Ty1, the most abundant retrotransposon in Saccharomyces cerevisiae, integrates preferentially upstream of genes transcribed by RNA polymerase III (Pol III). Targeting is likely due to interactions between the Ty1 integration complex and a feature of chromatin characteristic of sites of Pol III transcription. To better understand Ty1 targeting determinants, >150,000 Ty1 insertions were mapped onto the S. cerevisiae genome sequence. Logistic regression was used to assess relationships between patterns of Ty1 integration and various genomic features, including genome-wide data sets of histone modifications and transcription-factor binding sites. Nucleosomes were positively associated with Ty1 insertions, and fine-scale mapping of insertions upstream of genes transcribed by Pol III indicated that Ty1 preferentially integrates into nucleosome-bound DNA near the H2A/H2B interface. Outside of genes transcribed by Pol III, Ty1 avoids coding sequences, a pattern that is not due to selection, but rather reflects a preference for nucleosome-rich sites flanking genes. Ty1 insertion sites were also mapped in four mutant lines that affect Ty1 transposition frequency or integration specificity (rrm3Δ, hos2Δ, rtt109Δ, and rad6Δ). Patterns of integration were largely preserved in the mutants, although significantly more insertions into coding sequences were observed in the rad6Δ strain, suggesting a loosening of target specificity in this mutant that lacks an enzyme involved in ubiquitinating H2A. Overall, our data suggest that nucleosomes are necessary for Ty1 integration, and that a secondary factor, likely a histone modification or nucleosome-bound factor enriched at sites of Pol III transcription, determines preferred target sites.

The DNA helicase Rrm3 promotes replication fork progression through .1000 discrete genomic regions and represses the cDNA-mediated mobility of the Ty1 retrotransposon. We explored the connection between DNA replication and Ty1 retromobility by investigating the basis of increased retromobility in an rrm3 mutant. Even though Ty1 cDNA levels are increased in the absence of RRM3, neither the level nor target-site specificity of cDNA integration was altered. Instead, cDNA was incorporated into the genome by a Rad52-dependent mechanism that did not involve gene conversion of genomic Ty1 sequences. In rrm3 isolates, incorporated cDNA was often present in tandem arrays. Multimeric cDNA arrays probably arise during chromosomal break repair, since their appearance was strongly correlated with the formation of gross chromosomal rearrangements. Moreover, Ty1 multimers were invariantly located on rearranged chromosomes, when present. Overexpression of a cellular RNase H, which degrades RNA in an RNA:DNA hybrid, completely suppressed the increase in Ty1 multimer formation in an rrm3 mutant. We propose that RNA:DNA hybrid regions within nascent retrotransposition events block replication in an rrm3 mutant, leading to chromosome breaks within Ty1 sequences. Multiple extragenomic Ty1 cDNA molecules are then used as donors in recombinational repair of the break before it is healed.

The intra-S phase checkpoint protein complex Tof1/Csm3 of Saccharomyces cerevisiae antagonizes Rrm3 helicase to modulate replication fork arrest not only at the replication termini of rDNA but also at strong nonhistone protein binding sites throughout the genome. We investigated whether these checkpoint proteins acted either antagonistically or synergistically with Rrm3 in mediating other important functions such as maintenance of genome stability. High retromobility of a normally quiescent retrovirus-like transposable element Ty1 of S. cerevisiae is a form of genome instability, because the transposition events induce mutations. We measured the transposition of Ty1 in various genetic backgrounds and discovered that Tof1 suppressed excessive retromobility in collaboration with either Rrm3 or the F-box protein Dia2. Although both Rrm3 and Dia2 are believed to facilitate fork movement, fork stalling at DNA-protein complexes did not appear to be a major contributor to enhancement of retromobility. Absence of the aforementioned proteins either individually or in pair-wise combinations caused karyotype changes as revealed by the altered migrations of the individual chromosomes in pulsed field gels. The mobility changes were RNase H-resistant and therefore, unlikely to have been caused by extensive R loop formation. These mutations also resulted in alterations of telomere lengths. However, the latter changes could not fully account for the magnitude of the observed karyotypic alterations. We conclude that unlike other checkpoint proteins that are known to be required for elevated retromobility, Tof1 suppressed high frequency retrotransposition and maintained karyotype stability in collaboration with the aforementioned proteins.

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