, TOP2. Chromosomal integrants of the clone were derived by homologous recombination in strains lacking the 2 mu circle plasmid. Genetic analysis of these integrants indicates that we have cloned the TOP2 gene and not an extragenic suppressor. A YEp13-TOP2 hybrid plasmid integrant was used to localize the TOP2 gene to the left arm of chromosome XIV by the 2 mu circle-directed marker loss method. Results from standard meiotic mapping experiments indicate that TOP2 is about 16 centi-Morgans to the centromere proximal side of MET4. Northern blot analysis of TOP2 RNA isolated from a wild-type strain and from an rna2 mutant shows the RNA to be 4.5 kb long in both cases, thus indicating that the TOP2 gene has no large introns.

Recombination-stimulating sequences in yeast ribosomal DNA correspond to sequences regulating transcription by RNA polymerase I.

A DNA sequence (HOT1) from the repeated ribosomal RNA gene cluster of Saccharomyces cerevisiae can stimulate genetic exchange when inserted at novel locations in the yeast genome. Localization of the sequences required for HOT1 activity demonstrates that two noncontiguous fragments of DNA are required for the stimulation of recombination. One of these fragments contains the transcription initiation site for the major 35S ribosomal RNA precursor. The other contains an enhancer of RNA polymerase I transcription. We suggest that transcription by RNA polymerase I initiating in the inserted rDNA and proceeding through the adjacent sequences is responsible for the stimulation of exchange. Consistent with this interpretation, insertion of the putative termination site for RNA polymerase I transcription between HOT1 and the adjacent recombining DNA abolishes the recombination stimulation. Transcription through both copies of the homologous recombining sequences appears to be necessary for enhanced exchange.

Meiosis-specific RNA splicing in yeast.

Previous studies have suggested that the differentiated state of meiosis in yeast is regulated primarily at the transcriptional level. This study reports a case of posttranscriptional regulation of a gene whose product is essential for meiosis. The MER2 gene is transcribed in mitosis as well as meiosis; however, the transcript is spliced efficiently to generate a functional gene product only in meiosis. Meiotic levels of splicing depend on the MER1 gene product, which is also essential for meiosis and which is produced only in meiotic cells. Therefore, at least one of the functions of the MER1 protein is to mediate splicing of the MER2 transcript. Genetic data suggest that the MER1 gene may also be responsible for splicing the transcript of at least one other gene.

DNA topoisomerase activity is required as a swivel for DNA replication and for ribosomal RNA transcription.

Yeast strains with mutations in the genes for DNA topoisomerases I and II have been identified previously. The topoisomerase II mutants (top2) are conditional-lethal, temperature-sensitive mutants defective in the termination of DNA replication and the segregation of daughter chromosomes. The topoisomerase I mutants (top1), including strains with null mutations, are viable and exhibit no obvious growth defects, demonstrating that DNA topoisomerase I is not essential for viability in yeast. In contrast to the single mutants, top1 top2 double mutants grow poorly at the permissive temperature and stop DNA and ribosomal RNA synthesis at the restrictive temperature. Transfer RNA synthesis remains relatively normal. The rate of polyA+ RNA synthesis is down about 3-fold in the double mutant at the non-permissive temperature but the synthesis of three specific RNA polymerase II transcripts is unaffected. The results suggest that DNA replication and at least ribosomal RNA synthesis require an active topoisomerase, presumably to act as a swivel to relieve torsional stress, and that either topoisomerase can perform the required function (except for termination of DNA replication where topoisomerase II is required).

Involvement of host DNA gyrase in growth of bacteriophage T5.

Bacteriophage T5 did not grow at the nonpermissive temperature of 42 degrees C in Escherichia coli carrying a temperature-sensitive mutation in gyrB [gyrB(Ts)], but it did grow in gyrA(Ts) mutants at 42 degrees C. These findings indicate that the A subunit of host DNA gyrase is unnecessary, whereas the B subunit is necessary for growth of T5. The necessity for the B subunit was confirmed by a strong inhibition of T5 growth by novobiocin and coumermycin A1, which interfere specifically with the function of the B subunit of host DNA gyrase. However, T5 growth was also strongly inhibited by nalidixic acid, which interferes specifically with the function of the A subunit. This inhibition was due to the interaction of nalidixic acid with the A subunit and not just to its binding to DNA, because appropriate mutations in the gyrA gene of the host conferred nalidixic acid resistance to the host and resistance to T5 growth in such a host. The inhibition by nalidixic acid was also not due to a cell poison formed between nalidixic acid and the A subunit (K. N. Kreuzer and N. R. Cozzarelli, J. Bacteriol. 140:424-435, 1979) because nalidixic acid inhibited growth of T5 in a gyrA(Ts) mutant (KNK453) at 42 degrees C. We suggest that T5 grows in KNK453 at 42 degrees C because its gyrA(Ts) mutation is leaky for T5. Inhibition of T5 growth due to inactivation of host DNA gyrase was caused mainly by inhibition of T5 DNA replication. In addition, however, late T5 genes were barely expressed when host DNA gyrase was inactivated.

Need for DNA topoisomerase activity as a swivel for DNA replication for transcription of ribosomal RNA.

Yeast strains with mutations in the genes for DNA topoisomerases I and II have been identified previously in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. The topoisomerase II mutants (top2) are conditional-lethal temperature-sensitive (ts) mutants. They are defective in the termination of DNA replication and the segregation of daughter chromosomes, but otherwise appear to replicate and transcribe DNA normally. Topoisomerase I mutants (top1), including strains with null mutations are viable and exhibit no obvious growth defects, demonstrating that DNA topoisomerase I is not essential for viability in yeast. In contrast to the single mutants, top1 top2 ts double mutants from both Schizosaccharomyces pombe and Saccharomyces cerevisiae grow poorly at the permissive temperature and stop growth rapidly at the non-permissive temperature. Here we report that DNA and ribosomal RNA synthesis are drastically inhibited in an S. cerevisiae top1 top2 ts double mutant at the restrictive temperature, but that the rate of poly(A)+ RNA synthesis is reduced only about threefold and transfer DNA synthesis remains relatively normal. The results suggest that DNA replication and at least ribosomal RNA synthesis require an active topoisomerase, presumably to act as a swivel to relieve torsional stress, and that either topoisomerase can perform the required function (except in termination of DNA replication where topoisomerase II is required).