Increase in Ty1 cDNA recombination in yeast sir4 mutant strains at high temperature

Transposition of the Ty1 element of the yeast Saccharomyces cerevisiae is temperature sensitive. We have identified a null allele of the silent information regulator gene SIR4 as a host mutant that allows for transposition at high temperature. We show that the apparent increase in transposition activity in sir4 mutant strains at high temperature is dependent on the RAD52 gene and is thus likely resulting from an increase in Ty1 cDNA recombination, rather than in IN-mediated integration. General cellular recombination is not increased at high temperature, suggesting that the increase in recombination at high temperature in sir4 mutants is specific for Ty1 cDNA. Additionally, this high-temperature Ty1 recombination was found to be dependent on functional Sir2p and Sir3p. We speculate that the increase in recombination seen in sir4 mutants at high temperature may be due to changes in chromatin structure or Ty1 interactions with chromosomal structures resulting in higher recombination rates.     

Ty3 requires yeast La homologous protein for wild-type frequencies of transposition

The Saccharomyces cerevisiae retrovirus-like element Ty3 inserts specifically into the initiation sites of genes transcribed by RNA polymerase III (pol III). A strain with a disruption of LHP1, which encodes the homologue of autoantigen La protein, was recovered in a screen for mutants defective in Ty3 transposition. Transposition into a target composed of divergent tRNA genes was decreased eightfold. In lhp1 mutants, Ty3 polyproteins were produced at wild-type levels, assembled into virus-like particles (VLPs) and processed efficiently. The amount of cDNA associated with these particles was about half the amount in a wild-type control at early times, but approached the wild-type level after 48 h of induction. Ty3 integration was examined at two genomic tRNA gene families and two plasmid-borne tRNA promoters. Integration was significantly decreased at one of the tRNA gene families, but was only slightly decreased at the second tRNA gene family. These findings suggest that Lhp1p contributes to Ty3 cDNA synthesis, but might also act at a target-specific step, such as integration.     

Nucleotide excision repair/TFIIH helicases RAD3 and SSL2 inhibit short-sequence recombination and Ty1 retrotransposition by similar mechanisms

Eukaryotic genomes contain potentially unstable sequences whose rearrangement threatens genome structure and function. Here we show that certain mutant alleles of the nucleotide excision repair (NER)/TFIIH helicase genes RAD3 and SSL2 (RAD25) confer synthetic lethality and destabilize the Saccharomyces cerevisiae genome by increasing both short-sequence recombination and Ty1 retrotransposition. The rad3-G595R and ssl2-rtt mutations do not markedly alter Ty1 RNA or protein levels or target site specificity. However, these mutations cause an increase in the physical stability of broken DNA molecules and unincorporated Ty1 cDNA, which leads to higher levels of short-sequence recombination and Ty1 retrotransposition. Our results link components of the core NER/TFIIH complex with genome stability, homologous recombination, and host defense against Ty1 retrotransposition via a mechanism that involves DNA degradation.     

The Rad27 (Fen-1) nuclease inhibits Ty1 mobility in Saccharomyces cerevisiae

Although most Ty1 elements in Saccharomyces cerevisiae are competent for retrotransposition, host defense genes can inhibit different steps of the Ty1 life cycle. Here, we demonstrate that Rad27, a structure-specific nuclease that plays an important role in DNA replication and genome stability, inhibits Ty1 at a post-translational level. We have examined the effects of various rad27 mutations on Ty1 element retrotransposition and cDNA recombination, termed Ty1 mobility. The point mutations rad27-G67S, rad27-G240D, and rad27-E158D that cause defects in certain enzymatic activities in vitro result in variable increases in Ty1 mobility, ranging from 4- to 22-fold. The C-terminal frameshift mutation rad27-324 confers the maximum increase in Ty1 mobility (198-fold), unincorporated cDNA, and insertion at preferred target sites. The null mutation differs from the other rad27 alleles by increasing the frequency of multimeric Ty1 insertions and cDNA recombination with a genomic element. The rad27 mutants do not markedly alter the levels of Ty1 RNA or the TyA1-gag protein. However, there is an increase in the stability of unincorporated Ty1 cDNA in rad27-324 and the null mutant. Our results suggest that Rad27 inhibits Ty1 mobility by destabilizing unincorporated Ty1 cDNA and preventing the formation of Ty1 multimers.     

Transfer RNA Genes Are Genomic Targets for De Novo Transposition of the Yeast Retrotransposon Ty3

Insertions of the yeast element Ty3 resulting from induced retrotransposition were characterized in order to identify the genomic targets of transposition. The DNA sequences of the junctions between Ty3 and flanking DNA were determined for two insertions of an unmarked element. Each insertion was at position -17 from the 5' end of a tRNA-coding sequence. Ninety-one independent insertions of a marked Ty3 element were studied by Southern blot analysis. Pairs of independent insertions into seven genomic loci accounted for 14 of these insertions. The DNA sequence flanking the insertion site was determined for at least one member of each pair of integrated elements. In each case, insertion was at position -16 or -17 relative to the 5' end of one of seven different tRNA genes. This proportion of genomic loci used twice for Ty3 integration is consistent with that predicted by a Poisson distribution for a number of genomic targets roughly equivalent to the estimated number of yeast tRNA genes. In addition, insertions upstream of the same tRNA gene in one case were at different positions, but in all cases were in the same orientation. Thus, genomic insertions of Ty3 in a particular orientation are apparently specified by the target, while the actual position of the insertion relative to the tRNA-coding sequence can vary slightly.     

Ty5 gag mutations increase retrotransposition and suggest a role for hydrogen bonding in the function of the nucleocapsid zinc finger

The Ty5 retrotransposon of Saccharomyces paradoxus transposes in Saccharomyces cerevisiae at frequencies 1,000-fold lower than do the native Ty1 elements. The low transposition activity of Ty5 could be due to differences in cellular environments between these yeast species or to naturally occurring mutations in Ty5. By screening of a Ty5 mutant library, two single mutants (D252N and Y68C) were each found to increase transposition approximately sixfold. When combined, transposition increased 36-fold, implying that the two mutations act independently. Neither mutation affected Ty5 protein synthesis, processing, cDNA recombination, or target site choice. However, cDNA levels in both single mutants and the double mutant were significantly higher than in the wild type. The D252N mutation resides in the zinc finger of nucleocapsid and increases the potential for hydrogen bonding with nucleic acids. We generated other mutations that increase the hydrogen bonding potential (i.e., D252R and D252K) and found that they similarly increased transposition. This suggests that hydrogen bonding within the zinc finger motif is important for cDNA production and builds upon previous studies implicating basic amino acids flanking the zinc finger as important for zinc finger function. Although NCp zinc fingers differ from the zinc finger motifs of cellular enzymes, the requirement for efficient hydrogen bonding is likely universal.